摘要:

Crystal structures of nucleic acids and their drug complexes Stephen Neidle and Christine M. Nunn The CRC Biomolecular Structure Unit, The Institute of Cancer Research, Sutton, Surrey, UK SM2 5NG 1 Introduction 2 DNA native duplexes 2.1 B-DNA structures—DNA close to the physiological state? 2.1.1 DNA bending 2.1.2 Sequence-dependent DNA structure 2.1.3 Non-standard B-DNA structures 2.2 A-DNA structures 3 DNA base-mismatched structures at the duplex level 4 Base triplets and triple-helical nucleic acids 5 DNA quadruplexes 6 RNA and DNA–RNA hybrid structures 6.1 RNA duplex structures 6.2 Mismatches in RNA structures 6.3 RNA as an enzyme 6.4 RNA–DNA hybrid structures 7 Drug–nucleic acid structures 7.1 Intercalation complexes 7.2 Groove-bound complexes 7.2.1 Complexes with Hoechst analogues 7.2.2 Complexes with netropsin and analogues 7.3 Covalent complexes 8 Acknowledgements 9 References 1 Introduction The proposal for the structure of the DNA double helix in 19531 is generally considered to be the point at which genetic phenomena started to be understandable in molecular and chemical terms.Indeed Watson and Crick were themselves directly dependent for their success on the chemical knowledge of nucleotides from the work of Todd and his school, as well as relying heavily on X-ray crystallographic studies of nucleobases and nucleosides.2 The Watson–Crick structure is a model based on the fibredi Vraction data of Franklin and Wilkins.As such, it represents a low-resolution structure averaged over all sequences and conformations. More detailed descriptions of DNA structure, and of its wide variability, had to await the availability of pure quantities of defined oligonucleotide sequences in the 1970s. These have enabled single crystals of a wide range of DNA (and now RNA) structural types to be obtained and their structures solved, in a large part without recourse to any assumptions about particular models.In addition, the crystal structures of a large number of biologically-relevant drug–nucleic acid and protein–nucleic acid complexes have now been solved. The latter is an area of especially rapid growth, and will not be discussed here.3 This review will survey the significant crystal structures of nucleic acids and their drug complexes published over the past few years, which have seen an exponential growth in the oligonucleotide structures reported in the literature. The attention of readers is drawn to the comprehensive and up-to-date compilation of all these nucleic acid single-crystal structures in the Nucleic Acid Database,4 with 618 entries as of February 1997.Background information on nucleic acid structures, together with reviews on such topics as fibre diVraction from polynucleotides and NMR studies in solution, is available elsewhere.5–8 As with all crystal structures, considerations of accuracy and reliability are important when analysing and comparing structures.Nucleic acid structures are generally not at atomic resolution, with 2.0–2.5 Å being typical. This means that their crystallographic refinement requires the use of constraints and restraints on geometry and conformation. Refinement procedures themselves, as well as the individual geometric parameters used, have significantly changed over the past few years, which aVects any detailed comparison of structures.9 The X-PLOR program10 is now almost universally the choice for all nucleic acid-containing structures, involving the use of an empirical force-field.This is generally considered to be well-parameterised for nucleic acid duplexes, but may not be adequate for the increasing number of structures with nonstandard base–base interactions and folds, especially of RNA molecules. Extra parameters are invariably required for nucleic acid–drug complexes; however these are rarely available in the primary literature so it is not straightforward to assess the validity of such parameterisations. 2 DNA native duplexes 2.1 B-DNA structures—DNA close to the physiological state? Fibre-diVraction studies of helical natural and synthetic polynucleotides have characterised their structures in terms of precisely-repetitious mono- or di-nucleotide units, with only small variations being observed with particular sequences. The structure of the first fully base-paired full turn of double helix to be determined by single-crystal methods,11 using multiple isomorphous phasing, was that of the sequence d(CGCGAAT TCGCG), at 1.9 Å resolution.This structure shows, by contrast, a number of sequence-dependent features, such as a narrow minor groove in the AT region, high propeller twists for the A·T base pairs and considerable variation in base and base-step helical parameters such as helical twist and roll. These features are sensitive to the protocols used in the crystallographic refinement,12 which further underlines the inadvisability of deriving general conclusions about the detailed features of sequence-dependent DNA structure from one of even a few crystal structures.The AT region of the minor groove in d(CGCGAATTCGCG) was found to contain an ordered array of water molecules, the ‘spine of hydration’, whose existence has more recently been confirmed by NMR spectroscopic13 and simulation14 studies. There is an increasing realisation of the importance of structured water molecules, not only in stabilising particular aspects of DNA (and RNA) structure, but in acting as probes for nucleic acid recognition.15 A large number of variants16 of this dodecamer sequence, mostly with small changes in the central 4–6 base pairs, have subsequently been analysed.Almost all crystallise in the same space group (P212121), with interdigitation of adjacent helices in the crystal lattice and involvement of the terminal two base pairs at each end of an individual duplex.The fact that the central 6–8 base pair region in this packing arrangement is unaVected by adjacent molecules in the lattice, suggests that it is suitable for systematic studies of features occurring in this region, such as base mismatches and drug binding.17 It can be argued that the dodecamer duplex is less suitable, at least on its own, for comparative studies of sequence-dependent features, since ideally one would wish to observe a particular sequence type in more than one crystallographic context in order for Neidle and Nunn: Crystal structures of nucleic acids and their drug complexes 1lattice packing eVects to be minimised.18 An initial result of surveys which included decamer as well as dodecamer structures (see below) was the finding that there appears to be considerable variability in the geometries of many base steps and individual short sequences, and therefore that a search for sequence-dependent features is pursuing an illusory goal.It has even been suggested that the variations in structural features merely represent distortions arising from crystal packing forces. However a more realistic interpretation is that the variability observed for many features in these B-DNA structures, are real eVects, and represent their degrees of flexibility. Analysis of structure and conformation for ensembles of structures via the Nucleic Acid Database is an eVective approach. It has been used to show19 that phosphodiester backbone torsion angles in the higher resolution sub-set of B-DNA oligonucleotide structures cluster in discrete regions.The majority of these have been previously noted in studies of individual structures, but several new clusters are now apparent, which suggest hitherto unsuspected structural correlations and modes of concerted flexibility between backbone torsion angles. 2.1.1 DNA bending The dodecamer duplexes reported to date (apart from those involving mismatches or bound drug), are almost all of the type d(CGCX6GCG), where X6 is an AT-containing sequence, and where the overall sequence is self-complementary. Structures with runs of both alternating and non-alternating A·T base pairs have been reported.The structure20 of the non-self-complementary sequence d(CGCGAAAAAACG), crystallised with the sequence d(CGTTTTTTCGCG), is of particular interest. It is non-isomorphous with almost all other dodecanucleotide structures, even though it is still in the same space group (P212121).The extended AT tract has typically narrow minor groove and high propeller-twisted A·T base pairs. The helix has an overall bend of 30) in the major groove direction, which is somewhat distinct from the bending towards the major groove observed in the crystal structures of the various members of the d(CGCGAATTCGCG) duplex family. All of these crystal structures appear to diVer from the phased bending of A-tracts in solution, usually within rather longer sequences, in which bending has been inferred to occur towards the minor groove direction.21 The definition of bending in molecular terms for DNA containing A-tracts, continues to be controversial.The contrast in interpretations from solution and crystallographic studies has been ascribed to the eVects of the hydrophobic alcohols such as 2-methylpentane-2,4-diol (MPD) commonly used in the crystallisation of oligonucleotides.22 It has been suggested that MPD has the ability to markedly decrease DNA curvature, in accord with its eVect of reducing the anomalous behaviour of A-tract DNA in gel retardation experiments.This interpretation has been challenged in a comprehensive study23 of all the crystal structures which show bending, where it is pointed out that, even though MPD does indeed have some eVect on bending, it does not involve simple dehydration around the DNA, and moreover does not actually shed any light on the molecular basis of the phenomenon.There is no correlation between the degree of bending observed in the crystal, and the MPD concentration. These authors conclude that the crystallographic observations are consistent with bending occurring outside the A-tracts themselves, which remain straight. The details of the structural changes at these interface sequences have been suggested by several crystal structures,24–26 although none with true phased A-tract sequences have as yet have been reported.A comparative study27 of four oligonucleotide crystal structures, combined with gel retardation experiments, has provided further support for A-tract models with a narrow minor groove, high propeller twist for the A·T base pairs, cross-strand major groove bifurcated hydrogen bonds. In all these structures, the A-tract itself is straight, with bending at the flanking sequences. It is clear that there is no unique direction of the bending, and DNA sequences will deform in ways that are dictated by environment and the nature of the molecules with which it interacts.This is shown on the one hand by the consistent bending towards major groove directions shown by the oligonucleotide crystal structures, indicating that this mode of bending is accessible to even the comparatively lattice forces in a crystalline environment. On the other hand, it is typical for the DNA in protein–DNA complexes27 to bend in the minor groove direction, suggesting that this type of bending requires the greater enthalpy of interaction with a protein.An extreme example of DNA bending has been observed in oligonucleotide complexes with the TATA-box binding protein,28 where the DNA is bent by ca. 80), but now towards the major groove. This is accompanied by unwinding so that the DNA most aVected assumes a non-B-DNA A-like conformation, which can be modelled by simple deformations involving local conformation changes.29 2.1.2 Sequence-dependent DNA structure The relevance of crystal structures of native oligonucleotides containing gene-regulatory protein recognition sequences, to the structures of the protein–DNA complexes, has been demonstrated in several studies.The crystal structure30 of the trp operator/repressor complex incorporates the six base-pair recognition site d(ACTAGT). The crystal structure31 of the native decanucleotide duplex d(CCACTAGTGG), shows features of structure and hydration directly analogous to those in the protein complex.For example, the depth and contours of the major groove are similar for this sequence in both native and protein-bound states, as is the pattern of base hydration. Ten hydration sites in the major groove are conserved in the two states; the three which mediate critical protein–DNA contacts also have conserved hydrogen-bonding geometries. It is suggested that these features are intrinsic to this particular DNA sequence.On the other hand, a study32 of a constituent of this sequence, the tetranucleotide d(CTAG), which occurs in both the trp and met repressor–operator complexes, has shown that when compared with its structure in the native sequence d(CTCTAGAG), its conformation is somewhat variable. This variability is most pronounced at the central TpA step. The implication of these comparative studies is that the details of conformation, helical parameters and hydration at particular sequences, as seen in native oligonucleotide crystal structures, can have relevance to the situations in protein complexes.However the relationship between sequence and structure is complex, and still only imperfectly understood at the native DNA level, in spite of much eVort over the past 15 years. There have been a large number of studies which have attempted to correlate sequence-dependent structural features in native oligonucleotide structures. This initially focused on the ten distinct dinucleotide steps, but as an increasing number of oligonucleotide structures have been determined, it has been realised that the essential minimal unit of structure description is the tetranucleotide sequence, of which there are 134 variants.33, 34 Even at the dinucleotide level, it is apparent that only some XpY steps show consistent behaviour.This has been observed where a particular sequence has been crystallised in more than one packing arrangement.For example, the sequence d(CCAACITTGG) (where I is the non-natural nucleoside inosine) occurs in both monoclinic and trigonal space groups.35 The helical twists at the CpA and TpG dinucleotide steps are nearly 15) greater for the monoclinic structure. The TpA step has also been found to have a variable geometry, as observed36 in two structures [of d(CGCTAGCG) and d(CGCTCTAGAGCG)]. These observations of variability in structural features have been interpreted as being due to crystal packing eVects, although there is little evidence to demonstrate this for 2 Natural Product Reports, 1998particular runs of sequence. Rather, these observations are showing the inherent flexibility of some dinucleotide and tetranucleotide steps, which when observed in diVerent structures, are sampled at diverse points on their broad energy surfaces.This interpretation suggests that consideration of the available ensemble of structures, as has been done for backbone torsion angles,18 could provide a fuller picture of the flexibility available to the diVerent steps in an oligonucleotide.An examination37, 38 of 60 native structures, on the basis solely of the ten dinucleotide steps, has derived an overall kinematic classification involving base-pair slide, roll and helical twist. This analysis has confirmed and extended predictions made on the basis of empirical force-field calculations.39 These emphasise,40 on the one hand, the importance of electrostatic contributions to those relative motions of bases (shift and slide) which retain them parallel to each other, and on the other, van der Waals interactions which primarily contribute to those motions (such as roll, tilt and slide) which do not retain base parallelism. A comparative analysis of dinucleotide steps is valid since, with the exception of the under-represented ApG step, the available database of structures is now suY- ciently representative of all nine other steps for statistically meaningful conclusions to be drawn.However, it should be borne in mind that such an analysis necessarily ignores nearestneighbour and longer-range eVects, since few of the 134 tetranucleotide steps are represented in the oligonucleotide database. The available evidence is that these eVects (for example, the influence of an adjacent A-tract) can sometimes dominate structure at a particular sequence locus.41 2.1.3 Non-standard B-DNA structures The crystal packing motifs of many B-DNA oligonucleotides involves either the end-to-end stacking of helices (many decanucleotide structures) or the inter-digitating of the ends of helices (in almost all dodecanucleotide structures).A notable sub-class of the former has the end-to-end helices packed not in side-by-side parallel arrangements with respect to each other, but inclined at 40–60) to form crossed helices. This motif has been recognised as being a model for four-way junctions envisaged to occur during genetic recombination.It involves groove–backbone intermolecular close contacts42 (Fig. 1), involving the major groove of one helix and phosphate groups from another.43 Molecular modelling has been used to generate plausible four-way junctions models from these structures.44 The sequence d(CGCAATTGCG) forms distinct crystal structures, dependent on environmental conditions. One is of a fully base-paired decamer duplex,44 whereas the other has the 3* and 5* terminal nucleotides swung out from the helix and hydrogen bonding in the grooves of symmetryrelated duplexes.45 The crystal structure of the non-selfcomplementary decamer sequence d(CGACGATCGT) shows a helical octamer duplex stem formed by d(ACGATCGT) and its complement, together with a 5*-d(CG) sticky end.46 2.2 A-DNA structures The A polymorph of double-helical DNA was identified in early fibre-diVraction studies, as being formed under conditions of low relative humidity.A-type structures are characterised by having base pairs inclined with respect to the helix axis and significantly displaced from it, thus changing the dimensions of both major and minor grooves compared to B-type structures. The biological significance of the A family of DNA structures continues to be controversial. The determination of the structure of the TATA-box protein–DNA complex, which has shown that deforming a region of a B-DNA structure into an A form produces a widened minor groove and an overall bend of the DNA.28, 29 This suggests a role in protein-induced bending, and that A-type structures are most likely to exist when forced to by external factors such as crystal packing or protein binding.It is striking that no NMR studies of native DNA duplexes have revealed the existence of the A form in solution, i.e. when removed from these factors. The A family has been observed in numerous crystal structures of self-complementary octanucleotides, most likely as a consequence of specific crystal packing constraints,47–49 as well as in a number of decanucleotides with a high proportion of C·G base pairs.Considerable variability, in particular, in groove width, has been observed in these structures. The packing motifs of A-DNA oligonucleotides invariably involve interdigitation of the terminal base pairs from one duplex into the minor groove of an adjacent one in the lattice. The importance of crystallographic studies on this family of oligonucleotides is that they provide insight into the range of structures, and hence the flexibility of the A-type structural class, which in turn illuminates the mechanisms whereby some gene-regulatory proteins deform DNA.Several polymorphic crystal structures of d(CCGGGC CCGG)50 and two cytosine-methylated analogues have been determined, all of which require the polycationic amine spermine for crystallisation, and the more compact show ordered spermine molecules in the crystal structures.In common with the A-DNA octamer crystal structures, conformations of most of these polymorphs diVer significantly from that of classic fibre-diVraction A-DNA. For the three polymorphs of d(CCGGCC5meCGG), the number of residues per complete turn of helix ranges from 10.7 to 11.6, and the average inclination of base pairs to the helix axis ranges from 10.7 to 18.2). The hydration of A-DNA structures has been found to Fig. 1 Groove–backbone interactions in the crystal structure of the DNA decamer d(CGCAATTGCG) (ref. 44) Neidle and Nunn: Crystal structures of nucleic acids and their drug complexes 3be sequence-dependent,51 and the superior hydration of CpG base pairs compared to TpA correlates with diVerences in helical twist and base-pair roll.52 The sequence d(AGGCATGCCT) forms an unprecedented structure,53 with the central eight nucleotides base-pairing with a symmetry-related strand to form an A-form octamer duplex.The terminal bases are swung away from this short helix to form A·T base pairs with other symmetry-related molecules, forming an overall infinite chain-like arrangement. 3 DNA base-mismatched structures at the duplex level DNA bases can be covalently modified by the alkylating eVects of a wide range of chemicals. The resulting base lesions are mutagenic, and can ultimately produce cell transformation and cancer, if they are not correctly repaired by cellular repair enzymes.The recognition of lesions by these enzymes is likely to be based on the structural diVerences resulting from them. A number of crystal structures have examined DNA sequences with guanine methylated at the O6 position, a consequence of exposure to carcinogenic agents such as N-nitroso-Nmethylurea and N-methyl-N-nitrosoguanidine. (O6Me)Guanine· · ·cytosine base pairing has been observed in a left-handed Z-DNA structure,54 with standard Watson– Crick rather than wobble-type hydrogen bonding between the aVected bases (Fig. 2). This arrangement can only occur if there is protonation of the guanine or the cytosine, or if one or other is in a non-standard tautomeric state. It is likely that this is a consequence of the particular base-stacking requirements in Z-DNA, which would not favour a wobble arrangement that would have the methylated guanine partially unstacked and protruding into the major groove. The O6 methylation event results in GC]AT transition mutations, and thus the formation of a O6G· · ·T base pair during replication.The structures of two dodecanucleotide duplexes containing this mismatch have been reported.55, 56 The base pairing in both is analogous to the normal Watson– Crick arrangement (Fig. 3), whereas NMR solution studies57 have suggested that the N1G· · ·N3T hydrogen bond is absent or very weak. The origin of this diVerence is not clear, other than that it may reflect diVerences between crystal and solution environments. 4 Base triplets and triple-helical nucleic acids It has long been known that a third nucleic acid strand comprising pyrimidines can associate with a stretch of duplex consisting of purines on one strand and pyrimidines on the other, to form a triple helix.57 This parallel triple helix involves Hoogsteen hydrogen-bonded base triplets of the form T–A·T and C+–G·C, and can be either inter- or intra-molecular. In both instances, the third strand occupies the major groove of the initial duplex.The stringent sequence requirements of triple helix formation are currently being explored for the artificial down-regulation of the expression of particular genes, ultimately for therapeutic purposes.58 The structural details of DNA triple helices remain elusive; fibre diVraction studies have only been at low resolution and have been interpreted in terms of both A- and B-type conformations. No single-crystal structure of a pure DNA triple helix is as yet available, in spite of much eVort in a number of laboratories.The structure of a mixed DNA–peptide nucleic acid (PNA) has been reported.59 It has a standard DNA purine strand together with PNA sequences forming the two pyrimidine strands having a hexapeptide linking them in a hairpin-like manner (Fig. 4). The resulting triple helix (Fig. 5) has helical features of both A- and B-DNA, with bases approximately perpendicular to the helix axis (i.e.B form) yet significantly displaced from it (i.e. A form). It is not clear to what extent features such as the very wide major groove, reflect the non-nucleic acid nature of the PNA backbone. A short two-triplet stretch of C+–G·C triplets have been observed in a drug–decanucleotide crystal structure where the terminal nucleosides do not form part of the duplex but interact with neighbouring duplexes in the lattice, to form this triplet.60 Anti-parallel triple helices can be formed with third-strand purines and involve, for example, G–G·C base triplets.Again, there is no crystal structure of such a triple helix. The triplet base arrangements have been observed in several crystal structures of duplexes with over-hanging ends [d(GCGAAT TCG)61, 62 and d(GGCAATTGG)63], or with mismatches [d(GGCAATTGCG)].64 In all instances the triplets are formed by intermolecular hydrogen bonding of bases. 5 DNA quadruplexes The richness of possibilities for DNA structures is seen at its most striking for sequences containing either runs of either G,T or C,A nucleotides.These sequences are of considerable biological importance in view of their occurrence as repeated sequences, predominantly at the ends of chromosomes, forming so-called telomeres. Telomeric sequences can form a variety of four-stranded structures, all of which necessarily contain non-standard base-pairings. Fig. 2 The (O6Me)G·C base pair (ref. 54) Fig. 3 The (O6Me)G·T base-pair in the crystal structure of the d[CGTGAATTC(O6Me)GCG]2 duplex (ref. 55) Watson–Crick base-pairing +H3N C T C T T C T T C 3¢–G A G A A G A A G–5¢ –OOC C T C T T C T T C His Gly Ser Hoogsteen base-pairing His Gly Ser Fig. 4 Schematic view of the PNA:DNA triplex (ref. 59) 4 Natural Product Reports, 1998The cytosine-rich sequences form four-stranded intercalated complexes.65 That formed by the sequence d(CCCT) is typical, with two parallel-stranded duplexes intercalated into each other (Fig. 6), and the arrangement as a whole being formed by four d(CCCT) strands.66 Each duplex has cytosine· · ·cytosine base pairs (Fig. 7), with one of them required to be protonated (even though crystallisations were successful at pH 6 or even 7). The thymines do not actively participate in the duplexes, although some of them are stacked onto the ends. The cytosine rings are not directly stacked on top of one another. Instead, amino and carbonyl substituent groups form stacks, separated by 3.1 Å rather than the normal 3.4 Å in oligonucleotide helices.The same arrangement of intercalated cytosine duplexes has been observed in the structure67 of the sequence d(TAACCC), corresponding to the human telomere repeat. The structure has four strands associating together so as to produce an intercalated four-stranded structure, still with parallel cytosine duplexes, but now with the 5* terminal d(TAA) sequences forming intermolecular loops held together by A· · ·T base pairings (Fig. 8). One of these has a Hoogsteen arrangement, whilst the other shows reverse Watson–Crick pairing. The crystal structure of the sequence d(CCCAAT) has the intercalation motif extended by adenine· · ·adenine base pairs as part of the arrangement of parallel duplexes. The 3* terminal thymines participate in a variety of intermolecular A·A·T base triplets, which serve to stabilise the crystal structure. Although these C-rich sequences all appear to occur in telomeres within duplexes (in contrast to the G-rich repeats— see below), the fact that stable four-stranded structures can be formed by them with individual short sequences, suggests that if biological C-rich sequences became looped-out of the duplex they might then be stabilised by an intramolecular four-stranded arrangement.Such loops could occur as a result of negative supercoiling. The G-rich single strand of telomeres has repetitive sequences of the type d(TTTTGGGG)n (in Oxytricha), d(TGGG)n (in budding yeast) and d(TTAGGG)n (in Homo sapiens).Four-stranded structures can be formed in a variety of ways, by intramolecular folding of several consecutive repeats, by intermolecular association of four strands or by a combination of both.68 The crystal structure69 of the sequence d(GGGGTTTTGGGG) shows a stack of sets of the G-quartet motif (Fig. 9), four guanine bases hydrogen-bonded together in one plane (Fig. 10). The four thymines form loops between the stacks, as shown.A subsequent NMR spectroscopy study70 has reported a diVerent arrangement for the four strands, whilst retaining the same arrangement of stacked G-quartets. The structure of the sequence d(TGGGGT) has been determined at 0.95 Å resolution,71 the highest resolution to date of any nucleic acid structure. It consists of four individual strands associated by means of a stack of four G-quartets. Sodium ions stabilise the structure, between and within the G-quartets.An unconventional four-stranded structure is formed72 by the sequence d(GCATGCT), with two strands associating together Fig. 5 The crystal structure of the PNA:DNA triplex (ref. 59) Fig. 6 The four-stranded intercalated structure formed by d(CCCT) (ref. 66) Fig. 7 A C+·C base pair, of the type found within the C-rich quadruplex structures Neidle and Nunn: Crystal structures of nucleic acids and their drug complexes 5(Fig. 11), each folding back so that two quartets of (CG)2 are formed.This structure suggests that quartet-type structures may be more prevalent than hitherto supposed, since there is no longer a restriction on quartets to solely contain guanines. 6 RNA and DNA–RNA hybrid structures Early X-ray crystallographic investigations into RNA structure in the 1970s were concerned, on the one hand, with transfer RNA73 and on the other with dinucleoside monophosphate mini-helices both with and without bound drug molecules.74 These short helices form right-handed antiparallel duplexes with an A-form conformation. Investigations into RNA structure over the last few years has accelerated to produce a plethora of new and exciting structures.This results in a large part from recent developments in RNA synthesis, both chemical and enzymatic, which allows for the production of large quantities of high-quality RNA for crystallisation studies. Specific RNA sequences can now be synthesised, crystallised and their three-dimensional structures determined.75 This section will survey recent X-ray crystallographic structural studies on them.In addition to pure RNA structures RNA–DNA hybrids will also be discussed. 6.1 RNA duplex structures The octamer duplex r(CCCCGGGG) has been crystallised in both rhombohedral and hexagonal lattices.76 There is little diVerence between the two diVerent crystal forms. When compared with the structure of the DNA sequence d(CCCCG GGG)77 the RNA is seen to be more extensively hydrated than the analogous DNA sequence with the ribose 2*-hydroxy groups propagating stable and conserved water networks in both grooves of the RNA duplex.Crystallographic studies of ribosomal RNA are still in their infancy. The E. coli Shine–Dalgarno consensus ribosome binding site r(UAAGGAGGUGAU):r(AUCACCUCCUUA) has Fig. 8 The crystal structure of the four-stranded intercalated structure formed by d(TAACCC) (ref. 67) Fig. 9 The crystal structure of d(GGGGTTTTGGGG) (ref. 69) N NH N N O NH2 N HN N N O H2N N HN N N O NH2 N NH N N O NH2 Fig. 10 The hydrogen-bonding arrangement in the guanine quadruplex Fig. 11 The crystal structure of d(GCATGCT) (ref. 72) 6 Natural Product Reports, 1998been found to form a duplex structure with Watson–Crick base-pairing interactions along its length.78 Two unique duplexes for this sequence show very similar conformations and both resemble calf thymus A-DNA as found from X-ray fibre diVraction studies.X-Ray crystallographic studies for the entire 5S rRNA from the thermophilic bacterium Thermus flavus79 have only produced crystals to date which exhibit very poor resolution quality (8 Å). By subdividing the entire 5S ribosomal RNA into subunits it has been possible to obtain good-quality diVracting crystals. 5S Ribosomal RNA has been subdivided into five subdomains A–E. Subdomain A comprises two RNA strands which form a double helix involving Watson–Crick base-pairing interactions in addition to being stabilised by two U·G and G·U base-pairs.80 Domain E of Thermus flavus 5S rRNA contains the very stable and highly conserved 5*-GCGA tetraloop.Crystallisation and preliminary diVraction studies have been reported81 on it and structure determination is underway. The structure of the self-complementary tetradecamer sequence r[U(UA)6A] is an A-type duplex with two kinks along its length.82 The kink angles are 8.5 and 13.8) with an overall angle between the two distal ends of the duplex of 21.7).The structure of r(UUCGCG)83 has a central (CGCG)2 duplex formed by two RNA strands with an overhang of the 5*-UU bases from each strand. These overhanging uracil bases interact with the overhang from a symmetry-related duplex within the unit cell to form novel Hoogsteen-like U·U basepairs (Fig. 12). There is one hydrogen bond between atoms O4 and N3 of two uracil bases, while there is a second, less conventional hydrogen bond between C5–H and O4.This base-pairing arrangement results in a trans U·U pair on antiparallel strands in contrast to the usual cis base-pairs. It diVers from that found in RNA dodecamer duplexes with non-Watson–Crick base-pairs. The potential importance of C–H· · ·O hydrogen bonding interactions in nucleic acid structure has been discussed.84 6.2 Mismatches in RNA structures In addition to the U·U base-pairing interactions which exist within the structure of r(UUCGCG),83 base-pairing mismatches have been observed in a number of other RNA structures [Fig. 13(a)–(e)]. Mismatched regions in RNA structure are a common secondary structural motif and termed internal loops. These loop regions act as potential protein recognition sites. The structure of r(GGCGCUUGCGUC)85 contains tandem U·U base-pairs within a dodecamer duplex in addition to two G·U mismatches. As a result, the duplex has an overall bend of 11–12). The structure of r(GGACUUUGG UCC)86 similarly contains tandem U·U base pairs.The cis U·U wobble pairs observed in both of these structures have two hydrogen bonds, with potential sites for the binding of water molecules in the major and minor grooves. Both also contain, in addition to U·U base pairs, two G·U pairs along the length of the dodecamer duplexes. In the case of r(GGACUUUGGUCC) there is a run of four mismatched base pairs. The structure of r(GGACUUCGGUCC)87 has G·U and C·U mismatches in the central third of the structure. The structure has a central two-fold symmetry axis and two unique mismatches. The G·U mismatch is stabilised by a solvent molecule in the minor groove which also interacts with the ribose hydroxy group.The structure of r(CGCGAAUUA GCG)88 has two separated G·A mismatches within the dodecamer duplex structure. The structure of r(GGCCGAA AGGCC)89 has an internal loop with G·A and A·A mismatches. Again the dodecamer duplex has a two-fold symmetry axis, with two unique mismatches.The G·A mismatches in this structure involve reverse Hoogsteen hydrogen bonding and have been termed sheared G·A base-pairs. Both G·A and A·A base-pairs are very common in internal loops of RNAs, including ribosomal RNA and ribozymes. This structure has a 34) end-to-end curvature for the helix and its diameter is narrowed by 24% in the internal loop. The large number of structures of non-self-complementary RNA sequences forming duplex structures with mismatched base-pairs has resulted from attempts to crystallise RNA sequences which have the potential to form stem-loop structures.Studies using NMR spectroscopy for such sequences have shown them to form hairpin structures under the conditions used. However under the conditions required for the crystallisation of these sequences there is a preference for duplex RNA to be formed. 6.3 RNA as an enzyme Certain RNA sequences, termed ribozymes, have the ability to either cleave other RNA molecules, or to self-cleave, by means of a catalytic mechanism.The best studied ones are the hammerhead ribozymes, which consist of three double helical regions joined by 15 highly conserved nucleotides. These 15 central nucleotides are essential for ribozyme activity and form a complex structure which mediates RNA folding and catalysis. Hammerhead ribozymes comprise two strands, one corresponding to the ‘enzyme’ and the other to the ‘substrate’. A divalent metal ion such as Mg2+ is required by the ribozyme to mediate catalytic cleavage of an RNA species.Two crystallographic studies for hammerhead ribozymes (Fig. 14) have been carried out. The first structure reported has an RNA ‘enzyme’ strand and a DNA ‘substrate’ strand.90 The DNA strand was employed in order to prevent catalytic cleavage. The second hammerhead ribozyme structure (Fig. 15) is composed entirely of RNA with a single 2*-methoxy group modification at the active site to prevent cleavage.91 The two structures have very similar structures for the catalytic core region and results from these two structural studies have led to proposals for the mechanism of RNA-cleavage catalysis.92, 93 In addition to the two hammerhead ribozyme structures the crystal structure of a self-splicing Group I ribozyme domain has been recently determined.94 This 160 nucleotide P4–P6 domain from the Tetrahymena group I intron has been reported to a resolution of 2.8 Å.The structure consists of two extended helical regions that pack side by side with longrange interactions between them. These interactions involve an A-rich bulge of one helix and the minor groove of another, together with a GAAA tetraloop at the end of one helix and a tetraloop receptor in the minor groove of the other. This structure has close packing of the ribose–phosphate backbones, which is mediated by hydrated magnesium ions. 6.4 RNA–DNA hybrid structures Short stretches of RNA–DNA hybrid structures are formed during both replication and transcription, and are important in antisense therapeutic applications where a deoxyoligonucleotide sequence is targeted to a mRNA.One RNA–DNA hybrid crystal structure determined to date is left-handed and of Z-type,95 while the remainder are all right-handed. This hybrid consists of alternating purine and pyrimidine bases Fig. 12 U·U base-pair in the crystal structure of r(UUCGCG) (ref. 83) Neidle and Nunn: Crystal structures of nucleic acids and their drug complexes 7d(CG)r(CG)d(CG) and assumes the conformation seen for all DNA sequences of this type. The octameric sequence r(GUAUAUA)d(C)96 has a 3*-terminal deoxycytidine residue at the end of a heptamer RNA sequence.This structure forms a right-handed A-form double helix. The structure of the two octamer sequences d(I)r(C)d(ICICIC) and d(I)r(C)d(I)r(C) d(ICIC) have both been determined bound to the minor groove binding drug distamycin A.97 These structures adopt a B-form structure with two distamycin A molecules lying within the minor groove of the B-form duplex (see below for a further discussion of these two structures).These are the first examples of RNA duplexes adopting the B-form family of helices, which have been considered to be less stable for RNA compared to DNA. The presence of drug binding may induce the transition from A to B form in this structure which would suggest that upon protein binding both duplex RNA and RNA–DNA chimeric structures may be able to access a B form conformation if required.The nonamer sequence r(GCUUCGGC)dBrU forms an octamer duplex structure98 with two diVerent types of base Fig. 13 Mismatched base-pairs in dodecamer duplex RNA structures: (a) r(CGCGAAUUAGCG), (b) r(GGACUUCGGUCC), (c) r(GGACUU UGGUCC), (d) r(GGCCGAAAGGCC) and (e) r(GGCGCUUGCGUC) 8 Natural Product Reports, 1998mismatches. It has a G·U base pair with wobble hydrogen bonding, while the C·U mismatch involves just one hydrogen bonded contact, in addition to a bridging water molecule.The terminal BrU-3* bases pair with another BrU via interactions involving two hydrogen bonds. A number of decamer chimeric (i.e. RNA and DNA residues in the same strand) structures have been reported. The structure of r(GCG)d(TATACGC) is of an A-type decamer duplex.99 The three decamer structures d(GGGTATACGC)/ r(GCG)d(TATACCC) (which is an Okazaki fragment), r(G)d- (CGTATACGC) and d(GCGT)r(A)d(TACGC)100 all assume A-type duplex conformations. For d(GGGTATACGC)/ r(GCG)d(TATACCC) there is no diVerence in backbone conformation between the r(GCG)·d(CGC) portion of the structure and the d(TATACCC)·d(GGGTATA) all-DNA helical region.The three decamer structures d(CCGGC)r(G) d(CCGG),101 r(C)d(CGGCGCCG)r(G)102 and r(GC)d(GTAT ACGC),103 with one or two ribose bases along the decamer length, all adopt an A-type conformation.One structure of a DNA duplex with a 2*-O-methylribonucleotide insert104 has been reported. The decamer sequence r(UUCGGGCGCC)/d(GGCGCCC GAA)105 is specifically recognised by the ribonuclease H function of HIV reverse transcriptase. The structure of this sequence has neither an A- or B-type conformation but instead has characteristics of both. The structure of r(GCG) d(ATATA)r(CGC) has been determined in two diVerent crystal forms.106 This structure (Fig. 16) contains a single adenosine bulge—a common secondary structural motif in RNA. 7 Drug–nucleic acid structures A wide range of drugs are known to exert their biological eVects by means of interactions with cellular nucleic acids, especially with chromosomal DNA.107–108 The majority are anticancer agents, but antibacterial, antiviral, antifungal and antiparasitic activities have also been found for some of them. The DNA sequence selectivities shown by most of these drugs is usually modest, and even those which covalently bind to particular bases have selectivity for only short runs of sequence.Thus the biological selectivities shown, for example by the clinically-useful anthracycline anticancer drugs, cannot be solely ascribed to DNA binding. There is increasing evidence that interference with particular protein–DNA interactions is critical for biological activity.109–111 For example, the anthracyclines and related drugs have been shown to stabilise the cleavable complex between DNA and the enzyme DNA topoisomerase II.A number of covalent and noncovalent DNA minor-groove agents compete with transcription factors, and so interfere with gene regulation. There is as Fig. 14 Schematic representation of the two hammerhead ribozyme structures (refs. 90 and 91) Fig. 15 The crystal structure of the all-RNA hammerhead ribozyme (ref. 91). The two RNA strands are shown in diVerent colours Fig. 16 The crystal structure of r(GCG)d(ATATA)r(CGC) (ref. 106) Neidle and Nunn: Crystal structures of nucleic acids and their drug complexes 9yet no structural data on any drug–DNA–protein ternary complex, raising the question of the relevance of structural studies on drug–DNA complexes alone. It is reassuring that these have been at least partly successful in rationalising structure–activity behaviour, suggesting that the structures of the binary complexes have at least some relevance to those of ternary complexes. Crystallographic studies have concentrated on the intercalative and minor-groove categories of complexes, with few exceptions.Very few covalent complexes have been reported, in spite of the biological and medicinal importance of drugs such as mitomycin and the nitrogen mustards. The problems associated with obtaining significant quantities of pure oligonucleotide adducts of these drugs have been largely overcome, as attested by the numerous NMR studies on them. The experience in this and other laboratories points to the problem being largely of diYculties in obtaining crystals suitable for studies even at medium (ca. 2.5 Å) resolution. 7.1 Intercalation complexes The first drug–DNA crystal structures to be determined were of structurally-simple intercalating molecules, typified by the acridine proflavine,74 bound to dinucleoside monophosphate mini-duplexes. These structures showed the planar drug chromophore sandwiched between the two base pairs of the dinucleoside duplex, but were unable to address issues of conformational change in a nucleic acid beyond the immediate intercalation site.This has to some extent been addressed by subsequent structural studies on a large number of complexes involving the clinically-important anthracycline anticancer drug daunomycin (Fig. 17) and many of its derivatives, all complexed with hexanucleotide duplexes.112 As of May 1997, there were 22 entries for anthracycline complexes in the Nucleic Acid Database.4 Most crystallise in the tetragonal space group P41212.In general, these structures have two drug molecules bound per hexamer duplex, one each at the terminal base-pair sites, and with the daunosamine sugars lying in the minor groove. The semi-synthetic derivative idarubicin cocrystallises with the sequence d(CGATCG) in the trigonal space group P31. The resulting structure is essentially identical to the tetragonal ones, with invariance in the orientation of bound drug chromophore and in the drug–DNA hydrogenbond contacts being observed.113 A novel bis-daunomycin (Fig. 18) has recently been designed and synthesised114 using the established anthracycline-hexamer crystal structures as a starting-point. The semi-rigid linker between the two anthracycline chromophores was chosen so as to preserve the position and orientation of each, as seen in these monomer structures. The new compound bis-intercalates into duplex DNA with high aYnity and shows promising ability in cytotoxicity studies to circumvent multi-drug resistance in tumour cells.The crystal structure115 of the bis-daunomycin compound complexed with the sequence d(CGTACG) (Fig. 19) shows that many of the predictions are realised, with the linker positioned in the minor groove of the hexamer duplex. It is surprising that the linker appears to have very few close van der Waals contacts with the groove surface, suggesting that modifications to the structure of the linker might result in further enhancements of DNA aYnity.The related antitumour antibiotic nogalamycin (Fig. 17) also preferentially intercalates at pyrimidine-3*,5*-purine sites, but via a threading mechanism. Crystallographic studies, again on hexanucleotide duplex complexes, have shown that this anthracycline binds with the nogalose and aminoglucose groups lying in the minor and major grooves, respectively (Fig. 20). A complex with the sequence d(TGTACA) has shown the drug bound in the two high-aYnity TpG sites,116 and highlights the contribution of solvent-mediated contacts to the observed sequence selectivity of this drug. The complex between nogalamycin and the sequence d(CCCGGG) is notable117 in that it uniquely shows a 1:1 anthracycline–duplex complex, with the one drug molecule bound at the central CpG site (Fig. 20). The conformation of the unwound DNA has features of both A- and B-type helices. This structure is thus representative of anthracyclines bound in extended lengths of DNA sequence.Intercalation at the centre of extended sequences has also been observed118 for the antitumour antibiotic actinomycin (Fig. 21), showing this antitumour antibiotic bound at the central GpC site of the sequence d(GAAGCTTC). The two cyclic pentapeptide rings lie in the minor groove, and there is a predicted set of hydrogen bonds from threonine residues to N2 atoms of the two central guanines. The same DNA sequence O O O OH O O Me OH +NH3 O O OH OH O O Me O Me O OH Me O OH OH O Me O O N Me Me O Me Me Me Me HO OH Me O ( a) ( b) Me Fig. 17 (a) Daunomycin and (b) nogalamycin + + O O Me OH NH2 OMe O OH O OH Me O OH H2N O HO Me O Me O HO OH OH O O OMe Fig. 18 The bis-daunomycin molecule (ref. 115) 10 Natural Product Reports, 1998has also been cocrystallised with N8-actinomycin, where the 8-position in the phenoxazone ring has been replaced by a nitrogen atom.119 The interactions of meso-substituted porphyrins with DNA have been much studied.The crystal structure of the tetrapyridyl porphyrin TMPy with the sequence d(CGTACG) shows120 that intercalation of the central planar porphyrin ring system is accompanied by flipping-out of one nucleoside from the intercalation site. This is not unexpected, given the stringent steric requirements of the bulky porphyrin system with respect to intercalation. 7.2 Groove-bound complexes A large group of drugs bind non-covalently in the minor groove of AT-rich regions of B form DNA duplexes.Structural studies have focused on the nature of this sequence selectivity, which is currently being extensively exploited in the design of analogues with selectivities for mixed-sequence DNA.121–125 Particular use has been made of the selfcomplementary dodecamer duplex sequences d(CGCXAATT YGCG)2, where X=A or G and Y=T or C, since the central regions are largely unaVected by intermolecular contacts in the crystal, other than with water molecules.The pattern of hydrogen bonding in these complexes has been systematically examined.126 The crystal structures of a number of complexes with drugs typified by pentamidine, berenil and their analogues, have been reported.127–130 These have shown that hydrophobic interactions with hydrogen atoms from the nucleotide backbone (H1*, H4* and H5*, which line the walls of the minor groove), are in large part responsible for the AT-selectivity of these drugs.The minor groove is narrow at AT sequences and its width is close to the cross-sectional diameter of these drugs. Hydrogen bonding to A·T base-pair edges is, by contrast, relatively weak and variable. 7.2.1 Complexes with Hoechst analogues A second category130 of non-covalent minor-groove drugs, typified by netropsin, distamycin and Hoechst 33258 (Fig. 22), tend to have fixed patterns of hydrogen bonding to base pair edges, to N3 of adenines and O2 of thymines. Thus the two benzimidazole groups in Hoechst 33258 and its analogues consistently form two bifurcated pairs of hydrogen bonds to base edges (Fig. 23), extending over three base pairs in total.131–133 The analogue with three linked benzimidazole groups134 follows the same pattern of hydrogen bonding, with now three pairs of hydrogen bonds, covering four A·T base pairs. This ligand has an overall binding site of ca. 7.5 bases, so that it covers three quarters of a complete turn of a B-DNA double helix.In order for the three benzimidazole groups to be in register (in phase) with the four successive A·T base pairs, one end of the ligand is not in close van der Waals contact with the floor of the minor groove, so that the ligand is overall not isohelical with the contour of the groove. The isohelicity principle135 is also seen to be violated in the crystal structure of an amidinium analogue of Hoechst 33258 bound to d(CGCGAATTCGCG)2, where there is eVective binding to DNA, yet with the drug not having a concave inner curvature to match minor groove isohelicity.136 Here, the dominance of hydrogen bonds from one amidinium group is suYcient to overcome the usual requirement for eVective van der Waals contacts along the length of the ligand.Altering AT selectivity for GC by simple replacement of a hydrogen bond Fig. 19 Crystal structure of a bis-daunomycin–d(CGTACG) complex (ref. 115) Fig. 20 Crystal structure of nogalamycin complexed with d(CCCGGG) (ref. 117) O N Me CO OC O Me HN NH L-Thr L-Thr D-Val O L-Pro L- N-MeVal Sar O D-Val L-Pro Sar L- N-MeVal Fig. 21 Actinomycin Neidle and Nunn: Crystal structures of nucleic acids and their drug complexes 11donor in the minor groove by an acceptor (for the N2 group of guanine), has been shown to be ineVective in the case of the meta-hydroxy analogue of Hoechst 33258, contrary to theoretical predictions.137 The hydroxy group is positioned insufficiently deeply into the groove for such an interaction to occur, and the analogue is thus not able to actively recognise G·C base pairs. 7.2.2 Complexes with netropsin and analogues The antibiotic netropsin (from Streptomyces netropsis) is in many ways the paradigm for minor groove binding drugs, although several crystal structures have shown it bound in subtly diVerent ways within AT sequences. A careful study138 has been made of optimal refinement and electron-density map interpretation for the complex with d(CGCGAATTCGCG)2 has confirmed the original assignments of bifurcated hydrogen bonds to the AT base pairs, analogous to those formed by the benzimidazole drugs.That this result is not an artefact of dodecamers was shown by the crystal structure60 of netropsin bound to the decamer sequence d(CGCAATTGCG)2, where the same pattern of drug–DNA hydrogen bonding was observed as with the dodecamer complex. The lexitropsins have been developed as netropsin analogues, where methylpyrrole ring(s) have been replaced by methylimidazole rings in order to attempt to switch recognition from AT to GC sequences. As with the Hoechst analogue above, the lexitropsins have not fulfilled these predictions, and a crystal structure of a lexitropsin–dodecanucleotide complex139 shows the methylimidazole ring to be lying in the AT region, as in netropsin itself.An alternative way of recognising GC sequences has been developed, based on the observations from oligonucleotide crystal structures, that such sequences tend to have a wide minor groove.The ability of the monocationic antibiotic distamycin to form side-by-side dimers in such regions, has been the basis of fruitful studies on the design of ligands which can select mixed DNA sequences with high aYnity and specifi- city.123, 125 Several crystal structures have been reported for distamycin complexes with sequences such as d(ICICICIC)140 and d(ICITACIT),141 as well as variants containing DNA/ RNA chimers.97 In all cases, two distamycin molecules are bound per duplex (Fig. 24), which assumes a B-form structure, with a minor groove widened to 7.8 Å to accommodate the two drug molecules. Each distamycin hydrogen bonds to the nearest strand, with backbone amide nitrogen atoms acting as donors to thymine O2 and adenine N3 atoms, in a manner analogous to the hydrogen seen in distamycin 1:2 complexes. 7.3 Covalent complexes The crystal structures of only two covalently-linked drug– DNA complexes involving significant lengths of oligonucleotide, have been reported to date.That of the clinicallyimportant antitumour drug cis-platinum has the platinum linked to N7 atoms of adjacent guanine bases in the sequence d(CCTCTG*G*TCTCC):d(GGAGACCAGAGG), where G* represents platinated guanine,142, 143 i.e. with cis-platinum bound only on one strand. There are two duplexes in the asymmetric unit, and both show significant bending at + Netropsin Distamycin + Hoechst 33258 Anthramycin N NH N HN Me O Me O N NH N HN NH2 NH O Me O Me O NH2 N NH O Me NH N N NH N NH N HN Me OMe O CONH2 H H OH NH O HN H2N NH2 + + NH2 H2N H Fig. 22 Several groove-binding molecules Fig. 23 Crystal structure of the complex between the DNA duplex d(CGCGAATTGCGC) and a bis-benzimidazole compound (ref. 133) 12 Natural Product Reports, 1998the platination site, of 26) towards the major groove side. The minor groove is opened up, in a manner reminiscent of the DNA bending observed in the structures of TFIIIA–DNA complexes,27–29 with backbone geometry having features of both A- and B-form DNA.It is suggested that the platinuminduced bending of DNA could provide a signal for protein binding, especially for those involved in DNA damage repair. 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ISSN:0265-0568    

DOI:10.1039/a815001y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

The biosynthesis of shikimate metabolites Paul M. Dewick School of Pharmaceutical Sciences, University of Nottingham, Nottingham, UK NG7 2RD Covering: 1995 and 1996 Previous review: 1995, 12, 579 1 Introduction 2 The shikimate pathway 2.1 DAHP synthase 2.2 3-Dehydroquinate synthase 2.3 3-Dehydroquinase 2.4 The quinate utilization pathway: quinate dehydrogenase and 3-dehydroshikimate dehydratase 2.5 Shikimate kinase 2.6 EPSP synthase 2.7 Chorismate synthase 2.8 Chorismate mutase 2.9 Phenylalanine and tyrosine 2.10 Aromatic amino acid hydroxylases 2.11 Tyrosinase and melanin 2.12 p-Aminobenzoic acid, anthranilic acid and related compounds 2.13 Isochorismate synthase 3 Tryptophan and related compounds 3.1 Tryptophan 3.2 Tryptophanase 3.3 Indole-3-acetic acid and related metabolites 3.4 Indigo and indirubin 4 Phenols and phenolic acids 4.1 Phenol and tyrosine phenol-lyase 4.2 4-Hydroxybenzoic acid 4.3 Salicylic acid 4.4 Homogentisic acid 5 Phenylpropanoids 5.1 General 5.2 Phenylalanine ammonia-lyase 5.3 Hydroxycinnamic acids and esters 5.4 Coumarins 5.5 Lignins 5.6 Lignans 5.7 Tropic acid 5.8 Phenylpropenes 6 Flavonoids 6.1 Chalcones 6.2 Flavanones, flavonols, anthocyanidins and related flavonoids 6.3 Flavonoid sulfates 6.4 Carthamin 6.5 Diels–Alder-type adducts 6.6 Isoflavonoids 7 Stilbenes and dihydrophenanthrenes 8 Xanthones 9 Quinones 9.1 Naphthoquinones 9.2 Ubiquinones 9.3 Shikonin 9.4 Tocopherols 10 Cyanogenic glycosides, glucosinolates and related compounds 11 Miscellaneous shikimate metabolites 11.1 Sphagnum acid 11.2 4-(4-Hydroxyphenyl)butan-2-one 11.3 Phenylacetylenes from Asparagus 11.4 Diarylheptanoids and phenylphenalenones 11.5 Antibiotic LL-C10037· 11.6 Brominated tyrosine metabolites from sponges 11.7 Cyclohexanecarboxylic acid 11.8 3-Amino-5-hydroxybenzoic acid and mC7N units 11.9 3-Amino-4-hydroxybenzoic acid 11.10 Betalains 12 References 1 Introduction This report reviews the literature that was published during 1995 and 1996 on the biosynthesis of compounds, mainly non-nitrogenous, that are derived wholly or partly from shikimate, and continues the coverage described in Volume 12 of Natural Product Reports1 and earlier reports.2, 3 This review includes descriptions of the biosynthetic pathways, the enzymes and enzyme mechanisms involved, and brief information about the genes encoding for these enzymes.An authoritative review on the enzymology of the shikimate pathway has been published.4 Other recent reviews describe early steps in the shikimate pathway5, 6 and the molecular organization of the pathway in higher plants.7 A natural isomer of shikimic acid, namely 3-epishikimic acid 1, has been isolated along with shikimic acid from Sequoiadendron giganteum.8 The synthesis of (6R)- 2 and (6S)-fluoroshikimic acids 3 from the corresponding isomers of 3-fluorophosphoenolpyruvate has been achieved by the use of a mixture of the recombinant enzymes DAHP synthase, dehydroquinate synthase, dehydroquinase and shikimate dehydrogenase, together with the appropriate cofactors.9 2 The shikimate pathway 2.1 DAHP synthase The first reaction in the shikimate pathway involves the aldol-like condensation of phosphoenolpyruvate (PEP) 4 with D-erythrose 4-phosphate 5 giving 3-deoxy-D-arabinoheptulosonate- 7-phosphate (DAHP) 6 (Scheme 1), a reaction catalyzed by the enzyme DAHP synthase (phospho-2-dehydro- 3-deoxyheptonate aldolase).DAHP synthase enzymes fall into two distinct classes according to whether they are plantderived or microbial in origin. In microorganisms, isozymes of DAHP synthase are characterized by diVerent sensitivities towards feedback inhibition by the aromatic amino acids L-phenylalanine, L-tyrosine and L-tryptophan. In the methylotrophic actinomycete Amycolatopsis methanolica, a single DAHP synthase activity identified in the wild-type strain is inhibited by all three aromatic amino acids.10 However, a leaky phenylalanine-requiring auxotroph possessed an additional isozyme which was strongly inhibited OH OH HO CO2H OH OH HO CO2H R2 R1 1 2 R1 = F; R2 = H 3 R1 = H; R2 = F Dewick: The biosynthesis of shikimate metabolites 17by tyrosine. Tryptophan-sensitive DAHP synthase enzymes from Streptomyces coelicolor, S.rimosus and Neurospora crassa have been purified to homogeneity and their amino acid sequences have been determined.11 From the sequence data, these three enzymes appear unrelated to other microbial DAHP synthases, but are actually similar to plant enzymes.Thus, the distinction between plant and microbial enzymes now becomes less clear, with examples from bacterial and eukaryotic organisms falling into the plant class. The phenylalanine-regulated isozyme from Escherichia coli has been crystallized and preliminary crystallographic data have been reported for binary and tertiary complexes with substrates and/or phenylalanine.12 Isozymes of DAHP synthase in plants are diVerentiated by their requirements for the divalent cations Mn2+ or Co2+.The former isozyme type is typically located in the chloroplast, the latter in the cytosol. Cytosolic DAHP synthase–Co from cell cultures of carrot (Daucus carota) has been isolated, purified to homogeneity, and characterized.13 This isozyme has previously only been obtained in a partially purified form, and its properties have not been fully described.An antisense DNA construct encoding part of the DAHP synthase sequence in potato (Solanum tuberosum) has been inserted into potato cells.14 Some, but not all, of the derived transgenic plants expressing antisense RNA showed reduced levels of woundinduced DAHP synthase activity. 2.2 3-Dehydroquinate synthase 3-Dehydroquinate synthase catalyzes the conversion of DAHP 6 into 3-dehydroquinate 7 by a sequence of reactions requiring an oxidation, a ‚-elimination, a reduction and an intramolecular aldol condensation.Some of these steps undoubtedly occur non-enzymically, and current thinking is that dehydroquinate synthase is probably responsible only for the oxidation and reduction steps, whilst the other changes are brought about by spontaneous reactions occurring on the intermediates generated (see ref. 1). In recent studies,15 a cDNA clone encoding for dehydroquinate synthase has been isolated from tomato (Lycopersicon esculentum) and identified by complementation of a dehydroquinate synthase-deficient strain of E.coli. The deduced amino acid sequence resembles those of bacterial enzymes more than it does those of fungal systems, and it contains a putative N-terminal plastid-specific transit peptide. A close analogy to the dehydroquinate synthase reaction is found in the biosynthesis of 2-deoxy-scyllo-inosose, an intermediate between glucose and 2-deoxystreptamine in the formation of aminoglycoside antibiotics (see ref. 2). The full paper describing these studies has been published.16 2.3 3-Dehydroquinase The dehydration of 3-dehydroquinate 7 to 3-dehydroshikimate 8 (Scheme 1) is catalyzed by 3-dehydroquinase (3-dehydroquinate dehydratase). This enzyme activity has been identified as a monofunctional activity in bacteria, as part of a bifunctional system with shikimate dehydrogenase in plants, as one of the five activities of the AROM pentafunctional enzyme complex in fungi and yeasts, and as part of an inducible multifunctional system in fungi which is produced to metabolize quinate 10.Two diVerent classes of dehydroquinase enzymes having quite diVerent physical and biochemical properties are now recognized (Scheme 2). Type I enzymes have a biosynthetic role and use a SchiV base mechanism between the substrate and an active site lysine (see ref. 1). On the other hand, Type II enzymes can function as either biosynthetic or catabolic enzymes and do not use a SchiV base mechanism.They also carry out the elimination with opposite stereochemistry to Type I enzymes, involving an anti dehydration with loss of the axial pro-S hydrogen from C-2. With the use of electrospray mass spectrometry, an essential arginine residue in Type II dehydroquinases of Streptomyces coelicolor and Aspergillus nidulans has been identified.17 The technique was used to characterize inactivated protein modi- fied with the Arg-specific reagent phenylglyoxal, and demonstrated that reaction had occurred predominantly with a single conserved arginine residue, Arg-23 in the S.coelicolor enzyme, and Arg-19 in the case of A. nidulans. There was no evidence for reaction with a further conserved arginine during inactivation of the enzyme. Mutant enzymes derived by replacing Arg-23 in the S. coelicolor protein with Lys, Gln or Ala all had drastically reduced catalytic activities, though with stronger CO2 – PO O PO HO OH CO2 – PO HO OH OH O OH OH O CO2 – HO OH OH O CO2 – OH OH HO CO2 – OH OH HO CO2 – HO 4 PEP 5 Erythrose 4-phosphate 6 DAHP 7 3-Dehydroquinate NAD+ ii 8 3-Dehydroshikimate 10 Quinate iv iii v i 9 Shikimate NADH NADH 2 P = phosphate 6 Scheme 1 Enzymes: i, DAHP synthase; ii, 3-dehydroquinate synthase; iii, 3-dehydroquinase; iv, shikimate dehydrogenase; v, quinate dehydrogenase HO OH O HR HS OH –O2C HO OH O H H OH –O2C HO OH O H OH –O2C H A HO OH –O2C HN H H HO HO OH –O2C HN H HO H B OH OH O CO2 – OH OH O CO2 – H H B B 8 3-Dehydroshikimate 7 3-Dehydroquinate · * * * * * · · · · Type I Type II 11 + + Scheme 2 Enzyme: 3-dehydroquinase 18 Natural Product Reports, 1998substrate binding.18 A role for Arg-23 in stabilizing a carbanion intermediate was proposed.Comparison of the amino acid sequences around the essential arginine residues in both Type II and Type I (from E. coli) enzymes indicates a conserved structural motif that may reflect common substrate binding at the active centre.Evidence from a series of kinetic isotope studies supports a proposal for an enolate intermediate and an E1cb mechanism for Type II dehydroquinases (Scheme 2).19 Labelled (2R)- and (2S)-[2-2H]dehydroquinates in the presence of H2O or 2H2O were employed with Type I enzyme from E. coli and Type II enzymes from A. nidulans and Mycobacterium tuberculosis, and appropriate isotope eVects were measured. For all three enzymes, proton abstraction was partially rate-limiting.Substrate and solvent isotope eVects for the Type I enzyme were small, consistent with a complex multistep mechanism. Significant substrate isotope eVects were observed for the two Type II enzymes, but only the A. nidulans enzyme showed a large solvent isotope eVect. All of the results for the Type II enzymes could be rationalized by the action of a stepwise E1cb mechanism involving an enolate intermediate 11, formation of which is partially rate-limiting. It is proposed that there is an electrostatic interaction of the dehydroquinate with the essential arginine residue, and that this residue may also moderate the basicity of a histidine general base responsible for deprotonation (Scheme 3).There is no evidence that Type II dehydroquinases require any metal cofactors for activity.20 The dodecameric enzyme from A. nidulans adopts an arrangement in which two hexameric ring-like structures stack on top of each other. However, there are data to indicate that a ligand-induced conformational change occurs which increases modification of the active site arginine by phenylglyoxal.Binding also partially protects against proteolysis in the highly conserved N-terminal region which contains this arginine residue. The gene encoding Type II dehydroquinase in the stomach pathogen Helicobacter pylori has been isolated and sequenced.21 The gene, designated aroQ, has strong sequence identity to other members of the Type II family.The 5-deoxy 12 and 4,5-dideoxy 13 analogues of dehydroquinate have been tested as substrates of Type I (E. coli) and Type II (M. tuberculosis) enzymes.22 Results suggest that the C-4 hydroxy group is essential for imine formation on the Type I enzyme, only the 5-deoxy compound acting as a modest substrate through an imine-bound species. Both compounds were poor substrates for the Type II enzyme, but since the dideoxy compound was a competitive inhibitor, the 4-hydroxy group does not contribute significantly to specificity. 2.4 The quinate utilization pathway: quinate dehydrogenase and 3-dehydroshikimate dehydratase An inducible quinate-utilizing (QUT) catabolic pathway is observed in certain fungi, and a Type II dehydroquinase functions in this pathway. Quinate 10 enters the pathway by the action of quinate dehydrogenase (quinate:oxidoreductase) giving 3-dehydroquinate 7, and is subsquently transformed to protocatechuate (3,4-dihydroxybenzoate) by the action of 3- dehydroshikimate dehydratase on dehydroshikimate.All three QUT pathway enzymes, quinate dehydrogenase, dehydroquinase and dehydroshikimate dehydratase from Aspergillus nidulans can now be purified in bulk via overexpression in E. coli.23 The isolation and purification of quinate dehydrogenase from Phaseolus mungo sprouts24 and dehydroshikimate dehydratase from Vigna mungo cells cultured in the presence of shikimate25 have been described. A cofactor-independent quinate hydrolyase activity capable of converting quinate directly into shikimate has been reported in pea (Pisum sativum) roots.26 This activity may function in channelling quinate into the shikimate pathway. 2.5 Shikimate kinase Phosphorylation of shikimate 9 to shikimate 3-phosphate 14 is brought about by shikimate kinase in the presence of ATP (Scheme 4). Recent sequence data for the aroK-encoded isozyme of shikimate kinase from E. coli have indicated that sequences published earlier contained a number of errors, and now establish that the two isozymes aroK and aroL are of comparable length and share 30% identity.27 2.6 EPSP synthase EPSP synthase (3-phosphoshikimate 1-carboxyvinyltransferase) catalyzes the condensation of shikimate 3-phosphate 14 with PEP to produce the enol ether 5-enolpyruvylshikimate HO OH O H H OH –O2C N HN H N NH2 H2 N 7 3-Dehydroquinate + Scheme 3 Enzyme: 3-dehydroquinase (Type II) OH O CO2 – HO O CO2 – HO 12 13 4 5 5 OH OH HO CO2 – OH OH PO CO2 – OH O PO CO2 – CO2 – OH O CO2 – CO2 – 9 Shikimate 15 EPSP 14 Shikimate 3-phosphate 16 Chorismate i ii iii iv ATP PEP OH –O2C 17 Prephenate CO2 – O Scheme 4 Enzymes: i, shikimate kinase; ii, EPSP synthase; iii, chorismate synthase; iv, chorismate mutase Dewick: The biosynthesis of shikimate metabolites 193-phosphate (EPSP) 15 (Scheme 4).Gene cloning and expression has allowed the enzyme from Bacillus subtilis to be studied and modified through site-directed mutagenesis.28 The native Bacillus enzyme exhibited allosteric behaviour, a property not yet reported amongst other bacterial and plant EPSP synthases so far investigated.It was established via the mutant enzymes that both shikimate 3-phosphate and PEP have multiple interaction sites. There are two sites for PEP binding, a catalytic one and a non-catalytic one. A shikimate 3-phosphate interacting site has been located in the N-terminal domain.29 A mutant strain of E.coli EPSP synthase in which His-385 is replaced with asparagine is much less catalytically competent (6% activity) than the corresponding glutamine mutant, previously shown to retain some 25% activity.30 Accordingly, the Â-nitrogen of histidine is suggested to play a role in catalysis, perhaps by hydrogen-bonding to another residue that complexes the substrates or products. The broad spectrum herbicide glyphosate [N-(phosphonomethyl) glycine] 18 is a powerful inhibitor of EPSP synthase, and is generally considered to bind to the PEP binding site as an analogue of the PEP oxonium ion 19 formed transiently during catalysis.A stable ternary complex of the enzyme with glyphosate and shikimate 3-phosphate is produced. Recent studies now question the concept that glyphosate acts as a transition state analogue inhibitor.31 Thus, in the reverse reaction involving EPSP and phosphate, there should be little chance of glyphosate interacting with the enzyme or enzyme– substrate complex, but it was found that glyphosate can also be trapped on the enzyme–EPSP to give a ternary complex.Therefore the carboxyvinyl group of EPSP does not prevent glyphosate binding, and it is actually found to strongly facilitate binding. Results of rotational-echo double-resonance NMR and molecular dynamics simulations also support these results, and suggest glyphosate is unlikely to bind in the same fashion as PEP.32 A new, very potent inhibitor 20 of EPSP synthase from E.coli has been designed by eVectively joining together shikimate 3-phosphate with glyphosate.33 Although this should have a specific interaction with the shikimate 3-phosphate binding site, there is likely to be incomplete overlap at the PEP/ glyphosate subsite. The highly substituted shikimate ring can be replaced by structurally simple pyrrole systems to produce shikimate 3-phosphate analogue inhibitors of EPSP synthase. Thus, compound 21 is a surprisingly good inhibitor of the enzyme.34 2.7 Chorismate synthase The elimination of phosphoric acid from EPSP 15 yields chorismate 16 and is catalyzed by the enzyme chorismate synthase (5-enolpyruvylshikimate 3-phosphate-lyase) (Scheme 4).The enzyme achieves a stereochemically ambiguous anti 1,4-elimination, and has a requirement for a reduced flavin for activity, despite the reaction involving no overall change in oxidation state (Scheme 5). Some enzymes, e.g.those from Neurospora crassa and Bacillus subtilis, are bifunctional and have an associated flavin reductase that utilizes NADPH to produce the reduced flavin (Scheme 5). Others, e.g. enzymes from E. coli and the plants Pisum sativum and Corydalis sempervirens, are monofunctional and must be supplied with a reduced flavin. Using a recombinant chorismate synthase from E. coli, only a small isotope eVect was noted when (6R)-[6-2H]EPSP was employed as substrate and no isotope eVect occurred with the (6S)-[6-2H]-labelled substrate.35 Similar isotope eVects were observed for the decay of a spectroscopically characterized flavin semiquinone intermediate.This is consistent with the main rate-limiting step being distinct from the loss of the 6-pro-R hydrogen, and indicates that the flavin intermediate forms before C–H bond cleavage. (6S)-6-FluoroEPSP 22 is a competetive inhibitor of chorismate synthase, but is also converted into 6-fluorochorismate by the E.coli enzyme and at a rate some two orders of magnitude slower than EPSP.36 Of various possible mechanisms for the conversion, the decreased rate of reaction is most consistent with destabilization of an allylic cationic intermediate 23 (Scheme 6). However, this mechanism assigns no role for the reduced flavin cofactor, unless its electron rich character could play a role in stabilizing a cationic intermediate. Alternatively, an allylic radical intermediate 24 may also be destabilized by a fluorine substituent, and its formation and decay would provide a direct role for the cofactor. Extensive kinetic studies following substrate consumption, product formation, phosphate dissociation, and the formation and decay of the flavin intermediate also support these proposals.37 The overall reaction is non-concerted, and the flavin intermediate forms before the substrate is consumed so it is not simply associated with the conversion of substrate into product.A cDNA from Neurospora crassa encoding bifunctional chorismate synthase/flavin reductase has been isolated, sequenced, and expressed in E. coli.38 Its deduced amino acid sequence was found to be 79% similar to that of the Saccharomyces cerevisiae enzyme. Two regions unique to the N. crassa protein were deleted, but the protein was still able to 18 Glyphosate + 19 + CO2 – N P O O– O– H H CO2 – H H O H P O O– O– 21 20 OH NH PO CO2 – P O O– O– CO2 – NH CO2 – P O O– –O OH OH O PO CO2 – CO2 – OH O CO2 – CO2 – H H6 R H6 S 15 EPSP 16 Chorismate i · · NADPH NADP+ FMN FMNH2 Scheme 5 Enzyme: i, chorismate synthase OH O PO CO2 – CO2 – H F 22 6 20 Natural Product Reports, 1998complement a chorismate synthase-deficient mutant of E.coli. Domains responsible for the flavin reductase activity appear to lie within regions of homology with monofunctional chorismate synthases. The cDNA responsible for chorismate synthase in the plant Corydalis sempervirens had earlier been shown to encode a precursor polypeptide containing an N-terminal plastid transit peptide domain.Coding regions of a cDNA for the precursor peptide, and for the mature chorismate synthase have been expressed in E. coli.39 Only the mature chorismate synthase, and not the precursor protein, was enzymically active. Three plastidic isozymes were identified via their cDNAs in tomato (Lycopersicon esculentum), though these were derived from only two genes.40 Two of these isozymes were expressed in E.coli (the third was unstable in E. coli) both as precursor proteins with transit peptides, and as mature proteins. Again, only mature proteins were enzymically active. 2.8 Chorismate mutase Chorismate mutase catalyzes the Claisen-like rearrangement of chorismate 16 to prephenate 17, during which the PEP-derived side-chain becomes directly bonded to the carbocycle (Scheme 4). This is achieved by binding the unfavoured pseudoaxial conformer 25 of chorismate, achieving a chair-like transition state (Scheme 7).The uncatalyzed thermal rearrangement of chorismate to prephenate is also possible, but chorismate mutase increases the rate of reaction by a factor of 2#106. All of the experimental data favour a simple pericyclic process for the enzyme-catalyzed reaction. From a combined quantum mechanical–molecular mechanical study of chorismate mutase from Bacillus subtilis it was concluded that catalysis may be rationalized in terms of a combination of substrate strain and transition state stabilization, and the enzyme is not functioning by a chemical catalysis mechanism.41 Consideration of several simple model systems has indicated that selective transition state binding is achieved by appropriately positioned H-bond donors in the protein.42 Determination of activation barriers for the B.subtilis enzyme shows that the entropy barrier is nearly as unfavourable as for the uncatalyzed reaction so that factors other than entropy are important.43 The enzyme exerts considerable conformational control over the molecule and is highly complementary to the pseudoaxial conformer via multiple hydrogen-bonding and electrostatic interactions.The active site of yeast (Saccharomyces cerevisiae) chorismate mutase has been located by comparison with the mutase domain of the bifunctional chorismate mutase–prephenate dehydratase (the P-protein) from E. coli.44 The active site domains of the two enzymes are very similar, and of the seven active site residues, four are conserved.There are two Arg residues which bind to carboxylates, Lys which binds to the ether oxygen, and Glu which binds to the hydroxy group (see Scheme 8 which shows binding to the transition state analogue and inhibitor 26). Crystal structures of the yeast enzyme bound to the allosteric inhibitor tyrosine and the allosteric activator tryptophan have also been established and interpreted.45 The OH O PO CO2 – CO2 – OH O CO2 – CO2 – OH O CO2 – CO2 – OH O CO2 – CO2 – 15 EPSP 16 Chorismate FMNred FMNsq Pi FMNred 24 FMNsq 23 Pi H+ + Scheme 6 Enzyme: chorismate synthase CO2 – HO O CO2 – OH CO2 – O –O2C CO2 – –O2C O OH 17 Prephenate 16 Chorismate 25 Scheme 7 Enzyme: chorismate mutase OH CO2H O HO2C 26 – – + + + + – – + – – O O H O O O O H2N HN N OH O O H O O NH O NH3 NH NH2 H2N O O H O O O O NH2 HN NH2 O NH2 H2N NH O O S H H Glu-198 (Glu-52) Asn-194 (Asp-48) Glu-246 (Gln-88) Arg-16 (Arg-11) Lys-168 (Lys-39) Thr-242 (Ser-84) Arg-7 Arg-28 (Arg-157) Amino acid residues as shown: Saccharomyces cerevisiae chorismate mutase Amino acid residues in brackets: E.coli chorismate mutase Arg-90 Glu-78 Cys-75 Tyr-108 Bacillus subtilis chorismate mutase Scheme 8 Dewick: The biosynthesis of shikimate metabolites 21crystal structure of the E. coli enzyme shows it to be a homodimer with two equivalent active sites, each having contributions from both monomers.46 Site-directed mutagenesis of a fully functional chorismate mutase engineered from the E.coli P-protein has been employed to demonstrate that Lys-39 and Gln-88 play critical catalytic roles, in that mutant proteins with these residues replaced suVered a major loss of catalytic activity compared to the wild type enzyme.47 In parallel studies,48 Lys-39 and Gln-88 were shown to be critical for enzyme activity, Arg-11 and Arg-28 to be important, whilst Glu-52 played only a minor role.These results stress the importance of hydrogen bonding to the enolpyruvyl oxygen and carboxylate. Thermodynamic parameters for the E. coli enzyme and a Gln88Glu mutant enzyme have been determined and used to provide a direct mechanistic link between the chorismate mutase enzymes of E. coli and S. cerevisiae.49 The active site in Bacillus subtilis chorismate mutase (Scheme 8) has rather low similarity to those in E. coli and S. cerevisiae. Again, site-directed mutagenesis has been used to establish the relative importance of the various residues.50 Critical roles have been identified for Arg-7 and Arg-90, whilst Tyr-108, Arg-116, Phe-57, and Cys-75 do not feature significantly in catalysis.Glu-78 positioned near the 4-hydroxy group contributes to binding, and its replacement reduces activity. In other studies,51 a positively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential for activity, strongly supporting the hypothesis that a positive charge is required to stabilize a dipolar transition state resulting from scission of the C–O bond.Essential active site residues in the bifunctional chorismate mutase–prephenate dehydrogenase (the T-protein) from E. coli have been identified by residue-specific chemical reactions.52 A single lysine (Lys-37) is essential for the mutase activity, whilst His-131 is essential for dehydrogenase activity. A cDNA clone encoding a cytosolic chorismate mutase isozyme has been identified in the plant Arabidopsis thaliana and shown to encode a protein with a deduced amino acid sequence 50% identical to that of the plastidic isozyme previously isolated.53 The plastidic isozyme expressed in E.coli was activated by tryptophan, and inhibited by phenylalanine and tyrosine, whilst the cytosolic enzyme was insensitive to these amino acids. Transformed roots of Datura stramonium also contain two chorismate mutase isozymes, only one of which is present in intact plastids.54 At least two chorismate mutase activities were identified in the methylotrophic actinomycete Amycolatopsis methanolica.10 One of these was a bifunctional protein which also carried DAHP synthase activity.Both chorismate mutase activities were inhibited by the amino acids phenylalanine and tyrosine. Reviews describing chorismate mutase in microorganisms and plants,55 and in Escherichia coli,56 have been published. 2.9 Phenylalanine and tyrosine Several pathways for the conversion of chorismate 16 into the aromatic amino acids L-phenylalanine 28 and L-tyrosine 31 are known to exist (Scheme 9) and the pathway employed is dependent on the organism.Intermediates phenylpyruvate 27, 4-hydroxyphenylpyruvate 30, or L-arogenate 29 may be involved, and often, more than one route may operate in a particular species as a result of the enzyme activities available. Phenylalanine- and tyrosine-requiring auxotrophs of the actinomycete Amycolatopsis methanolica have been identified and shown to be deficient in prephenate dehydratase or arogenate dehydrogenase, respectively.57 These strains thus have single pathways for the production of phenylalanine and tyrosine.Extracts of the organism yielded two fractions with aminotransferase activity towards phenylalanine and tyrosine, and three fractions acting on prephenate. Mutant and biochemical studies identified two aminotransferase activities that were dominant in phenylalanine and tyrosine biosynthesis.The prephenate aminotransferase has been partially purified.58 Prephenate dehydratase from this organism has also been purified, and shown to be a homotetramer.59 Tyrosine aminotransferase in humans is concerned primarily with tyrosine catabolism; a cDNA encoding this enzyme in the liver has been cloned and expressed in HeLa cells.60 A recombinant tyrosine aminotransferase from Trypanosoma cruzi has been shown to also possess alanine aminotransferase activity.61 Purification of one of the two aromatic amino acid aminotransferases found in Azospirillum brasiliense showed it was able to utilize all three amino acids.62 Enzymes of the shikimate pathway involved in aromatic amino acid biosynthesis in the unicellular chlorophyte alga Chlorella sorokiniana have been characterized and shown to be typical of higher plants rather than photosynthetic prokaryotes such as cyanobacteria.63 Thus, the following distinctive enzymes were identified: aMn2+-stimulated isozyme of DAHP synthase, a bifunctional dehydroquinase-shikimate dehydrogenase, an allosterically controlled isozyme of chorismate mutase, a thermotolerant prephenate aminotransferase, tyrosine-inhibited arogenate dehydrogenase, and arogenate dehydratase.The biocatalytic conversion of glucose into aromatic compounds in E. coli can be enhanced considerably by modification of the chromosome.64 Insertion of a multigene cassette carrying the genes aroA, aroC and aroB (encoding for EPSP synthase, chorismate synthase and dehydroquinate synthase, respectively) into an E.coli strain where transcriptional repression of aroL (shikimate kinase) is not operative resulted in a doubling of the yields of L-phenylalanine, prephenic acid and phenyllactic acid. The enzyme phenylalanine dehydrogenase catalyzes the reversible NAD+-dependent oxidative deamination of Lphenylalanine to phenylpyruvate 27 and ammonia.The gene encoding the enzyme in Bacillus badius has been sequenced.65 The deduced amino acid sequence for the enzyme was found to be similar to those reported earlier from B. sphaericus and Thermoactinomyces intermedius. For the enzyme from 16 Chorismate 17 Prephenate i 29 L-Arogenate 28 L-Phenylalanine 27 Phenylpyruvate 30 4-Hydroxyphenylpyruvate 31 L-Tyrosine iii PLP ii iv PLP v vii NAD+ vi NAD+ viii PLP CO2 – O CO2 – O OH OH O CO2 – CO2 – OH –O2C CO2 – O OH –O2C CO2 – NH2 CO2 – NH2 CO2 – NH2 OH Scheme 9 Enzymes: i, chorismate mutase; ii, prephenate dehydratase; iii, phenylpyruvate aminotransferase; iv, prephenate aminotransferase; v, arogenate dehydratase; vi, arogenate dehydrogenase; vii, prephenate dehydrogenase; viii, 4-hydroxyphenylpyruvate aminotransferase 22 Natural Product Reports, 1998B. sphaericus, amino acid residues Gly-124 and Leu-307 have been suggested to play an important role, probably contributing towards the substrate specificity of the enzyme.These residues have been altered by site-specific mutagenesis to Ala and Val, respectively, which are corresponding residues in the related enzyme leucine dehydrogenase.66 Single and double mutants displayed lower activity towards phenylalanine and enhanced activity towards aliphatic amino acid substrates. 2.10 Aromatic amino acid hydroxylases Direct hydroxylation of L-phenylalanine to L-tyrosine and of L-tyrosine to L-(3,4-dihydroxyphenyl)alanine (L-DOPA) may be achieved in certain organisms via aromatic amino acid hydroxylases.This route is particularly important in mammals, which lack the shikimate pathway. These enzymes are members of a group of tetrahydropterin-dependent hydroxylases and include phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase. The mixed oxidation reaction consumes molecular oxygen and one equivalent of reduced pterin cofactor to hydroxylate the substrate. Most of the enzymes contain a metal at the active site which is reduced by the tetrahydropterin cofactor, though an exception has been found in phenylalanine hydroxylase from Chromobacterium violaceum.To help identify the function of metals in mammalian phenylalanine hydroxylases, the Fe-dependent rat liver enzyme has been compared with the metal-independent C. violaceum enzyme using 2H-labelled phenylalanine substrates. 67 Both enzymes displayed similar kinetic isotope eVects with [4-2H]phenylalanine as substrate, and an almost identical retention of 2H (85%) via NIH shift to position 3.NIH shifts with [2,3,5,6-2H4]phenylalanine were also identical (84% 2H4). This suggests that the NIH shift is probably not directly mediated by the enzyme. Results with other phenylalanine substrates and analogues indicated that both rat liver and C. violaceum phenylalanine hydroxylases must utilize a very similar oxygenating intermediate. Recombinant human phenylalanine hydroxylase has been produced in E. coli and purified to homogeneity.68 The recombinant enzyme exists as a mixture of tetramers and dimers.By expression as a fusion protein, proteolytic degradation can be circumvented.69 Rat liver phenylalanine hydroxylase can be activated by cAMP-dependent protein kinase-mediated phosphorylation of Ser-16, and it has been suggested that this is a consequence of the introduction of the negatively charged phosphate group. By site-directed mutagenesis, Ser-16 has been replaced with a variety of other amino acids.70 Activation was only noted when Ser-16 was replaced with the negatively charged amino acids glutamine or aspartic acid.The mechanism of action of recombinant rat liver tyrosine hydroxylase, an Fe-containing enzyme, has been studied using a variety of 4-substituted phenylalanines as substrates.71 The enzyme accepts phenylalanine, 4-halogenated phenylalanines, and several other compounds, giving multiple hydroxylated products in most cases (Scheme 10).It was found that as the size of the substituent increases, the site of hydroxylation switched from position 4 to position 3 in the aromatic ring. The substituent at position 4 was sometimes eliminated, yielding tyrosine as product, or it could be migrated via an NIH shift, producing 3-substituted tyrosine. From the observations it was concluded that the transition state for hydroxylation must be highly electron-deficient or cationic in nature.Iron– oxo type intermediates 32 and 33 may be involved (Scheme 10). Purified recombinant rat liver tyrosine hydroxylase contains 0.5–0.7 Fe3+ atoms per subunit.72 During turnover, this is reduced by the tetrahydropterin to the Fe2+ form, though a fraction becomes reoxidized by O2. Five histidine residues are highly conserved in all tyrosine hydroxylase proteins that have been sequenced, and also in related phenylalanine hydroxylase and tryptophan hydroxylase.By site-directed mutagenesis, these residues in tyrosine hydroxylase have been replaced separately.73 Mutant proteins without histidine at positions 331 or 336 contained no iron and had no detectable activity; others contained wild-type levels of iron, and somewhat lower enzymic activity. This is consistent with His-331 and His-336 being ligands for the active site iron atom. Sequence data for the human tryptophan hydroxylase gene show considerable similarity to those for tyrosine hydroxylase and phenylalanine hydroxylase.74 Purification techniques for tryptophan hydroxylase from rat brain have been reported,75 and the tryptophan hydroxylase gene has been expressed in a human cell line.76 Deletion of the regulatory domains from the full-length sequence has allowed cloning and expression of the catalytic core, which displays high levels of trytophan hydroxylase activity.77 The structure and function of aromatic amino acid hydroxylases, 78 and human tyrosine hydroxylase79 have been reviewed. An extensive review on tyrosine hydroxylase has been published.80 2.11 Tyrosinase and melanin Melanin, the main pigment in mammalian skin, hair and eyes, is a heterogeneous polymer derived by oxidation of L-tyrosine (Scheme 11). The principal enzyme involved is tyrosinase, which catalyzes both the hydroxylation of L-tyrosine to L-DOPA 34, and the subsequent oxidation of L-DOPA to DOPAquinone 35.Several of the steps beyond DOPAquinone are apparently spontaneous chemical transformations.In recent studies, crystalline tyrosinase has been obtained from the skin of white silky fowl (Gallina lanigera),81 and partially purified enzyme has been isolated from potato (Lycopersicon esculentum).82 Tyrosinase isoforms have been identified in fruit bodies of the fungus Agaricus bisporus,83 and a cDNA encoding the protein has also been isolated from this source.84 The deduced amino acid sequence for the protein has significant homology to tyrosinase from Neurospora crassa.Six isozymes have been reported in the browned gills of Lentinus edodes,85 each isozyme consisting of two types of polypeptide. In the later stages of melanin biosynthesis, L-DOPAchrome 37 is transformed into either 5,6-dihydroxyindole-2-carboxylic acid (DHICA) 39 or 5,6-dihydroxyindole (DHI) 38 (Scheme 11). Polymerization of DHICA to melanin by a glycoprotein from mouse melanoma cells has been reported.86 An enzyme D-DOPAchrome tautomerase which converts the non-natural D-isomer of DOPAchrome into DHI has been isolated from human blood erythrocytes.87 A cDNA encoding this enzyme in rat liver has been cloned and sequenced.88 A comprehensive review of the mechanism of tyrosinase has been published.89 Other reviews cover its role in mammalian pigmentation,90 physiological implications of kinetic properties91 and molecular structure.92 The chemistry of melanins and melanogenesis has also been reviewed.93 R = 32 33 + + X R OFe X R OH R OH R X X OFe H R X OH R CO2H NH2 Scheme 10 Enzyme: tyrosine hydroxylase Dewick: The biosynthesis of shikimate metabolites 232.12 p-Aminobenzoic acid, anthranilic acid and related compounds The biosynthetic pathways to p-aminobenzoate 41 and anthranilate (o-aminobenzoate) 43 share many features (Scheme 12).A glutamine amidotransferase activity provides ammonia from glutamine, and this then reacts with chorismate either at the 4 or 2 positions, yielding 4-amino-4-deoxychorismate 40 or 2-amino-2-deoxyisochorismate 42.Lyase enzymes then release p-aminobenzoate or anthranilate from these respective intermediates. The two subunits PabA and PabB required for 4-amino-4-deoxychorismate synthesis in E. coli appear to act as a complex, but only recently has the successful isolation of the intact complex been reported.94 The association of the two subunits is enhanced by the presence of glutamine.Mutations in PabB can aVect its ability to associate with PabA, as well as its ability to aminate chorismate. Both PabA and PabB have cysteine residues essential for catalytic activity and/or subunit interaction.95 Anthranilate synthase from Ruta graveolens plants and cell cultures has been purified.96 Glutamine- and ammoniadependent activities copurified in all steps. cDNA cloning and complementation of an E. coli deletion mutant defective in anthranilate synthase showed young Ruta plants express two genes for functional ammonia-dependent AS· subunits.An AS‚ subunit is required for the glutamine-dependent reaction. The two AS· isozymes vary in function, one being elicitorinducible and involved in alkaloid biosynthesis, whilst the other is constitutive and strongly feedback inhibited by tryptophan. 97 The genes are diVerentially expressed according to the plant’s requirements for tryptophan or for defence-related alkaloid biosynthesis. Two anthranilate synthase isozymes have been detected in cell cultures of Ailanthus altissima.98 A glutamine-dependent isozyme was purified and characterized.An Arabidopsis thaliana mutant containing anthranilate synthase which shows enhanced feedback-resistance towards tryptophan also accumulates levels of tryptophan three-fold higher than the wild type plant.99 Tryptophan resistance was traced back to the single amino acid substitution Asp-341] Asn in the mutant. The enzymes anthranilate synthase, p-aminobenzoate synthase and isochorismate synthase (see Section 2.13) exhibit significant amino acid sequence homology and may thus share common mechanistic features.To probe this hypothesis, three compounds 44–46 designed to mimic, in their all-axial conformers 47, the putative transition state 25 have been synthesized and tested against the three E. coli-derived enzymes.100 All three were competitive inhibitors, binding HO CO2H NH2 HO CO2H NH2 O CO2H NH2 HO O HO HO NH CO2H HO O N CO2H HO HO NH O O NH HO HO NH CO2H O O NH CO2H 34 L-DOPA Melanins 31 L-Tyrosine 35 DOPAquinone 37 DOPAchrome 38 DHI 39 DHICA O2 O2 O2 –CO2 i i ii iii i 36 CycloDOPA Scheme 11 Enzymes: i, tyrosinase; ii, DOPAchrome tautomerase; iii, DHICA oxidase OH O CO2 – CO2 – NH2 O CO2 – CO2 – CO2 – NH2 O CO2 – 16 Chorismate OH O CO2 – CO2 – 16 Chorismate 40 4-Amino-4-deoxychorismate 41 p-Aminobenzoate Glu O CO2 – CO2 – 42 2-Amino-2-deoxyisochorismate Gln + CO2 – NH3 43 Anthranilate O CO2 – ii iii + Glu Gln + NH3 iv v vi NH2 NH2 + i Scheme 12 Enzymes: i, glutamine amidotransferase (PabA); ii, 4-amino-4-deoxychorismate synthase (PabB); iii, 4-amino-4- deoxychorismate lyase (PabC); iv, glutamine amidotransferase (anthranilate synthase II, AS‚, TrpG); v, 2-amino-2- deoxyisochorismate synthase; vi, 2-amino-2-deoxyisochorismate lyase; [v and vi/anthranilate synthase I (AS·, TrpE)] º 44 X = Y = OH 45 X = NH3 +; Y = OH 46 X = OH; Y = NH3 + 47 Y O CO2 – CO2 – X O CO2 – Y X –O2 C 24 Natural Product Reports, 1998strongly to anthranilate synthase and isochorismate synthase, but weakly to p-aminobenzoate synthase.The aYnity of the 6-amino-4-hydroxy isomer 45 was some ten times greater than the 4-amino-6-hydroxy isomer 46, though this is largely due to their conformational equilibria and the relative proportion of the all-axial conformer. The similarity between anthranilate synthase and isochorismate synthase is extended by the observation that both enzymes catalyze the formation of 2-amino- 2-deoxyisochorismate 42 in the presence of ammonia.The findings support a mechanism of direct substitution of the C-4 hydroxy group with a C-6 substituent via the proposed Mg-coordinated transition state 48 in the case of these two enzymes (Scheme 13). For p-aminobenzoate synthase, the mechanism must be somewhat diVerent, perhaps by a sequential mechanism (Scheme 14) rather than by direct substitution.The remarkable hydrolysis of an amine to an alcohol in the reverse reactions involving anthranilate synthase and p-aminobenzoate synthase has been studied and discussed.101 The enzymology of anthranilate synthase in microorganisms and plants has been reviewed.102 In the pathway to anthranilate by oxidative degradation of L-tryptophan 50 (Scheme 15), the first reaction is catalyzed by tryptophan 2,3-dioxygenase, producing N-formylkynurenine 51. The reaction is believed to involve addition of oxygen across the C-2–C-3 bond to form a dioxetane with subsequent decomposition. In a recent report,103 tryptophan 2,3-dioxygenase from yeast (Saccharomyces cerevisiae) has been purified.Hydrolytic cleavage of L-kynurenine 52 and L-3-hydroxykynurenine 53 to anthranilate 43 and 3- hydroxyanthranilate 54, respectively, is brought about by kynureninase, a PLP-dependent enzyme. The amino acid sequence of kynureninase from rat liver has been established, 104 and a cDNA clone encoding human kynureninase has been isolated.105 The predicted amino acid sequence of the human enzyme has high similarity to that of the rat liver enzyme and also to a S.cerevisiae gene product tentatively ascribed to kynureninase. A PLP-binding site could be defined. 4-Aminobenzoate hydroxylase is an FAD-, O2- and NAD(P)H-dependent monooxygenase responsible for the decarboxylative hydroxylation of p-aminobenzoate 41 to produce the 4-hydroxyaniline 55 moiety of N-(„-L-glutamyl)-4- hydroxyaniline 56 in the mushroom Agaricus bisporus (Scheme 16).A cDNA encoding this enzyme has been cloned and sequenced.106 Two regions on the protein share homology with other flavoproteins such as salicylate hydroxylase and 4-hydroxybenzoate hydroxylase. 48 25 42 2-Amino-2-deoxyisochorismate Y = N 49 Isochorismate Y = O O CO2 – OH –O2C O CO2 – HnO –O2C YHn Mg Enz O CO2 – –O2C YHn Scheme 13 Enzymes: anthranilate synthase/isochorismate synthase 40 4-Amino-4-deoxychorismate 16 Chorismate NH3 + OH O CO2 – CO2 – O CO2 – CO2 – Nu Enz NH2 O CO2 – CO2 – Enz Nu Scheme 14 Enzyme: p-aminobenzoate synthase NH CO2 – NH2 CO2 – O NH NH2 CHO CO2 – O NH2 NH2 CO2 – O NH2 NH2 OH CO2 – NH2 OH 50 L-Tryptophan CO2 – NH2 52 L-Kynurenine 51 N-Formylkynurenine 53 3-Hydroxykynurenine 43 Anthranilate i O2 ii iv Ala iii Ala iii 54 3-Hydroxyanthranilate Scheme 15 Enzymes: i, tryptophan 2,3-dioxygenase; ii, kynurenine formamidase; iii, kynureninase; iv, kynurenine 3-hydroxylase HO2C NH2 HO NH2 HO N H O CO2H NH2 56 i NAD(P)H O2 41 55 ii Glu Scheme 16 Enzymes: i, 4-aminobenzoate hydroxylase; ii, „-glutamyltransferase Dewick: The biosynthesis of shikimate metabolites 25Some cyclic hydroxamic acids based on 2,4-dihydroxy-2H- 1,4-benzoxazin-3(4H)-one (DIBOA) 58 found in maize, wheat, rye and other members of the Graminae, serve as major defence compounds against bacteria, fungi and insects.It has been established that part of the tryptophan biosynthetic pathway from anthranilate is used in their biosynthesis, though tryptophan itself is not a precursor (see ref. 1). A suggested pathway is via oxidative decarboxylation of 1-(2- carboxyanilino)-1-deoxyribulose 5-phosphate 61 (see Scheme 19) and subsequent cleavage of glyceraldehyde 3-phosphate. In more recent studies, it has been shown that maize (Zea mays) shoots grown in the presence of labelled indole 57 accumulate labelled DIMBOA 59 (Scheme 17).107 In particular, retention of label from [2-14C]indole suggested this compound was on the direct pathway; incorporation via anthranilic acid derived by cleavage of indole would involve loss of this label.Also, feeding of [2H6]indole gave DIMBOA with equal deuterium labelling at the three aromatic positions and at the acetal. The mechanism of benzoxazinone formation from indole has yet to be established. The chemistry of biologically active benzoxazinoids has been reviewed.108 2.13 Isochorismate synthase The formation of isochorismate 49 from chorismate 16 (Scheme 18, see also Scheme 13) creates a branchpoint away from the main shikimate pathway leading to the aromatic amino acids, and is utilized for the biosynthesis of some quinones (see Section 9) and simple phenolic acid derivatives such as 2,3-dihydroxybenzoic acid, a precursor of the siderophore enterobactin. The reaction is catalyzed by isochorismate synthase (isochorismate hydroxymutase) and has been shown to proceed via a double SN2* sequence.In independent studies109–111 it has been demonstrated that diVerent genes encode for isochorismate synthase according to its involvement in menaquinone (menF) or in enterobactin (entC) biosynthesis. An entC mutant of E. coli deficient in enterobactin biosynthesis grows normally and synthesizes wild-type levels of menaquinone under anaerobic conditions in iron-suYcient media.109 The gene responsible (menF) has been identified and sequenced, and shown to encode a protein 23.5% identical and 57.8% similar to EntC.110 The involvement of menF in menaquinone biosynthesis was proved via selective mutation studies and complementation of entC deletion mutants.Duplicate genes menF and dhbC for isochorismate synthase have also been implicated in Bacillus subtilis.111 MenF deletion mutants are able to produce wild-type levels of menaquinone and 2,3-dihydroxybenzoate, via utilization of the dhbC gene product.On the other hand, deletion of dhbC results in synthesis of only menaquinone, and menF is unable to compensate for the loss of 2,3-dihydroxybenzoate. It is shown that expression of dhbC, but not of menF, is regulated by iron concentration, and this repression can be abolished by mutations within the dhb promoter. Transgenic root cultures of Rubia peregrina containing the entC gene from E. coli have been established.112 This leads to increased isochorismate synthase activity and higher anthraquinone production. 3 Tryptophan and related compounds 3.1 Tryptophan L-Tryptophan 50 is formed from chorismate 16 via anthranilate 43 as shown in Scheme 19. A feature of this pathway is the Amadori rearrangement on phosphoribosylanthranilate 60 catalyzed by the enzyme phosphoribosylanthranilate isomerase. It has been shown using the enzyme from Saccharomyces cerevisiae that the primary product of a single turnover of 60 is NH NH O O NH O O OH NH O O OH MeO N O O OH N O O OH MeO OH OH Anthranilate 57 Indole Tryptophan 59 DIMBOA 58 DIBOA O2, NADPH O2, NA DPH O2, NADPH Scheme 17 OH O CO2 – CO2 – O CO2 – CO2 – OH OH2 16 Chorismate 49 Isochorismate Scheme 18 Enzyme: isochorismate synthase CO2 – NH2 CO2 – NH CH2OP OH HO O CO2 – NH O OP OH OH NH OH HO OP NH CO2 – NH2 NH 61 1-(2-carboxyanilino)-1-deoxyribulose 5-phosphate ii 50 L-Tryptophan 62 Indole-3-glycerol phosphate iii iv 43 Anthranilate v b2 Ser PLP Ser PLP 60 Phosphoribosylanthranilate Chorismate 57 Indole i v a v a2b2 Scheme 19 Enzymes: i, anthranilate synthase (TrpE, TrpG); ii, anthranilate phosphoribosyltransferase (TrpD); iii, phosphoribosylanthranilate isomerase (TrpF); iv, indole-3-glycerol phosphate synthase (TrpC); tryptophan synthase (TrpA, TrpB), · and ‚ refer to subunits of tryptophan synthase 26 Natural Product Reports, 1998fluorescent, but it slowly isomerizes to a stable non-fluorescent product, which is then the competent substrate for the next enzyme, indole-3-glycerol phosphate synthase.113 The fluorescent product is tentatively assigned as the enol amine 63, whilst the non-fluorescent product is the keto amine 1-(2- carboxyanilino)-1-deoxyribulose 5-phosphate 61 (Scheme 20).Conversion of 63 into 61 is not eVected by any of the three enzymes anthranilate phosphoribosyltransferase, phosphoribosylanthranilate isomerase or indole-3-glycerol phosphate synthase, and it appears that tryptophan biosynthesis is thus rate-limited by an uncatalyzed enol–keto tautomerism.Escherichia coli contains a bifunctional enzyme phosphoribosylanthranilate isomerase–indole-3-glycerol phosphate synthase. The individual monofunctional proteins have now been obtained through genetic engineering as stable monomeric proteins possessing full catalytic activity.114 The two monofunctional enzymes did not associate in vitro, nor in vivo by coexpression of the domains in the same cells. There appear to be no obvious selective advantages for the bifunctional enzyme over the monofunctional enzymes.In Arabidopsis thaliana, three genes encoding phosphoribosylanthranilate isomerase have been characterized.115 Two genes were virtually identical, whilst the third shared about 90% identity. The phosphoribosylanthranilate isomerase protein is not associated with indole-3-glycerol phosphate synthase as in most microorganisms. Transgenic plants expressing antisense phosphoribosylanthranilate isomerase RNA showed a significantly reduced level of protein and enzymic activity.A cDNA encoding indole-3-glycerol phosphate synthase has been isolated from A. thaliana, and deduced sequences for the encoded protein showed 22–38% identity to reported microbial sequences.116 Crystal structures for this enzyme isolated from the hyperthermophilic archaeon Sulfolobus solfataricus have been reported.117, 118 This monomeric enzyme shows only 30% sequence identity with the indole-3-glycerol phosphate synthase domain of the bifunctional enzyme from E.coli, but there is a high degree of structural similarity. Tryptophan synthase catalyzes the final reaction in the sequence, transformation of indole 3-glycerol phosphate 62 plus L-serine into L-tryptophan. It is a multienzyme complex (the ·2‚2 complex), comprising two · subunits and a ‚2 dimeric subunit. The · subunits also catalyze aldolytic cleavage of indole 3-glycerol phosphate, and, by use of the cofactor PLP, the ‚ subunits catalyze reaction of L-serine and indole 57, giving L-tryptophan.The overall reaction is catalyzed only by the ·2‚2 complex, and indole is not normally released as an intermediate, but is channelled between the active sites via a hydrophobic tunnel. In a single enzyme turnover of the ·2‚2 reaction, indole cannot be detected, but by site-directed mutagenesis, the size of the tunnel can be restricted thus impairing eYcient channelling of indole and allowing the direct observation of indole.119 This has been achieved in the Salmonella typhimurium enzyme by replacing Cys-170 in the ‚ subunit with tryptophan or phenylalanine.Evidence for an ·-amino acrylate SchiV base intermediate 65 in the ‚-reaction (Scheme 21) has been available only through absorption and fluorescence spectroscopy. Direct solid state NMR observation of 65 has now been obtained, using L-[3-13C]serine with the ·2‚2 complex from S. typhimurium.120 The ·-amino acrylate SchiV base is in equilibrium with its methyl keto amine tautomer 66 at the enzyme active site in the ‚ subunit.Conformational changes associated with formation of the ·-amino acrylate SchiV base 65 are believed to play a part in the mechanism by which the tryptophan synthase ‚ subunit accelerates cleavage of indole 3-glycerol phosphate catalyzed by the · subunit. This allosteric activation has been demonstrated by producing a mutant enzyme in which the ‚-active site lysine (Lys-87) was replaced with threonine.121 This enzyme failed to catalyze the ‚-reaction, though there was normal activity for the ·-reaction.A SchiV base between PLP and serine could be formed at the ‚-active site, and using a chemical rescue method, this was converted into an ·-amino acrylate type intermediate using a high concentration of ammonia. This led to a significant enhancement in ·-reaction activity, showing the allosteric interaction in the absence of tryptophan formation. From a series of kinetic studies and isotope eVects it is concluded that the chemical reaction in which indole attacks the enzyme-bound ·-amino acrylate SchiV base is best described as occurring in two steps, a binding step followed by a Michael addition onto the conjugated double bond.122 Again, conversion of the serine external aldimine 64 into the ·-amino acrylate SchiV base 65 is implicated as the ‚-site process which activates the ·-site.cDNAs encoding the ·-protein of tryptophan synthase have been isolated from Arabidopsis thaliana123 and maize (Zea mays).124 In both cases, the cDNA clone complemented an E.coli trpA mutant. Several lines of evidence suggest the active tryptophan synthase enzyme from A. thaliana is a heterosubunit complex, probably analogous to the prokaryotic ·2‚2 complex. The tryptophan biosynthetic enzymes phosphoribosylanthranilate isomerase and the · subunit of tryptophan synthase from A.thaliana are synthesized as higher molecular weight precursors, are then imported into chloroplasts and processed into their mature forms.125 Two DNA fragments from Buchnera, the prokaryotic symbiont of the aphid Schlechtendalia chinensis have been cloned and sequenced and shown to contain the trpEG genes in the smaller fragment, and trpDCFBA genes in the larger.126 Both gene clusters are present as a single copy and probably constitute two transcriptional units. The structure, function and genetic aspects of tryptophan synthase from Salmonella typhimurium have been covered in recent reviews.127, 128 The genetics of tryptophan biosynthesis in plants has also been reviewed.129 Water-stressed leaves of tomato (Lycopersicon esculentum) accumulate increased amounts of L-tryptophan and N-malonyl-L-tryptophan.130 Crude leaf fractions were able to malonylate both L- and D-tryptophan.A partially purified tryptophan N-malonyltransferase was obtained which catalyzed malonyl CoA-dependent malonylation of both enantiomers, 131 the same protein being responsible for utilization of both L- and D-forms of the amino acid.A tryptophan 2*,3*-oxidase from Chromobacterium violaceum catalyzes double bond formation in the side-chain of L-tryptophan and related substrates.132 The product from L-tryptophan has been established to have the Z configuration CO2 – NH POCH2 HO OH O CO2 – PO OH HO OH NH CO2 – PO O HO OH NH 63 (enol amine) 60 Phosphoribosylanthranilate 61 (keto amine) 62 Indole-3-glycerol phosphate i ii Scheme 20 Enzymes: i, phosphoribosylanthranilate isomerase; ii, indole-3-glycerol phosphate synthase Dewick: The biosynthesis of shikimate metabolites 2767.The reaction, for which no cofactor was required, could also occur if tryptophan was part of a short peptide (5–24 residues) irrespective of its position in the sequence. 3.2 Tryptophanase Tryptophanase [tryptophan indole-lyase (deaminating)] catalyzes the synthesis of tryptophan from indole, pyruvate and ammonia in a PLP-dependent reaction, though its physiological role is cleavage of tryptophan rather than its synthesis. The gene for tryptophanase from Enterobacter aerogenes has been highly expressed in E.coli, giving cells capable of synthesizing L-tryptophan from indole, pyruvate and ammonia.133 The enzyme was subsequently purified and shown to have higher stability towards heat than tryptophanase from E.coli. A kinetic study of tryptophanase from E. coli using ·-deuteriated L-tryptophan has suggested that proton transfer to carbon to form the indoleninium intermediate 68 (Scheme 22) is relatively slow, and probably at least relatively ratelimiting. 134 With aza and thia analogues of L-tryptophan, the rate-determining step changes according to the position and type of heteroatom. Structural, spectral and catalytic aspects of tryptophanase have been reviewed.135 3.3 Indole-3-acetic acid and related metabolites The plant growth hormone indole-3-acetic acid (IAA) 69 can be produced from tryptophan by several diVerent pathways according to species.Intermediates indole-3-pyruvic acid, tryptamine, indole-3-acetamide or indole-3-acetaldoxime may be involved (Scheme 23). Further metabolism may then yield a variety of indole or oxindole derivatives. The indole-3-acetamide pathway is not usually found in plants, but is typical of many plant pathogenic and symbiotic bacteria.Plants usually gain the capacity for converting indole- 3-acetamide into IAA after transformation with organisms such as Agrobacterium tumefaciens and incorporation of the gene encoding indoleacetamide hydrolase. Results of experiments with suspension cultures of scorzonera (Scorzonera hispanica) and carrot (Daucus carota) have demonstrated that this enzyme is absent from normal cells, but is present after transformation with A. tumefaciens.136 Conversion of tryptophan into indole-3-acetamide is catalyzed by the flavoprotein tryptophan 2-monooxygenase.The gene encoding this enzyme in Pseudomonas savastanoi has been isolated and expressed in E. coli.137 The purified enzyme contained tightly bound indole- 3-acetamide which could be removed by dialysis. The enzyme would accept phenylalanine and methionine, amino acids having large hydrophobic side-chains, as alternative substrates. Kinetic studies138, 139 on the enzyme have indicated that many features of the reaction resemble those of D-amino acid oxidase, and a mechanism involving the same initial steps is proposed (Scheme 24).Cleavage of the C–H bond is likely to produce a carbanion 70 which interacts with the flavin. The tryptophan 2-monooxygenase reaction then diVers in that a rapid decarboxylation of the imino acid occurs. Indole-3-acetonitrile features in the pathway to IAA in Arabidopsis thaliana. This is converted into IAA by the hydrolytic enzyme nitrilase, which is known to exist in at least four NH PO O– N+ Lys H Enz NH PO O– N+ H CO2 – HO NH PO O– N+ H CO2 – HO NH PO O– N+ H CO2 – NH PO O N+ H CO2 – NH PO O– N+ H CO2 – HN + NH PO O– N+ H CO2 – NH NH PO O– N+ H CO2 – NH Ser Enz-Lys H+ H+ Indole H+ Enz-Lys Trp Internal aldimine 64 External aldimine 66 Methyl keto amine tautomer 65 a-Amino acrylate Schiff base + + + + + H H H Scheme 21 Enzyme: tryptophan synthase NH CO2H NH2 67 28 Natural Product Reports, 1998isoforms.The nucleotide sequence of the nitrilase I gene has been reported.140 The gene encoding nitrilase II has been integrated into the genome of Nicotiana tabacum, and catalytic activity of the expressed protein demonstrated.141 Hydrolysis of indole-3-acetonitrile to IAA has also been reported for the first time in the plant-associated bacteria Agrobacterium and Rhizobium, and the enzyme has been purified.142 The indole-3-pyruvate route rather than the indole-3- acetamide pathway is operative in a plant-associated strain of Erwinia herbicola.143 Indole-3-pyruvate and indole-3- acetaldehyde, but not indole-3-acetamide or other potential intermediates could be converted into IAA, and these compounds were also detected in the cultures.Similar conclusions are reported for Bradyrhizobium elkanii,144 and some species of Rhizoctonia.145 The enzyme activity indolepyruvate decarboxylase was detected in cell-free extracts of B. elkanii, and indole-3-ethanol (tryptophol) was a further metabolite from both tryptophan and indole-3-acetaldehyde in R.solani. The soil bacterium Azospirillum brasiliense normally employs the indole-3-acetamide pathway for IAA synthesis, but under microaerophilic conditions, it can convert indole-3-pyruvic acid, indole-3-lactic acid and indole to give IAA.146 Two potential indole-3-acetaldehyde dehydrogenases catalyzing the NAD+-dependent conversion of indole-3-acetaldehyde into IAA have been identified in the phytopathogenic fungus Ustilago maydis.147 However, one activity appeared to be primarily involved in oxidation of ethanol to acetic acid.The second activity was concerned with conversion of indole-3- acetaldehyde derived from tryptamine, and was induced by arabinose, but repressed in the presence of glucose. A soluble auxin-binding protein from mung bean (Vigna radiata) was shown to be an indole-3-acetaldehyde reductase activity catalyzing NADPH-dependent conversion of indole-3- acetaldehyde into indole-3-ethanol.148 It was found to have high amino acid sequence homology with alcohol dehydrogenase enzymes, and indole-3-acetaldehyde was actually a rather poor substrate compared with several other aldehydes.An indole-3-ethanol oxidase enzyme has been isolated from Internal aldimine External aldimine B1 HB1 B1 68 Indoleninium intermediate B1 gem-Diamine iminoacrylate Internal aldimine + + B1 B1 Amino acrylate Schiff base + HN CO2 – H NH2 Lys-270 HN PLP N CO2 – H +NH PLP N CO2 – +NH PLP H B2 H B2 N+ CO2 – +NH PLP H B2 H N H B2 CO2 – NH PLP H2 N Lys-270 N H CO2 – +NH2 PLP HN Lys-270 N CO2 – +NH PLP H B2 Lys-270 NH2 Scheme 22 Enzyme: tryptophanase NH CO2H Indole-3-acetaldoxime Tryptophan Indole-3-acetonitrile Indole-3-acetamide Tryptamine Indole-3-pyruvic acid Indole-3-lactic acid Indole-3-acetaldehyde Indole-3-ethanol (Tryptophol) 69 IAA Scheme 23 Dewick: The biosynthesis of shikimate metabolites 29seeds of bean (Phaseolus vulgaris).149 This enzyme required molecular oxygen and produced H2O2. An alternative pathway to IAA via indole but not tryptophan has also been suggested, particularly in maize (Zea mays) (see ref. 1). Clear-cut confirmation of this has yet to be achieved. Tryptophan was a precursor in dark grown maize seedlings,150 and results from labelling experiments on seedlings grown in 2H2O were complicated by exchange reactions and the randomization of label during MS analysis.151 Although indole appeared to be converted into IAA without the intermediacy of tryptophan in maize endosperm, there was evidence to suggest that several diVerent pathways to IAA coexisted.152 Catabolism of IAA by some strains of Bradyrhizobium japonicum leads to oxindole derivatives 71–73, isatinic acid 74 and eventual formation of anthranilic acid (Scheme 25).153 The homologue of IAA, indole-3-butyric acid (IBA) 75, is found naturally in some plants, and has been thought to arise from IAA and acetyl CoA via a typical acetate chain extension process.Recent investigations on the biosynthesis of IBA in maize (Zea mays) suggest a two-step reaction.154 A microsomal membrane preparation from dark grown shoots and roots converted 14C-labelled IAA and acetyl CoA in the presence of ATP into an unknown product, but not into IBA. The labelled product was then eYciently transformed into IBA when supplied to other maize cell fractions along with NADPH.On the other hand, microsomal membranes from light grown shoots and roots converted IAA into IBA when supplied with acetyl CoA, and the reaction was enhanced by the addition of ATP and NADPH. In this system, the unknown product did not accumulate. The product hydrolyzes to IAA, is not formed in the absence of acetyl CoA and is not IAA CoA. It is believed to be an intermediate in the reaction, possibly a conjugate 76 of IAA with ADP. In further studies,155 the indole-3-butyric acid synthetase activity has been partially purified.When obtained from dark grown seedlings, this consisted of two proteins, one of which was responsible for producing the intermediate. A single protein obtained from light grown seedlings appeared to be analogous to the combined dark grown proteins, and light may induce combination of the proteins to form a single enzyme which catalyzes the conversion without release of an intermediate. Recent reviews discuss IAA biosynthesis and metabolism, 156–158 the synthesis of phytohormones by plantassociated bacteria,159 and the structure and function of indolepyruvate decarboxylase.160 3.4 Indigo and indirubin There is growing evidence that the pigment indigo 77 is derived from indole by a sequence not involving tryptophan (compare DIMBOA, Section 2.12). In recent studies,161 2H-labelled indole supplied to shoot cultures of Polygonum tinctorium was incorporated specifically into both indigo and indirubin 78. In N N NH N O O R R CO2 – NH3 + H N N NH N O O R R CO2 – NH3 + N N X N O O R R CO2 – NH2 + H NH N NH N O O R R CO2 – NH2 + R O NH2 NH – – – B B H+ CO2 O2 R = 70 Scheme 24 Enzyme: tryptophan 2-monooxygenase NH CO2H NH CO2H HO O NH OH O NH O O NH2 CO2H O CO2H NH2 69 IAA 72 Dioxindole 71 Dioxindole-3-acetic acid 73 Isatin 43 Anthranilic acid 74 Isatinic acid Scheme 25 NH CO2H NH O P OH O P O OH O CO.SCoA OH O O OH OH Ad 75 IBA 76 NH HN O O HN NH O O 77 Indigo 78 Indirubin 30 Natural Product Reports, 1998suspension and root cultures, however, the feeding of indole suppressed indigo biosynthesis, and only indirubin accumulated.Feeding of tryptophan did not lead to the production of either labelled product. 4 Phenols and phenolic acids 4.1 Phenol and tyrosine phenol-lyase Tyrosine phenol-lyase is found in various bacteria and catalyzes the PLP-dependent elimination of phenol from L-tyrosine, giving pyruvate and ammonia. The crystal structure of the enzyme from Erwinia herbicola has recently been established.162 The enzyme exists as a tetramer.Site-directed mutagenesis of the Citrobacter freundii enzyme has been used to establish that Tyr-71, an invariant residue in all known tyrosine phenol-lyase sequences, has a dual role.163 It acts as a general acid catalyst in the reaction mechanism (Scheme 26), and is also involved in binding the cofactor PLP. All enzymic activity is lost when Tyr-71 is replaced with phenylalanine.Histidine-343 is also conserved in all known sequences of both tyrosine phenol-lyase and the related tryptophan indole-lyase (tryptophanase, see Section 3.2). However, site-directed mutagenesis experiments have established that this residue is not an essential basic group in the C. freundii enzyme, although it does appear to play an important function in catalysis, perhaps facilitating a conformational change when the substrates bind.164 Replacement of His-343 with Ala resulted in lower rates of elimination. 4.2 4-Hydroxybenzoic acid 4-Hydroxybenzoate 79 is produced in bacteria from chorismate via the enzyme chorismate lyase, but in plants it is synthesized by side-chain degradation of cinnamic acids (Scheme 27). Chorismate lyase is not normally present in plants; in E. coli it is encoded by the gene ubiC and provides entry to ubiquinone biosynthesis (see Section 9.2). Expression of ubiC in tobacco (Nicotiana tabacum) has led to transgenic plants showing high chorismate-lyase activity, and in which 4-hydroxybenzoate was accumulated as a glucoside.165 4-Hydroxybenzoate content was some 1000 times higher than in untransformed plants.Feeding experiments with [1,7- 13C2]shikimic acid yielded double-labelled 4-hydroxybenzoate and therefore proved that the chorismate-lyase pathway was now operative rather than the route via cinnamic acids. 4-O-Glucosylbenzoic acid 80 is characteristically accumulated in shikonin-free cell cultures of Lithospermum erythrorhizon, and is located mainly in the vacuoles. Enzymes involved in its synthesis and subsequent hydrolysis, namely 4-hydroxybenzoate glucosyltransferase and ‚-glucosidase, respectively, are located exclusively in the cytosol.166 This suggests the glucoside is synthesized in the cytosol, but is then transported and stored in the vacuole.It is then again transported to be hydrolyzed and subsequently utilized in shikonin biosynthesis (see also Section 9.3). 4.3 Salicylic acid Salicylic (2-hydroxybenzoic) acid has long been known as a plant constituent, but in recent years it has been identified to have plant hormone activity as a natural inducer of disease resistance. Its biosynthetic origins appear to be from cinnamic acid via a chain shortening mechanism, but two routes by way of the intermediates 2-coumaric acid or benzoic acid may be involved, depending on the sequence of ortho-hydroxylation and chain shortening.In infected cucumber (Cucumis sativus) plants, the pathway cinnamic acid]benzoic acid]salicyclic acid is implicated.167 Both labelled phenylalanine and benzoic acid were incorporated into salicyclic acid, and 2-aminoindane- 2-phosphonic acid, a specific inhibitor of PAL (see Section 5.2), completely inhibited the incorporation of phenylalanine but not that of benzoic acid. A soluble benzoic acid 2-hydroxylase has been obtained from tobacco (Nicotiana tabacum) and has been partially purified.168 The enzyme had characteristics of a P450-dependent system, and was strongly induced in tobacco leaves by inoculation with tobacco mosaic virus, or by infiltration with benzoic acid.Shoots of rice (Oryza sativa) also use the same pathway, readily converting cinnamic acid and benzoic acid into salicylic acid.169 Although salicyclic acid is present largely in the free form in rice shoots, endogenously supplied salicyclic acid is conjugated as a glucoside by the action of an inducible glucosyltransferase.An analogous enzyme activity has been reported in salicylic acid-treated tobacco leaves.170 This enzyme may play an important role in regulating the levels of free salicylic acid. The isolation of the isomeric mixture 2-carbomethoxyoxepin 81 and 1-carbomethoxybenzene 1,2-oxide 82 from cultures of the fungus Phellinus tremulae has suggested that arene oxides CO2 – H +NH PLP O H CO2 – +NH PLP O H HO Tyr-71 CO2 – +NH PLP O –O Tyr-71 H B2 H CO2 – +NH PLP O HO Tyr-71 B2 H CO2 – H +NH PLP O H B1 B2 HB1 B2 HB1 B1 L-Tyrosine + PLP Scheme 26 Enzyme: tyrosine phenol-lyase OH O CO2 – CO2 – OH CO2 – CO2 – OH CO2 – NH2 16 Chorismate 79 i OGlc CO2 – L-Phe 80 Scheme 27 Enzyme: i, chorismate-lyase Dewick: The biosynthesis of shikimate metabolites 31may be intermediates in the biosynthesis of methyl salicylate 83 and salicyclic acid in this organism.171 [7-13C]Benzoic acid was eYciently incorporated into methyl salicylate, methyl benzoate and 2-carbomethoxyoxepin, supporting a sequence benzoic acid]methyl benzoate]oxepin 81, then rearrangement to methyl salicylate (Scheme 28).The acid-catalyzed rearrangement of 82 to methyl salicylate has been shown to occur readily and it involves an NIH shift with migration of the carbomethoxy group. The biosynthesis and metabolism of salicylic acid has been reviewed.172 4.4 Homogentisic acid Homogentisic acid 85 is a tyrosine-derived phenylacetic acid derivative formed by the action of 4-hydroxyphenylpyruvate dioxygenase on 4-hydroxyphenylpyruvate 30 in the presence of oxygen.Molecular oxygen is incorporated into the carboxy and new hydroxy groups, and a mechanism involving oxidative decarboxylation to a peroxy acid 84, which then oxygenates the ring and allows migration of the side-chain has been proposed (Scheme 29). Homogentisic acid is of importance as a source of quinonoid derivatives, e.g. tocopherols and plastoquinones (see Section 9.4).The enzyme has also been implicated in the biosynthesis of melanin-like pigments (pyomelanin) in microorganisms.173 Thus, pigment formation correlated with homogentisic acid production and expression of the enzyme 4-hydroxyphenylpyruvate dioxygenase in three disparate marine species, Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana. 4-Hydroxyphenylpyruvate dioxygenase isolated from rat liver also displays a related oxidative decarboxylation reaction on ·-ketoisocaproate 86 giving ‚-hydroxyisovalerate 87 (Scheme 30).174 In fact, isolation and sequence determination of the enzyme catalyzing the reaction of Scheme 30 indicated the enzyme to be analogous to liver-specific rat F antigen, which is believed to be a species variant of 4-hydroxyphenylpyruvate dioxygenase.Both activities are provided by a single enzyme. The enzyme required the cofactors molecular oxygen, Fe2+, ascorbate and dithiothreitol for activity.Molecular cloning and expression of the protein in E. coli has allowed further studies of its substrate specificity.175 The C-terminal 14 amino acid portion is indispensible for catalytic activity; truncation results in complete loss of both activities.176 5 Phenylpropanoids 5.1 General Stress-induced phenylpropanoid metabolism has been reviewed.177 5.2 Phenylalanine ammonia-lyase Phenylalanine ammonia-lyase (PAL) catalyzes the stereospecific elimination of ammonia from L-phenylalanine to give (E)-cinnamic acid 89 (Scheme 31).Recent sources from which the enzyme has been isolated and purified include the plant basil (Ocimum basilicum),178 and microorganisms Rhodosporidium toruloides,179 Rhodotorula glutinis,180, 181 Sporidiobolus pararoseus182 and Ustilago maydis.183 The biological function of the enzyme in yeasts and fungi is still somewhat unclear. Gene sequences encoding PAL in Arabidopsis thaliana,184 O CO2Me O CO2Me CO2Me 82 81 H+ 83 CO2H CO2 Me OH Scheme 28 85 Homogentisate 30 4-Hydroxyphenylpyruvate CO2 O2 i, Fe2+ 84 O CO2 – OH O O OH O H O O– OH O O CO2 – OH OH CO2 – OH Scheme 29 Enzyme: i, 4-hydroxyphenylpyruvate dioxygenase 86 87 O2, Fe2+ i CO2 – O CO2 – HO Scheme 30 Enzyme: i, 4-hydroxyphenylpyruvate dioxygenase L-Phe 88 89 Cinnamate + + + + + + + + HN NH OH CO2 – NH3 HR HS CO2 – NH3 HR HS OH HN NH B Enz HN NH OH CO2 – H B Enz CO2 – NH3 HO HN NH H B Enz Scheme 31 Enzyme: phenylalanine ammonia-lyase (PAL) 32 Natural Product Reports, 1998Bromheadia finlaysoniana,185 lemon (Citrus limon),186 tobacco (Nicotiana tabacum),187 rice (Oryza sativa),188 parsley (Petroselinum crispum),189 hybrid aspen (Populus kitakamiensis), 190, 191 Stylosanthes humulis192 and wheat (Triticum aestivum)193 have been reported, and shown to be highly similar to those known for other plants.Small gene families are frequently reported, e.g. in Arabidopsis (three PAL genes),184 tobacco,187 rice,188 parsley (four genes),189 aspen (four genes),190, 191 and wheat (two genes).193 A PAL gene from poplar (Populus trichocarpa#deltoides) has been expressed in cell cultures from the insect Spodoptera frugiperda.194 Sitedirected mutagenesis and expression of a mutant enzyme demonstrated that Ser-202 was essential for catalytic action.Transgenic tobacco plants containing a PAL gene from bean overexpress PAL, and synthesize increased levels of chlorogenic (5-O-caVeoylquinic) acid and 4-coumaric acid glucoside.195 The active site of PAL is known to contain a dehydroalanine residue which participates in the elimination of ammonia.This dehydroalanine is produced post-translationally by modification of a serine residue, Ser-202. By site-directed mutagenesis, Ser-202 in parsley PAL has been replaced with alanine or threonine.196 Mutant proteins and recombinant wild-type enzyme were then treated with NaBH4. All three proteins were virtually inactive with L-phenylalanine, but catalyzed deamination of L-(4-nitrophenyl)alanine.L-Tyrosine was a poor substrate, whilst DL-m-tyrosine was converted at a rate comparable to phenylalanine. These findings are consistent with a mechanism in which the crucial step is an electrophilic attack of the dehydroalanine prosthetic group at position 2 or 6 of the phenyl group (Scheme 31). In the resulting carbocation 88, the ‚-HSi atom is activated in a similar way as it is in the nitro analogue 90.This mechanism contrasts with that previously proposed which invokes attack of the phenylalanine nitrogen onto dehydroalanine, then removal of the ‚-HSi to generate a carbanion intermediate (Scheme 32). 5.3 Hydroxycinnamic acids and esters Hydroxycinnamic acids are usually obtained by further aromatic substitution of the cinnamic acid formed by the action of PAL, sequentially building up the oxygenation and methylation pattern. Cinnamic acid 4-hydroxylase (CA4H) is a cytochrome P450-dependent enzyme transforming cinnamic acid into 4-coumaric acid.Genes encoding for CA4H in Populus tremuloides,197 P. kitakamiensis198 and Catharanthus roseus,199 have recently been isolated and sequenced. A small gene family is present in hydrid aspen (P. kitakamiensis).198 Translational fusions of CA4H with cytochrome P450 reductase allow production of an enzyme that requires no additional cofactors.199 Methods for the isolation of CA4H from plant microsomes have been described.200 Ferulate 5-hydroxylase from Arabidopsis thaliana has a maximum 34% sequence identity with other P450 enzymes, much less with CA4H, and is considered to belong to a new subfamily of P450 monooxygenases.201 A cinnamic acid O-methyltransferase activity in wheat (Triticum aestivum) seedlings showed a preference for caVeic acid as substrate (giving ferulic acid) in the early stages of development prior to lignin deposition.202 After lignin deposition began, the O-methyltransferase activity then showed a preference for 5-hydroxyferulic acid (giving sinapic acid).The substrate specificity of a bispecific caVeic acid–5- hydroxyferulic acid O-methyltransferase from aspen (Populus tremuloides) has been investigated.203 Although a wide range of phenolic substrates were accepted, none was as eVective as the natural substrates. Site-directed mutagenesis experiments demonstrated that conserved cysteine residues played no important role in the catalytic mechanism.Nucleotide sequences for the aspen gene,204 and a caVeic acid O-methyltransferase gene from almond (Prunus amygdalus)205 have been established. There is growing evidence that in many plants, methylation may occur with a cinnamoyl CoA substrate rather than on the free cinnamic acid. Both types of enzyme are found in Zinnia elegans, and they are diVerentially expressed.206 Nucleotide sequences for caVeoyl CoA 3-Omethyltransferase genes in long-stalked chickweed (Stellaria longipes)207 and aspen (Populus tremuloides)208 have been established.Activation of cinnamic acids to coenzyme A esters is accomplished by ligase enzymes. Gene sequences for 4- coumarate:CoA ligase from Loblolly pine (Pinus taeda),209 Arabidopsis thaliana,210 tobacco (Nicotiana tabacum)211 and Lithospermum erythrorhizon212 and for a ferulate–sinapatespecific CoA ligase from aspen (Populus tremuloides)213 have been reported. Most of the genes display high homology.Multiple genes were isolated from tobacco and Lithospermum. The 4-coumarate:CoA ligase proteins typically accept other hydroxycinnamic acids, e.g. ferulate and caVeate, as substrates. Coenzyme A esters are involved in the acylation of shikimic acid in young green fruits of date (Phoenix dactylifera) and of quinic acid in leaves of chicory (Cichorium endivia).214 The enzymes were partially purified, then treated with amino acid-specific reagents to demonstrate that histidine, cysteine and lysine residues were important for catalytic activity.Four hydroxycinnamoyltransferases in gametophytes and sporophytes of horsetail (Equisetum arvense) have been partially purified and studied.215 These enzymes utilize caVeoyl CoA 91 to acylate meso-tartaric acid (giving mono- then di-esters 94 and 95), shikimic acid (giving the 5-ester dactylifric acid 93) and quinic acid (giving the 5-ester chlorogenic acid 92) (Scheme 33). The enzymes varied according to their ability to accept caVeoyl CoA and 4-coumaroyl CoA as substrates; cinnamoyl CoA and feruloyl CoA were less eVective donors.In the case of tartaric acid as an acceptor, only the meso isomer was utilized. An enzyme from tobacco (Nicotiana tabacum) 90 + + + + + CO2 – NH2 N –O O– CO2 – NH2 N O O– O NH H2N HS CO2 – H Ph HR B+ H OH NH B H2N HS CO2 – H Ph HR B B OH NH B H2N CO2 – H B H Ph H O NH B NH2 B H Ph CO2 – O NH NH3 B+ H H B H Ph CO2 – H+ + + – + + + Scheme 32 Enzyme: phenylalanine ammonia-lyase (PAL) Dewick: The biosynthesis of shikimate metabolites 33that transfers hydroxycinnamoyl groups from their CoA esters to the ¢-hydroxy group of 16-hydroxypalmitic acid has been purified.216 Feruloyl CoA was the most eVective donor, though sinapoyl CoA and 4-coumaroyl CoA could also be utilized; a number of long chain 1-alcohols and ¢-hydroxyfatty acids would function as acceptors.This type of enzyme activity has been detected in a wide range of plant systems.217 Acylation of the amine tyramine is catalyzed by a pathogen elicitorinducible enzyme preparation from cell suspension cultures of potato (Solanum tuberosum).218 Feruloyl CoA was the preferred acyl donor, and tyramine the acceptor, though octopamine and dopamine were also utilized less eVectively.Purification of the enzyme showed it to be a heterodimer with the subunits non-covalently associated.219 Cell-free extracts of Aphelandra tetragona stem are able to acylate the polyamines spermine 96 and spermidine, but not putrescine, with 4-coumaroyl CoA acting as a better donor than feruloyl CoA.220 The product from reaction of spermine with 4-coumaroyl CoA is a monoamide, though the position of acylation has not been confirmed.Roots of Aphelandra contain the macrocyclic polyamine alkaloid aphelandrine 97 which is composed of spermine with two 4-coumaroyl residues, and the demonstrated enzyme activity may be involved in the early stages of its biosynthesis. A variety of hydroxycinnamic acid conjugates are known to be formed by processes involving glucose esters rather than the coenzyme A esters as the activated form of the acid.An enzyme purified from Brassica napus has been shown to transfer glucose from UDPglucose to sinapic acid in the synthesis of 1-O-sinapoyl glucose.221 A short review describes the biosynthesis of rosmarinic acid, the ester of caVeic acid with 3,4-dihydroxyphenyllactic acid, in Coleus blumei.222 5.4 Coumarins The isolation of a glucosyltransferase activity from Duboisia myoporoides converting scopoletin 98 into scopolin 99 has been reported.223 5.5 Lignins Lignins are natural phenolic polymers found in plant cells and are generally believed to arise by phenolic oxidative coupling of hydroxycinnamyl alcohol monomers (monolignols) brought about by peroxidase enzymes. The most important of these monolignols are 4-hydroxycinnamyl (4-coumaryl) alcohol, coniferyl alcohol and sinapyl alcohol which are derived, respectively, from 4-coumaric acid, ferulic acid and sinapic acid via the corresponding coenzyme A esters and aldehydes.However, recent observations have identified an additional pathway in which methylation occurs at the coenzyme A level rather than on the free cinnamic acids (see Section 5.3). Altered lignin composition in transgenic tobacco (Nicotiana tabacum)224 and Populus tremula x P. alba225 has been achieved by the expression of antisense gene constructs for the caVeic acid–5-hydroxyferulic acid bispecific O-methyltransferase.In certain lines, this led to a reduced O-methyltransferase activity, 92 Chlorogenic acid 93 Dactylifric acid meso-Tartaric acid 95 Quinic acid 94 Caffeoyl-CoA Shikimic acid 91 Caffeoyl-CoA O CO2H HO HO OH O OH OH O HO OH O OH OH CO2H COSCoA HO HO HO HO O CO2H H O HO CO2H H HO HO O CO2H H O CO2H H OH HO O O Scheme 33 97 Aphelandrine 96 Spermine O OH H H O N NH NH O HN H H2N N H H N NH2 98 Scopoletin R = H 99 Scopolin R = Glc O O MeO RO 34 Natural Product Reports, 1998a reduction in the syringyl (4-hydroxy-3,5-dimethoxyphenyl) to guaiacyl (4-hydroxy-3-methoxyphenyl) ratio in the lignin, and the appearance of a new component, namely 5-hydroxyguaiacyl residues.Introduction of chimeric sense– antisense gene constructs for 4-coumarate CoA ligase into tobacco plants also yielded lower enzyme activity, and a reduction in lignin content.226 Reduction of cinnamoyl CoA precursors to cinnamyl alcohols is catalyzed by two NADPH-dependent enzymes cinnamoyl CoA:NADPH oxidoreductase and cinnamyl alcohol dehydrogenase (CAD). Gene sequences encoding for CAD have recently been reported for alfalfa (Medicago sativa),227 poplar (Populus trichocarpa x P.deltoides),227 Arabidopsis thaliana,228, 229 Pinus radiata230 and Pinus taeda.231 Deduced amino acid sequences for the proteins were 50–80% identical to those established in other plants. Site-directed mutagenesis has been used to demonstrate the involvement of Ser-212 in Eucalyptus gunni CAD.232 This residue is conserved in the enzyme from several species, and it also corresponds to Asp-223 in horse liver alcohol dehydrogenase, an NADHdependent enzyme.A protein in which Ser-212 was replaced with aspartic acid was overexpressed in E. coli, and shown to have significantly decreased catalytic eYciency with NADP+, but an unchanged low activity with NAD+ (about 4% of the activity of wild-type enzyme using NADP+).This implicates Ser-212 in recognition of the phosphate grouping in NADPH. Introduction of an antisense construct for the cinnamyl alcohol dehydrogenase gene from Aralia cordata into tobacco has resulted in transgenic plants with much lower CAD enzyme activity.233 Lignin content was not significantly aVected, but there was an observed increase in the levels of 4-hydroxycinnamaldehyde groups incorporated into the lignin.Monolignols such as coniferyl alcohol and sinapyl alcohol may be synthesized and stored as glycosides, and then released enzymically when required for lignin biosynthesis. Coniferin 100 typically accumulates to high levels in gymnosperms prior to lignin deposition during the spring. Two ‚-glucosidase enzymes have been isolated from lodgepole pine (Pinus contorta var. latifolia) and shown to hydrolyze coniferin and syringin 101.234 One of these enzymes was purified, shown to be a homodimer, and had an N-terminal sequence with high homology to other known plant ‚-glucosidases.Coniferin labelled with 3H in its side-chain and 14C in the glucose residue was transformed into syringin in Magnolia kobus with loss of all 14C label.235 Thus, conversion of guaiacyl to syringyl substitution must occur by preliminary hydrolysis to the monolignol. EPR spectroscopy has been used to study the eVects of methoxy substitution on the unpaired electron distribution in lignin precursor radicals.236 It was deduced that methoxy substitution increases the unpaired electron density on the phenolic oxygen, and that this subsequently determines the nature of bond formation during polymerization, explaining the high degree of selectivity observed in the incorporation of phenylpropanoids into lignin.Thus, it is predicted that for 4-coumaryl alcohol, coupling is primarily 8–8 with a small amount of 8–5, and for coniferyl alcohol mainly 8–8 and 8–5 with some 8–O–4 (see Scheme 34). 8–O–4 Coupling becomes more important for sinapyl alcohol. These predictions are borne out by the couplings observed with in vitro dehydrogenation polymers produced from the precursors. The oxidizing abilities of a fungal laccase and horse radish peroxidase have been compared.237 The laccase enzyme oxidized sinapic acid better than ferulic or 4-coumaric acids, and sinapyl alcohol better than coniferyl alcohol. Horse radish peroxidase oxidized 4-coumaric acid better than ferulic or sinapic acids, and coniferyl alcohol better than sinapyl alcohol.It was also demonstrated that with both enzymes, ferulic acid was predominantly oxidized from a mixture of 4-coumaric acid and ferulic acid, sinapic acid from a mixture of 4-coumaric acid and sinapic acid or from a mixture of ferulic acid and sinapic acid, and sinapyl alcohol from a mixture of sinapyl alcohol and coniferyl alcohol. To explain these observations it was suggested that the 4-hydroxyphenyl radical can oxidize guaiacyl and syringyl groups to produce their respective radicals, and that the guaiacyl radical can oxidize the syringyl group.These proposals are supported by results using peroxidase isozymes from Vigna angularis.238 This peroxidase oxidized sinapic acid and sinapyl alcohol only very slowly, but rapidly transformed compounds with a 4-hydroxyphenyl or guaiacyl group. However, the oxidation of sinapyl alcohol could be greatly enhanced by adding 4-coumaric acid or ferulic acid.This indicates that there is indirect oxidation of sinapyl alcohol by the radicals generated from 4-coumaric acid or ferulic acid. Reviews on lignin biosynthesis have been published,239–241 including discussion of molecular biology240 and mechanisms of control.241 5.6 Lignans Lignans are essentially cinnamyl alcohol dimers (dilignols), though further cyclization and other modifications create a wide range of structural types.The coupling process is an example of phenolic oxidative coupling, analogous to that proposed for the biosynthesis of lignins. However, this coupling is not the result of H2O2-requiring peroxidases, and, in contrast to lignin biosynthesis, stereochemically controlled couplings operate leading to enantiomerically pure products. Oxidizing enzymes (laccases and peroxidases) from Forsythia intermedia convert coniferyl alcohol 102 into (&)-pinoresinol together with racemic lignans (&)-dehydrodiconiferyl alcohol and (&)-guaiacylglycerol-‚-O-coniferyl ether.242 However, in the presence of an additional 78 kDa protein, the only product is (+)-pinoresinol 103.This protein may thus function as a switching mechanism between lignin and lignan pathways. In the lignan pathway (Scheme 35), (+)-pinoresinol is successively reduced by NADPH-dependent reductase activities to (+)-lariciresinol 104 and (")-secoisolariciresinol 105. Two isoforms of a (+)-pinoresinol/(+)lariciresinol reductase have been purified from F.intermedia, both of which catalyze the sequential stereospecific reductions and have comparable physical properties.243 The stereospecificity was confirmed by incubating a preparation containing both isoforms with (&)-pinoresinol and [(4R)-2H]NADPH and observing the 100 Coniferin R = H 101 Syringin R = OMe OH OGlc MeO R Coupling sites: 2 3 5 4 6 7 9 8 OH OH OMe Scheme 34 Dewick: The biosynthesis of shikimate metabolites 35formation of (+)-[(7*R)-2H]lariciresinol (Scheme 36).Similarly, (+)-lariciresinol was transformed into (")- secoisolariciresinol. The gene encoding this enzyme activity has been cloned and expressed in E. coli as a functional ‚-galactosidase fusion protein. The deduced amino acid sequence for the protein revealed a strong homology (41–44% identity, 61–64% similarity) with the enzyme isoflavone reductase (see Section 6.6). Cell-free extracts obtained from Arctium lappa catalyze the enantioselective formation (20% enantiomeric excess) of (+)-secoisolariciresinol from coniferyl alcohol in the presence of NADPH and H2O2.244 Cell cultures of Pinus taeda under appropriate conditions can be induced to undergo cell wall lignification and production of an extracellular lignin precipitate.245 Addition of KI, an H2O2 scavenger, inhibits lignification, but results in the build-up of lignans derived from coniferyl alcohol, probably as part of the lignin assembly process.These include dehydrodiconiferyl alcohol 107 and dihydrodehydrodiconiferyl alcohol 108, guaiacylglycerol-‚-O-coniferyl ether 106, pinoresinol 103, shonanin 109 and isoshonanin 110. Some of these are probably related biosynthetically as shown in Scheme 37, but cannot be O O H Ar H H Ar H O H H Ar H D Ar H HO H H Ar D D Ar H HO H OH N CONH2 R H D N CONH2 R H D 7¢ 7 103 (+)-Pinoresinol 104 (+)-Lariciresinol 105 (–)-Secoisolariciresinol Scheme 36 OH OH MeO OH O MeO O O H OH OMe HO MeO H H O OMe O MeO H HO OH O H OH OMe HO MeO H OH H OH OMe HO MeO H OH HO 102 Coniferyl alcohol x 2 103 (+)-Pinoresinol 104 (+)-Lariciresinol O 105 (–)-Secoisolariciresinol NADPH NADPH Scheme 35 O OH OH OMe HO MeO O OH OH OMe HO MeO O O H OH OMe HO MeO H O MeO HO OH OMe O MeO HO OH OMe OH OMe OH OH OMe HO 107 Dehydrodiconiferyl alcohol 103 (+)-Pinoresinol 108 Dihydrodehydrodiconiferyl alcohol 106 Guaiacylglycerol-b- O-coniferyl ether 110 Isoshonanin 109 Shonanin Scheme 37 36 Natural Product Reports, 1998explained on the basis of simply phenolic oxidative coupling, requiring other processes, e.g.reduction. The biosynthesis of lignans is covered in a recent review.246 5.7 Tropic acid The biosynthetic origin of tropic acid 111 by a rearrangement process from phenylalanine has been appreciated for many years, but the mechanism of the rearrangement and the nature of intermediates still challenge investigators.From earlier labelling studies, an intramolecular migration of the carboxy group with retention of configuration at the benzylic centre of phenylalanine and a back migration of the 3-pro-S hydrogen to C-2 [Scheme 38(a)] has been the basis for speculation and experimentation. Phenyllactic acid 114 is now well established as an intermediate between phenylalanine and tropic acid, though the rearrangement takes place after esterification to tropine, the component heterocycle of the tropane alkaloids.However, the use of phenyllactic acid has now allowed the stereochemistry associated with the rearrangement to be reassessed. Incubation of (R)-D-3-phenyl[2-13C,2H]lactic acid with transformed root cultures of Datura stramonium gave hyoscyamine 113 with retention of the 13C–2H bonding.247 2H-Label was lost from a similar feeding of the enantiomeric substrate. This establishes that the R enantiomer of phenyllactic acid in littorine 112 is the one processed, and rules out phenylpyruvate as a precursor, since this would result in loss of 2H.A similar conclusion was reached based on feeding of the separate enantiomers of phenyllactic acid to the roots of Datura stramonium plants, though in the aerial parts, substantial incorporations of the S enantiomer were recorded.248 In further experiments,249 (RS)-DL-3-phenyl[2-3H]lactic acid was used as precursor, giving hyoscyamine with 3H located at C-3 of tropic acid. The chirality at the 3-hydroxymethyl was then established as S by reductive conversion to a chiral methyl and subsequent oxidation to acetate, indicating that any back migration of hydrogen from the benzylic position must involve inversion. However, by using several diVerent chirally labelled (R)-phenyllactate precursors with 13C at C-2 and 2H at C-2 or C-3, it was firmly demonstrated that the true fate of the C-2 and C-3 protons is as shown in Scheme 38(b).250 Thus, the 3-pro-S hydrogen is retained during the rearrangement whilst the 3-pro-R hydrogen is lost.The rearrangement proceeds with stereochemical inversion at the migration terminus and there is no migration of hydrogen from C-3 to C-2. A plausible mechanism for the rearrangement of the phenyllactate moiety in littorine 112 to the tropate residue in hyoscyamine 113 has been proposed as a result of feeding experiments with labelled phenyllactic acid in transformed root cultures of Datura stramonium and Brugmansia candida x B.aurea.251 (RS)-3-Phenyl[1,3-13C2]lactic acid was incorporated into hyoscyamine with the expected retention of doublelabelling as measured by MS, the incorporation being una Vected by concomitant feedings of tropic acid. In addition, the tropane alkaloids 3·-(2*-phenylacetoxy)tropane 119 and 3·-(2*-hydroxyacetoxy)tropane 118 were also labelled to a similar extent, 119 being double-labelled and 118 being singlelabelled (Scheme 39). This indicated that the phenylacetic acid group of 119 cannot be derived from free phenyllactic acid since decarboxylation would give a single-labelled species, but probably arises by loss of the hydroxymethyl from tropic acid.In confirmation, 3-phenyl[2-13C, 2-2H]lactic acid did not label 3·-(2*-phenylacetoxy)tropane but did produce labelled hyoscyamine and littorine. Similarly, it is suggested that the 2-hydroxyacetate residue is also produced from phenyllactate, by cleavage of the phenylmethyl group.Littorine labelled with 2H in the tropane methyl group and 1,3-13C2 in the phenyllactate system gave the predicted M+5 molecular ions for hyoscyamine and 119, and M+4 for 118. A mechanism for tropic acid biosynthesis allowing formation of these alternative acid residues is shown in Scheme 39. This begins with abstraction of a C-3 hydrogen to generate a free radical 115, and rearrangement occurring through the cyclopropane alkoxyl radical 116, via cleavage of the C-1–C-2 bond.The alternative acid groups are suggested to arise from radicals 115 and 117 by appropriate cleavage processes as indicated. 5.8 Phenylpropenes The overall pathway to epoxyisoeugenol 2-methylbutyrate (EPB) 125 in Pimpinella anisum has been delineated via feeding CO2H HS H HR NH2 H OH H HO2C CO2H HR H HS NH2 CO2R HR HO HS H H CO2R OH H H N Me O 28 L-Phenylalanine · · 111 Tropic acid ( a) ( b) R = tropyl: * · · ° * 2 3 2 3 112 Littorine 113 Hyoscyamine Scheme 38 CO2H HO H 114 ( R)-Phenyllactic acid 2 3 OH R O OH R O O OR OH OH O OR OH O OR O OR OH R O N Me O H H 1¢ 2¢ 3¢ 1¢ 3¢ Cleavage C-2¢–C-3¢ Cleavage C-2¢–C-3¢ 2¢ R = tropyl: 118 119 112 Littorine 113 Hyoscyamine 117 115 116 = 13C • • • • •• • • • • • • • • Scheme 39 Dewick: The biosynthesis of shikimate metabolites 37experiments (Scheme 40).Although superficially a phenylpropanoid, EPB contains the rare 2,5-dioxygenation and has been shown to arise from anethole 123 through pseudoisoeugenol 124 via a migration of the side-chain, initiated by a hydroxylation-induced NIH shift.On the basis of incorporation data, (4-methoxy)cinnamyl alcohol 122 rather than anol 121 was considered an intermediate. Recent experiments have reversed this conclusion, and anol is now implicated as an obligatory intermediate.252, 253 Although both 121 and 122 are incorporated into EPB in callus cultures of P. anisum, the incorporation of anol 121 is severely depressed by its rapid modification by peroxidases.When fed along with sodium azide, an inhibitor of peroxidases, incorporations were dramatically increased, and anol was substantially better than (4-methoxy)cinnamyl alcohol as a precursor. Furthermore, an anol O-methyltransferase activity was detected in a callus homogenate; this also methylated allyl phenols eugenol and chavicol, but not 4-coumaryl alcohol 120, coniferyl alcohol or hydroxycinnamic acids. There was also evidence for the presence of a 4-coumaryl alcohol reductase activity in the extracts.In further studies, the enzymes PAL (see Section 5.2) and cinnamic acid 4-hydroxylase (see Section 5.3) were also shown to be active in the cell-free extracts.254 Addition of the PAL inhibitor L-2-aminooxy-3-phenylpropionic acid inhibited incorporation of phenylalanine into EPB. 6 Flavonoids 6.1 Chalcones The condensation of 4-coumaroyl CoA 126 with three molecules of malonyl CoA to produce the chalcone naringeninchalcone 127 is catalyzed by the enzyme chalcone synthase [naringenin-chalcone synthase; malonyl CoA:4-coumaroyl CoA malonyltransferase (cyclizing)] (Scheme 41).Flavonoids containing a resorcinol substitution pattern in the acetate/ malonate-derived ring, rather than the phloroglucinol pattern as in naringenin-chalcone, are derived from the chalcone isoliquiritigenin 129, which arises as a result of a reductase acting concomitantly with chalcone synthase (Scheme 41) (see also Section 7).Multiple genes encoding chalcone synthase have now been reported, and sequenced, from several plant species. These include subterraneum clover (Trifolium subterraneum) (at least nine copies, of which six have been sequenced),255 Pueraria lobata (four genes)256 and soybean (Glycine max) (at least seven genes in gene clusters).257 Two members of a gene family (at least six members) from potato (Solanum tuberosum) have been sequenced,258, 259 as have thirteen genes from seven species of Ipomoea.260 A single chalcone synthase gene in rice (Oryza sativa) has been reported.261, 262 There is little detailed knowledge available concerning flavonoid biosynthesis in the lower plants, and information has only recently been presented demonstrating chalcone synthase activity in a representative liverwort Marchantia polymorpha.263 This yielded naringenin when incubated with 4-coumaroyl CoA and malonyl CoA, and the enzyme crossreacted with antibodies raised against chalcone synthase derived from three higher plants (rye, parsley and spinach) suggesting a close similarity in the proteins. Chalcone reductase is also encoded by multiple genes in alfalfa (Medicago sativa), and seven clones have been isolated from a cDNA library isolated from alfalfa leaves infected with the fungus Pseudomonas syringae pv.pisi.264 Three of the genes have been sequenced.265 In parallel studies, sequence data for two chalcone reductase genes from alfalfa,266 and one gene from Glycyrrhiza echinata267 have been reported.Although stress conditions stimulate flavonoid biosynthesis in M. sativa, OH OH OH OMe OMe OH OH OMe O OMe O O Phe 122 121 Anol 123 Anethole 124 Pseudoisoeugenol 120 125 EPB Scheme 40 O CoAS OH O OH O O SCoA O O OH O SCoA O H OH OH OH O OH HO O OH O OH HO O OH O HO OH OH O HO 126 4-Coumaroyl-CoA Malonyl-CoA 127 Naringenin-chalcone 128 Naringenin 130 Liquiritigenin i 129 Isoliquiritigenin ii NADPH iii iii Scheme 41 Enzymes: i, chalcone synthase; ii, reductase, iii, chalcone isomerase 38 Natural Product Reports, 1998the expression of chalcone synthase and chalcone reductase activities appears to be controlled diVerentially according to the various conditions.264 Chalcone synthase-like genes active during corolla development in Gerbera hybrida have been identified, and two of these encode for typical chalcone synthase enzymes.268, 269 Their gene expression appears to correlate with the production of flavonol and anthocyanin pigments.However, a third gene is significantly diVerent, its expression being unrelated to pigment production, and its deduced amino acid sequence deviating from those of typical chalcone synthase or stilbene synthase proteins. Furthermore, the protein is unable to transform 4-coumaroyl CoA, instead utilizing eVectively only benzoyl CoA to give as yet unidentified products. Relationships between chalcone synthase and stilbene synthase are discussed further in Section 7. 6.2 Flavanones, flavonols, anthocyanidins and related flavonoids The cyclization of chalcones to (2S)-flavanones is catalyzed by the enzyme chalcone isomerase (chalcone-flavanone isomerase) (Scheme 41). A chalcone isomerase cDNA from Pueraria lobata has been cloned and overexpressed in E. coli.270 Sitespecific mutagenesis showed that Cys-119 was not an essential active site residue. Elicited cultures of old man cactus Cephalocereus senilis synthesize a group of flavonoids characterized by an unsubstituted B-ring, e.g.the flavone baicalein 131 and the aurone cephalocerone 132. Preliminary studies (see ref. 3) had indicated that chalcone synthase from these cultures was active with cinnamoyl CoA, and that chalcone isomerase accepted the product 2*,4*,6*-trihydroxychalcone. In recent studies,271 two isoforms of chalcone isomerase were identified, and these demonstrated activity towards both naringenin-chalcone and 2*,4*,6*-trihydroxychalcone.A single form of cinnamate CoA ligase was active with both cinnamate and 4-coumarate. Therefore, the cultures contain enzyme activities for the synthesis of both B-ring hydroxy- and deoxy-flavonoids, though elicitation results in synthesis of only B-ring deoxy compounds. It is suggested that metabolic compartmentation may allow bypassing of cinnamate-4-hydroxylase activity, and that cinnamoyl CoA is then utilized by the nondiscriminating chalcone synthase and chalcone isomerase activities.Cell-free extracts from cell suspension cultures of Cassia didymobotrya contain polyphenol oxidase enzymes capable of transforming 2-hydroxychalcones into aurones.272 Thus, chalcones 133 were converted into a mixture of (&)-flavanone, (E)- and (Z)-aurones 134, and auronol 135, the latter proven to occur by further oxidation of the aurone. A free radical coupling sequence (Scheme 42) is proposed to account for these products.The relationship of flavanones to dihydroflavonols, flavonols, flavan-3,4-diols (leucoanthocyanidins) and anthocyanidins is outlined in Scheme 43. Flavanone 3-hydroxylases are 2-oxoglutarate-dependent dioxygenases requiring the cofactors oxygen, Fe2+ and ascorbate. Genes encoding for flavanone 3-hydroxylase have been identified in a number of plants and sequence data presented. Recently reported sources include Arabidopsis thaliana,273, 274 Bromheadia finlaysoniana,275 maize (Zea mays),276 alfalfa (Medicago sativa)277 and carnation (Dianthus caryophyllus).278 Most of the deduced amino acid sequences share 80–90% identity.A microsomal flavonoid 3*-hydroxylase in sweet orange (Citrus sinensis) has been demonstrated to be a typical cytochrome P450 system.279 It hydroxylated naringenin 128 to eriodictyol 136, and also dihydrokaempferol 138 to dihydroquercetin 139, and kaempferol 141 to quercetin 142. Dihydroflavonol 4-reductase is an NADPH-dependent enzyme converting dihydroflavonols into flavan-3,4-diols (leucoanthocyanidins), precursors of anthocyanidins.Using a Petunia hybrida-derived probe, a full length cDNA clone from petals of Rosa hybrida has been isolated and sequenced.280 Sequence identity at the amino acid level with other reported dihydroflavonol 4-reductase genes was 59–67%. A Petunia cultivar, pale pink in colour due to a deficiency in flavonoid 3*-hydroxylase and flavonoid 3*,5*-hydroxylase, was transformed with the Rosa dihydroflavonol 4-reductase DNA to give transgenic plants salmon-pink in colour and containing pelargonidin 146, an anthocyanidin rarely found in Petunia.280 The insertion of the Rosa gene enabled the conversion of dihydrokaempferol 138 into leucopelargonidin 143 to be achieved, a conversion not catalyzed by the Petunia dihydro- flavonol 4-reductase. A gene encoding dihydroflavonol 4-reductase from Gentiana triflora has also been characterized, along with those for flavonoid 3*,5*-hydroxylase and flavonoid 3-glucosyltransferase.281 Sequence identities with counterparts from other plants were 57–71, 62–77 and 31–52%, respectively.The latter two genes were expressed in E. coli to give active enzymes. Flavonoid 3*,5*-hydroxylase was eVective in hydroxylating naringenin 128 (to 136), eridictyol 136 (to 137), dihydrokaempferol 138 (to 139), dihydroquercetin 139 (to 140) and the flavone apigenin 149. The anthocyanidins pelargonidin 146, cyanidin 147, and delphinidin 148 were the preferred O O HO OH HO HO O O O O 131 Baicalein 132 Cephalocerone OH R O HO OH OH R O HO O OH R O HO O OH R O HO O O R O HO O HO O O O H HO O O OH R R HO O O OH R OH 133 R = H, OMe 134 R = H, OMe – H (±)-Flavanone – H 135 R = H, OMe + H O Scheme 42 Dewick: The biosynthesis of shikimate metabolites 39substrates for the glucosyltransferase.An anthocyanidin 3-Oglucosyltransferase from cell suspension cultures of grape (Vitis vinifera) had highest activity with cyanidin as acceptor, though delphinidin and pelargonidin were also excellent substrates. 282 Anthocyanin biosynthetic genes are expressed in all tissues of grape, except for that encoding flavonoid 3-Oglucosyltransferase which is expressed only in the fruit skin. White grapes lack anthocyanins because they are deficient in flavonoid 3-O-glucosyltransferase.283 Cell-free extracts of developing leaves of lemon (Citrus limon) contain a hesperetin 7-O-glucosyltransferase activity.284 As well as glucosylating the flavanone hesperetin 151, the enzyme also accepted naringenin 128, the flavones apigenin 149 and crysin 150, and the flavonols morin 152 and kaempferol 141 as substrates.Wild-type Petunia pollen accumulates high levels of flavonol 3-O-glycosides, and pollen germination appears to be dependent on these flavonoids. Conditionally male-fertile pollen contains no flavonols, and is unable to germinate, though this function can be restored if kaempferol or quercetin is added.Two glycosyltransferase activities have been isolated from Petunia pollen, and shown to be highly specific for flavonols.285 Both kaempferol and quercetin are O OH O OH HO O OH O OH HO OH O OH O OH HO OH O OH O OH HO OH OH O OH O OH HO OH OH OH O OH OH OH HO OH O OH OH OH HO OH OH O OH OH OH HO OH OH OH O OH OH HO OH O OH OH HO OH OH O OH OH HO OH OH OH O OH O OH HO OH O OH O OH HO OH OH O OH O OH HO OH OH 138 Dihydrokaempferol 136 Eriodictyol 128 Naringenin 139 Dihydroquercetin 140 Dihydromyricetin 144 Leucocyanidin 145 Leucomyricetin 146 Pelargonidin 147 Cyanidin 148 Delphinidin 141 Kaempferol 142 Quercetin i iii ii ii iv iv iv v vi vi vi iii 137 v ii + + + 143 Leucopelargonidin Scheme 43 Enzymes: i, flavonoid 3*-hydroxylase; ii, flavanone 3-hydroxylase; iii, flavonoid 3*,5*-hydroxylase; iv, dihydroflavonol 4-reductase; v, flavonol synthase; vi, leucoanthocyanidin oxidase/dehydratase O R O OH HO O OMe O OH HO OH O OH O OH HO OH OH 149 Apigenin, R = OH 150 Crysin, R = H 151 Hesperetin 152 Morin 40 Natural Product Reports, 1998transformed by the action of the enzymes into their respective 3-O-(2+-O-glucosyl)galactosides 153 and 154, identical to materials found in the wild-type pollen.The major anthocyanins found in an Afghan cultivar of carrot (Daucus carota) are cyanidin 3-O-xylosylglucosylgalactoside acylated with sinapic or ferulic acid.Two of the enzymic glycosylations involved in the biosynthesis of these compounds have been detected in protein preparations.286 A galactosyltransferase utilizes UDPgalactose to glycosylate the 3-hydroxy group of cyanidin 147, and a xylosyltransferase then transfers the xylosyl group from UDPxylose to give the 3-xylosylgalactoside 155 (Scheme 44). The galactosyltransferase activity was subsequently purified. Anthocyanin acyltransferases have been isolated from cell cultures of Ajuga reptans.287 Of two separable activities displaying rather broad specificities, one catalyzed the transfer of a hydroxycinnamic acid from its CoA ester to 3-O-glycosides and 3,5-di-Oglycosides of cyanidin and delphidin.The second activity transferred the malonyl group from malonyl CoA preferably to anthocyanidin 3-O-coumaroylglucoside-5-O-glucosides and anthocyanidin 3-O-glucosides. Compounds such as the acylated diglucoside 156 of cyanidin are typical of the Ajuga anthocyanins.The main anthocyanins can contain two hydroxycinnamoyl groups. Purple leaves of lettuce (Lactuca sativa) contain cyanidin 3-O-(6+-malonyl)glucoside and enzyme extracts are able to malonylate the 6-hydroxy group of the glucose moiety in cyanidin 3-O-glucoside. The enzyme showed broad specificity and also eYciently malonylated 3-O-glucosides of pelargonidin, delphinidin, peonidin 157, petunidin 158 and malvidin 159.288 Succinyl CoA, but not acetyl CoA, was able to substitute for malonyl CoA at lower eYciency.Green leaves contained about 20% of the enzyme activity found in the purple leaves. Succinyl esters of anthocyanins are found naturally in the flowers of cornflower (Centaurea cyanus). In blue flowers the main derivative is cyanidin 3-succinylglucoside-5-glucoside, whilst in pink flowers the corresponding pelargonidin derivative predominates. An enzyme preparation from blue flowers catalyzed succinyl CoAdependent succinylation of the 3-O-glucosides of cyanidin and pelargonidin, but not of the 3,5-di-O-glucosides.289 Similar results were obtained using extracts from pink flowers, and both enzymes would also malonylate the anthocyanidin 3-Oglucosides equally eVectively.It may be deduced that acylation of the anthocyanidin 3-O-glucosides precedes the 5-Oglucosylation step in forming the cornflower pigments, in keeping with similar observations on the biosynthesis of acetylated anthocyanidin diglucosides in other plants.The plant Scutellaria baicalensis contains a wide range of flavone glucuronides, and a flavone-specific glucuronidase enzyme has now been purified from callus cultures.290 The enzyme is a homotetramer and accepts as substrates luteolin 3-O-glucuronide 160, bacailin 161, wogonin 7-O-glucuronide 162 and oroxylin 7-O-glucuronide 163. O OH O OH HO O O O OH HO HO OH HO HO OH O R 153 R = H 154 R = OH O OH OH HO OH O OH HO OH O HO O OH OH HO O OH OH HO OH OH O OH OH HO OH O OH HO O O HO 147 Cyanidin UDPXyl 155 UDPGal + + + Scheme 44 O OH HO OH O HO HO OH O O O HO O HO HO OH O HO O O O 156 + O OH OH HO R OH OMe 157 Peonidin R = H 158 Petunidin R = OH 159 Malvidin R = OMe + Dewick: The biosynthesis of shikimate metabolites 41The flavanone sakuranetin 164 is synthesized as a phytoalexin on UV irradiation of rice (Oryza sativa).A methyltransferase catalyzing SAM-dependent methylation of naringenin has been detected in extracts of irradiated leaves.291 The flavone apigenin 149 and flavonol kaempferol 141 were also eYciently methylated, but surprisingly, the most eVective substrate was the flavone luteolin 165.An eriodictyol 136 4*-O-methyltransferase has been partially purified from Citrus aurantium.292 A gene encoding flavonol 3*,5*-O-methyltransferase in Chrysosplenium americanum has been isolated.293, 294 The deduced amino acid sequence for the encoded protein showed 67–85% similarity to other plant O-methyltransferase sequences. Recombinant protein was produced which showed strict specificity for positions 3* and 5* of partially methylated flavonols based on quercetin 142 and quercetagetin 166, but not quercetin itself, in keeping with the earlier enzymic studies and the pattern of flavonols found in Chrysosplenium.294 The genetics and biochemistry of anthocyanin biosynthesis have been reviewed.295 6.3 Flavonoid sulfates Sulfation of the flavonol quercetin in Flaveria chloraefolia is catalyzed by two sulfotransferases, giving firstly the 3-sulfate, and then the 3,4*-disulfate.These enzymes show strict substrate and position specificities, yet cDNA sequencing has indicated that the enzymes share 69% identity in their amino acid sequences. In recent studies,296 chimeric flavonol sulfotransferases have been constructed by reciprocal exchange of DNA fragments then expression in E. coli. The chimeric proteins were active, though at a reduced level, and a specific region in the sequence was deduced to be responsible for determining substrate and position specificity.Other regions were shown to be highly conserved in other plant and animal sulfotransferase amino acid sequences. Region IV in the plant 3-sulfotransferase has been demonstrated to be required for the binding of the cofactor PAPS (3*-phosphoadenosine 5*-phosphosulfate) with Arg-276 being a critical residue.297 Region I was not required for PAPS binding, though Lys-59 was critical for catalysis and may help to stabilize an intermediate.A flavonol sulfotransferase-like cDNA clone in Flaveria bidentis has been identified and sequenced.298 6.4 Carthamin Carthamin 169 is a red pigment from the saZower (Carthamus tinctorius) and has a bichalcone-type structure. A likely biosynthetic precursor 168 which is converted into carthamin by oxidative decarboxylation on exposure to air or oxidizing agents has been isolated, and was reported earlier (see ref. 1). The same conclusions are reached by an independent research group who have also isolated the unstable yellow compound 168.299 In addition, this group has reported the isolation of a further possible intermediate in carthamin biosynthesis, namely anhydrosaZor yellow B 167.300 This dark orange compound is transformed into carthamin by allowing a solution in methanol–pyridine (NMR solvent) to stand for about a month. Of particular interest is the additional glucose moiety in 167, which may ultimately supply the extra carbon atom linking the two chalcone fragments in the carthamin structure (Scheme 45).A precarthamin material which can be converted enzymically into carthamin is released from a bound form of precarthamin by treatment of powdered florets with enzymes such as ‚-glucosidase, cellulase or pectinase.301 6.5 Diels–Alder-type adducts Flavonoid-based molecules that appear to be derived by Diels–Alder-type reactions between a chalcone dienophile and a flavonoid with a modified prenyl group as the diene found in plants of the Moraceae are reviewed in a recent article.302 From the high incorporations of label and the labelling O O GlcAO R2 OH R1 O OH HO OH OGlcA 161 Bacailin R1 = H; R2 = OH 162 R1 = H; R2 = OMe 163 R1 = OMe; R2 = H 160 O O O OH MeO OH O O OH HO OH OH O O OH HO OH OH 164 Sakuranetin OH 165 Luteolin HO 166 Quercetagetin O HO Glc HO O O OH O O HO O O HO OH Glc Glc HO OH OH HO CO2H OH O O HO O O HO OH Glc Glc HO OH OH O OH O O Glc HO OH OH H H HO OH H H OH CH2OH OH 167 Anhydrosafflor yellow B 168 169 Carthamin Scheme 45 42 Natural Product Reports, 1998patterns obtained, it has been suggested that both phenylalanine and tyrosine can be utilized in supplying the C6C3 portions of chalcomoracin 170 and kuwanon J 171 in Morus alba cell cultures (see ref. 1). Further evidence has now been presented to indicate that two independent pathways to 4-coumaroyl CoA exist in this system.303 Simultaneous administration of [1-13C]phenylalanine and [3-13C]tyrosine gave chalcomoracin with incorporation of label into all of the appropriate positions.Furthermore, [2-13C]cinnamic acid as its N-acetylcysteamine thioester was also specifically incorporated. Thus, the pathways phenylalanine]cinnamic acid]4- coumaric acid and tyrosine]4-coumaric acid are both shown to be operative. 6.6 Isoflavonoids Isoflavonoids are structurally modified flavonoids in which the shikimate-derived aromatic ring has migrated to the adjacent carbon atom of the heterocycle.Rearrangement of flavanone precursors such as liquiritigenin 130 and naringenin 128 is brought about by the actions of isoflavone synthase, a cytochrome P450-dependent monooxygenase, and then a dehydratase, and yields initially the corresponding isoflavone. These can then be transformed into a range of other iso- flavonoid variants. The pterocarpan medicarpin 177 is derived in alfalfa (Medicago sativa) from reduction of 2*-hydroxyformononetin 174 to the isoflavanone vestitone 175, followed by a further NADPH-dependent reduction to the isoflavanol 176.Subsequent cyclization is achieved via the enzyme 7,2*- dihydroxy-4*-methoxyisoflavanol (DMI) dehydratase (Scheme 46). The penultimate enzyme in this sequence, vestitone reductase, has now been produced by cDNA cloning and expression in E. coli.304 The enzyme had strict substrate specificity for (3R)-vestitone and not (3S)-vestitone as also observed for the enzyme isolated from alfalfa.The pterocarpan maackiain 180 contains a methylenedioxy bridge which is formed early in the biosynthetic sequence at the isoflavone oxidation level. A microsomal preparation from elicited cell cultures of chickpea (Cicer arietinum) has been obtained which transforms the isoflavone calycosin 178 into pseudobaptigenin 179 (Scheme 46).305 This enzyme required O2 and NADPH cofactors and was characterized as a cytochrome P450- dependent system.It would also catalyze methylenedioxy bridge formation in pratensein (5-hydroxycalycosin) giving the corresponding 5-hydroxypseudobaptigenin. A cDNA clone encoding an isoflavone reductase-like protein has been characterized in white lupin (Lupinus albus).306 The pea (Pisum sativum) phytoalexin (+)-pisatin 185 has the opposite absolute configuration to (")-(6aR,11aR)-maackiain 180 from chickpea, and is produced by 6a-hydroxylation then O-methylation of (+)-(6aS,11aS)-maackiain 184 (Scheme 47).However, the reduction product of pea isoflavone reductase is (")-(3R)-sophorol 182, stereochemically equivalent to (")- (6aR,11aR)-maackiain 180. This implicates a change of stereochemistry at a late stage in the pathway to pisatin (see ref. 1). In investigating this, it has been found that isoflavone 181 and (")-sophorol 182 were more eYciently incorporated into pisatin than (+)-sophorol 183 or (+)-maackiain 184.307 This O OH OH HO HO O HO OH OH O OH OH HO HO HO OH O OH OH 170 Chalcomoracin 171 Kuwanon J O O HO OH O O HO OMe O O HO OMe OH O O HO O O O O HO O O H H O O HO OMe HO O O HO OMe HO H O HO HO OMe HO H O O HO H H OMe 172 Daidzein 180 Maackiain 179 Pseudobaptigenin 173 Formononetin 130 Liquiritigenin 7 4¢ 174 178 Calycosin 175 (3 R)-Vestitone 176 v NADPH i iii 177 Medicarpin vii NADPH iv vi O OH O OH HO ii O2, NADPH O2, NADPH O2, NADPH O2, NADPH Scheme 46 Enzymes: i, isoflavone synthase; ii, dehydratase; iii, iso- flavone reductase; iv, vestitone reductase; v, DMI dehydratase; vi, pseudobaptigenin synthase Dewick: The biosynthesis of shikimate metabolites 43suggests that isomerization at the isoflavanone stage, the most obvious timing, is not significant. Detection of the corresponding isoflav-3-ene 186 in extracts leads to a suggestion that this may be a non-chiral intermediate in the conversion of (")- (3R)-sophorol 182 into (+)-pisatin 185.A discrepancy in the detailed understanding of isoflavone biosynthesis relates to the formation of formononetin 173 from daidzein 172 (Scheme 46).The isoflavone O-methyltransferase activity found in yeast elicitor-treated cell suspension cultures of Medicago sativa preferentially methylates the 7-hydroxy group of daidzein in vitro, whereas in vivo, stress-related isoflavonoid biosynthesis requires and involves 4*-Omethylation to formononetin. To investigate this anomaly further, the enzyme was purified by a substrate-based aYnity system, and some internal peptide sequences were determined. 308 Sequence similarities to regions in catechol O-methyltransferase from barley, and 6a-hydroxymaackiain 3-O-methyltransferase from pea were noted, though there was little similarity to other O-methyltransferase enzymes in alfalfa acting on caVeic acid or isoliquiritigenin. The purified iso- flavone O-methyltransferase had substrate specifity towards isoflavones with a free 7-hydroxy group, converting daidzein into isoformononetin (7-O-methyldaidzein) and genistein 187 into prunetin 188, but could also methylate the 5-hydroxy group of genistein giving a compound tentatively identi- fied as 4*-hydroxy-5,7-dimethoxyisoflavone. Isoflavones with a 4*-methoxy group, e.g.formononetin and biochanin A, were not susceptible to 7-O-methylation. By far the best substrate however was 6,7,4*-trihydroxyisoflavone, which has no role in the formation of alfalfa stress-induced isoflavonoids.Despite the in vitro characteristics of this enzyme, it is still believed that its in vivo role is to catalyze 4*-O-methylation of daidzein to produce formononetin. The isoflavonoid phytoalexin pathway has been reviewed with emphasis on enzymes, genes, and transcription factors.309 7 Stilbenes and dihydrophenanthrenes Stilbenes are produced from the same cinnamoyl CoA/malonyl CoA substrates as are chalcones, but the enzyme-bound polyketide is folded diVerently and a decarboxylation occurs during cyclization, leading to resveratrol 190 rather than naringenin-chalcone 127 for example (Scheme 48).Accordingly, it has been found that stilbene synthases and chalcone synthases are closely related homodimeric enzymes with high amino acid sequence identity. Using stilbene synthase from peanut (Arachis hypogaea) and chalcone synthase from white mustard (Sinapis alba), protein cross-linking and site-directed mutagenesis studies have identified a subunit contact site close to the active site Cys-169.310 By producing heterodimers in which one monomer was inactive, it was demonstrated that in both stilbene synthase and chalcone synthase, each subunit acts independently and is able to synthesize the end product.The active sites of the two subunits are close together in the dimer. In the presence of a monomeric reductase (see Section 6.1), chalcone synthase also synthesizes a 6*-deoxychalcone, with the 6*-hydroxychalcone as a second product when using plant preparations.Both products were also synthesized by E. coli expressing the individual proteins, and both were also obtained with a chalcone synthase heterodimer containing a single active site. This indicates that 6*-deoxychalcone synthesis requires no other plant factor apart from the reductase, O O HO O O HO O O HO O O HO H O O HO O O HO H O O HO O O H H O O HO O O H H O O MeO O O H OH 182 (–)-(3 R)-Sophorol 181 183 (+)-(3 S)-Sophorol 180 (–)-(6a R,11a R)-Maackiain 6a 11a 184 (+)-(6a S,11a S)-Maackiain 185 (+)-(6a R,11a R)-Pisatin i ? NADPH ? Scheme 47 Enzyme: i, isoflavone reductase O HO O O HO O O R OH OH 186 187 Genistein, R = OH 188 Prunetin, R = OMe 7 4¢ 5 R CoAS O R O O COSCoA O R O O SCoA O O R O OH HO OH R HO OH 126 R = OH 189 R = H º 127 Naringenin-chalcone, R = OH 190 Resveratrol, R = OH 191 Pinosylvin, R = H 3 x Malonyl-CoA CO2 i ii i, ii Scheme 48 Enzymes: i, stilbene synthase; ii, chalcone synthase 44 Natural Product Reports, 1998and that formation of the two products is probably an intrinsic property of the interaction between dimeric chalcone synthase and the monomeric reductase.cDNAs representing two closely related stilbene synthase genes have been isolated from Pinus strobus using a probe derived from P. sylvestris.311 The encoded proteins showed only five amino acid diVerences. The proteins expressed in E. coli both preferred cinnamoyl CoA 189 to dihydrocinnamoyl- CoA, though P.strobus accumulates pinosylvin 191 and dihydropinosylvin 192. Otherwise, the proteins demonstrated quite significant variations in properties. In particular, one enzyme (STS1) had only 3–5% of the activity shown by the other (STS2), and with cinnamoyl CoA yielded a second, unknown product. Site-directed mutagenesis showed that a single arginine to histidine exchange in STS1 was responsible for all the observed diVerences. Bibenzyls are intermediates on the way to 9,10- dihydrophenanthrenes, phytoalexins of several orchid species.Bibenzyl synthase is an enzyme closely related to stilbene synthase, though it utilizes CoA esters of a dihydrocinnamic acid as substrate rather than the ester of a cinnamic acid (Scheme 49). By exploiting the homology between bibenzyl synthase and stilbene synthases, two full-length cDNA clones were isolated from elicitor-induced plants of Phalaenopsis species, and subsequently expressed in E.coli.312 Both proteins were catalytically active, with virtually the same selectivity towards dihydro-3-coumaroyl CoA 193 as had the enzyme isolated from plant tissues. Thus, this substrate was preferred over phenylpropionyl CoA, and cinnamoyl CoA and 4-coumaroyl CoA were rather poor substrates. A cDNA clone for (S)-adenosylhomocysteine hydrolase was also isolated in these studies; this enzyme is involved in the supply of SAM utilized in the methylation of the bibenzyl 194 to give batatasin III 195 (Scheme 49).Oxidative coupling then leads to the dihydrophenanthrene hircinol 196. 8 Xanthones The xanthone skeleton is derived from a benzoyl CoA and three malonyl CoA units via a benzophenone. A benzophenone synthase activity has been demonstrated in cell-free extracts obtained from cultures of Centaurium erythraea that accumulate 1,3,5-trihydroxyxanthone 199.313 The extracts catalyzed the synthesis of 2*,3,4,6-benzophenone 198 from 3-hydroxybenzoyl CoA 197 and malonyl CoA (Scheme 50). 9 Quinones 9.1 Naphthoquinones A range of naphthoquinone derivatives, including phylloquinone (vitamin K1) and menaquinone (vitamin K2), are produced from chorismate via isochorismate 49, 2-succinyl- 6-hydroxycyclohexa-2,4-diene-1-carboxylate (SHCHC) 200, o-succinylbenzoate (OSB) 201, and 1,4-dihydroxynaphthoate 203 (Scheme 51). The enzyme o-succinylbenzoyl CoA synthetase which activates OSB by conversion into its ‘aliphatic’ CoA ester 202 in E.coli has been overexpressed and puri- fied.314 The enzyme is a homotetramer, requires ATP and coenzyme A, and its activity is stimulated by Mg2+ ions. Neither benzoyl CoA nor benzoylpropionic acid (succinylbenzene) acts as a substrate for the purified enzyme stressing the requirement for both the benzoyl carboxy group and the succinyl side-chain. 9.2 Ubiquinones Ubiquinones (coenzyme Q) 209 are also derived from chorismate, but via 4-hydroxybenzoate 79, and an isoprenoid sidechain of length varying according to the organism is attached to this substrate early in the biosynthetic sequence.The generally accepted pathway is shown in Scheme 52 though not all intermediates are fully proven. 3-Hexaprenyl-4- hydroxybenzoic acid 204 (n=6) has been isolated as the predominant labelled lipid material from cultures of yeast (Saccharomyces cerevisiae) supplied with 14C-labelled 4-hydroxybenzoic acid and grown under log-phase conditions. 315 In stationary phase cultures, labelled 204 was also produced, but the major labelled product was coenzyme Q 209 (n=6).A yeast mutant (coq7-1) deficient in ubiquinone synthesis was found to synthesize the intermediate demethoxyubiquinone 207 (n=6).316 The corresponding wild-type coq7 gene was isolated and sequenced, and shown to restore growth and ubiquinone biosynthesis to the mutant. Several coq7 HO OH 192 Dihydropinosylvin CoAS O HO HO HO OH HO MeO OH HO MeO OH 3 x Malonyl-CoA 193 i SAM Homocysteine 194 195 Batatasin III S-Adenosylhomocysteine 196 Hircinol ii Scheme 49 Enzymes: i, bibenzyl synthase; ii, (S)-adenosylhomocysteine hydrolase OH CoAS O OH HO OH O OH O HO OH O OH 3 x Malonyl-CoA 199 197 198 Scheme 50 Dewick: The biosynthesis of shikimate metabolites 45deletion mutants then generated were shown to accumulate 3-hexaprenyl-4-hydroxybenzoic acid 204 rather than 207.The coq7 gene may thus encode a protein involved in one or more monooxygenase/hydroxylase steps in the biosynthetic sequence.In eukaryotes, the first O-methylation step is carried out by the Coq3 protein on 3,4-dihydroxy-5-polyprenylbenzoate 205, whilst in E. coli, the predicted substrate is 2-octaprenyl-6-hydroxyphenol 206 (n=8), with the enzyme yet to be identified. The second O-methylation, conversion of demethylubiquinone 208 (n=8) into ubiquinone, involves UbiG, an enzyme which is 40% identical in amino acid sequence to yeast Coq3. It has now been demonstrated that UbiG probably catalyzes both O-methylation steps in E.coli.317 The ubiG gene was able to restore respiration and ubiquinone synthesis in yeast coq3 mutants, provided a mitochondrial leader sequence was also inserted to target the activity to the mitochondria. In vitro assays showed the UbiG enzyme was able to methylate synthetic analogues such as the ‘eukaryotic’ 3,4-dihydroxy-5-farnesylbenzoate 205 (n=3) and the ‘prokaryotic’ 2-farnesyl-6-hydroxyphenol 206 (n=3).Methylation of 3,4-dihydroxy-5-farnesylbenzoate 205 (n=3) was also accomplished using mitochondria from yeast containing the coq3 gene, showing that the chain length is not critical for substrate recognition.318 The ubiquinone biosynthetic pathway in the protozoon Leishmania major probably shows similar characteristics to those in mammalian and bacterial systems. Thus in preliminary studies,319 4-hydroxybenzoic acid, acetate and mevalonate were established as precursors of ubiquinone-9 209 (n=9). 9.3 Shikonin Shikonin 211 is an example of another group of naphthoquinones, which have their origins in 4-hydroxybenzoate rather than OSB. The remainder of the skeleton of shikonin is derived from geranyl diphosphate (Scheme 53). Deoxyshikonin 210 has recently been established as a late intermediate in the pathway.320 Labelled phenylalanine was rapidly incorporated into deoxyshikonin in cell cultures of Lithospermum erythrorhizon and then into fatty acid esters of shikonin, e.g.acetylshikonin 212 and ‚-hydroxyisovalerylshikonin 213. Deoxyshikonin was also incorporated into the esters of shikonin, though shikonin itself was only poorly utilized. Similar results were obtained using a cell-free system from L. erythrorhizon. A sequence of hydroxylation and esterification via shikonin is postulated, the poor utilization of shikonin being accounted for if a membrane-bound multienzyme system is operative. The biosynthesis of acetylshikonin, 4-hydroxybenzoic acid O-glucoside 80 and rosmarinic acid in L.erythrorhizon cultures is completely suppressed if 2-amino-2-phosphonic acid, an inhibitor of PAL, is added.321 Use of mevinolin, an inhibitor of HMGCoA reductase, specifically blocked acetylshikonin biosynthesis, thereby increasing the formation of 4- hydroxybenzoic acid O-glucoside (see also Section 4.2). Shikonin biosynthesis has been reviewed.322 CO2H OH O HO2C CO2H OH O CO2H O CO2H CO2H O COSCoA CO2H OH OH CO2H 202 (49) Isochorismate Chorismate 200 SHCHC 201 OSB 203 Vitamin K Anthraquinones 2-Oxoglutarate TPP HSCoA ATP i ii, iii iv v vi Scheme 51 Enzymes: i, isochorismate synthase (entC); ii, 2- oxoglutarate decarboxylase; iii, SHCHC synthase [ii and iii/menD]; iv, OSB synthase (menC); v, OSB:coenzyme A ligase (menE); vi, naphthoate synthase (menB) CO2H OH CO2H OH R CO2H OH R HO CO2H OH R MeO OH R OH R HO OH R MeO O R MeO O O R MeO O Me O R MeO O Me HO O MeO O Me MeO H H OPP Chorismate i 79 204 205 206 207 208 n 209 Ubiquinone n ii Eukaryotes Prokaryotes iii iv v vi vii vi Saccharomyces cerevisiae: n = 6 E.coli: n = 8 Man: n = 10 viii ix Scheme 52 Enzymes: i, chorismate lyase (ubiC); ii, 4-hydroxybenzoate polyprenyltransferase (UbiA); iii, Coq3; iv, UbiD; v, UbiB; vi, UbiG; vii, UbiH; viii, UbiE; ix, UbiF/Coq7 46 Natural Product Reports, 19989.4 Tocopherols Plant chloroplasts accumulate a range of polyisoprenoid quinones, quinols and chromanols, e.g.plastoquinones and tocopherols, that have their origins in homogentisic acid (see Section 4.4) and mevalonic acid. These products contain methyl substituents on the aromatic ring, one of which is produced by the decarboxylation of the homogentisate sidechain, the rest originating by C-methylation from SAM. An enzyme responsible for the cyclization step giving a chroman ring in the biosynthesis of ·-tocopherol 214 in the cyanobacterium Anabaena variabilis was recently reported and its reaction mechanism has been probed (Scheme 54) (see ref. 1). In further studies on this system,323 the use of a range of synthetic substrate analogues has established that three major recognition sites are necessary for the enzyme to exert its action. These are, the hydroxy group at C-1 of the hydroquinone, the E configuration of the double bond in the side-chain, and the length of the lipophilic side-chain (3 or 4 isoprene units). 10 Cyanogenic glycosides, glucosinolates and related compounds The biosynthetic pathways to cyanogenic glycosides and glucosinolates appear to share many features, though there are considerably more experimental data available for the former group.The basic pathway to cyanogenic glycosides as exemplified by dhurrin 221 is shown in Scheme 55. Tyrosine N-hydroxylase, an enzyme catalyzing the transformation of tyrosine into 4-hydroxyphenylacetaldoxime 218 and 219 in the biosynthesis of dhurrin in Sorghum bicolor, is shown to be a multifunctional cytochrome P450-dependent enzyme of a type not previously demonstrated in plant biosynthetic pathways.324 It is proposed that a double N-hydroxylation is achieved giving N-hydroxytyrosine 215 then N,Ndihydroxytyrosine 216, and that this is converted into the oxime via dehydration to the nitroso compound 217 and subsequent decarboxylation.The dehydration and decarboxylation appear to be non-enzymic. The E to Z ratio in the oxime product was 69:31.The multifunctional nature of the enzyme was demonstrated in that binding of either L-tyrosine or N-hydroxy-L-tyrosine mutually excludes binding of the other substrate. A cDNA clone encoding the enzyme has been obtained from S. bicolor, and sequence data presented.325 The sequence showed highest identity with that of the flavonoid 3*,5*-hydroxylase from Petunia hybrida (see Section 6.2) and a P450 protein of unknown function from avocado. Recombinant protein has been expressed in E.coli and purified.326 The enzyme showed high specificity in that it would also bind phenylalanine, but not transform this substrate. The hydrolysis of cyanogenic glycosides is brought about by the action of ‚-glucosidases liberating cyanohydrins, which are then decomposed into aldehyde and HCN by hydroxynitrile lyases (Scheme 56). cDNAs encoding for the ‚-glucosidases hydrolyzing dhurrin 221 in Sorghum bicolor (dhurrinase)327 and amygdalin 222 in Prunus serotina (amygdalin hydrolase) 328 have been cloned and sequenced.(R)-Mandelonitrile lyase from the fern Phlebodium aureum has been purified to homogeneity and shown to be a homomultimer existing in at least three isoforms.329 In contrast to other hydroxynitrile lyases, this enzyme is not inhibited by sulfhydryl- or hydroxymodifying agents and may therefore employ a diVerent catalytic mechanism. It is not a flavoprotein and therefore diVers from the (R)-mandelonitrile 223 lyases of the Rosaceae which CO2H OH CO2H OGlc CO2H OH OH OH O O OH OH O O OH OH OH 79 O O OH OH 210 Deoxyshikonin 80 212 R = Me 213 R = Me2C(OH)CH2 OCOR 211 Shikonin GeranylPP Scheme 53 HO OH O HO D i 214 a-Tocopherol D+ 1 Scheme 54 Enzyme: tocopherol cyclase CO2H NH2 CO2H HN OH CO2H N OH HO CO2H N O CO2H N+ O –O N OH N HO CN HO H CN GlcO H HO HO HO HO HO HO HO HO CN HO HO S 221 Dhurrin 219 ( Z)-Oxime O2 218 ( E)-Oxime 215 NADPH 31 L-Tyrosine 217 220 4-Hydroxymandelonitrile 216 O2 NADPH NADPH O2 Scheme 55 Dewick: The biosynthesis of shikimate metabolites 47are FAD-dependent. X-Ray crystallographic studies on (S)-4- hydroxymandelonitrile 220 lyase from Sorghum bicolor have been reported.330 Recent reviews discuss the biosynthesis of cyanogenic glycosides with emphasis on the P450 enzymes involved,331 and the functions and properties of hydroxynitrile lyases.332 The biosynthetic pathway to glucosinolates 226 is outlined in Scheme 57.Glucosinolate synthesis can be induced in seedlings of Sinapis alba by treatment with jasmonic acid, leading to an accumulation of 4-hydroxybenzylglucosinolate 227.333 An NADPH-dependent microsomal system from S.alba seedlings converting L-tyrosine into 4-hydroxyphenylacetaldoxime (224, compare 218, 219) has been characterized, demonstrating similarities with cyanogenic glycoside biosynthesis in the early part of the pathway. A similar enzyme activity producing phenylacetaldoxime from phenylalanine is present in jasmonic acid-treated seedlings of nasturtium (Tropaeolum majus).334 Both these systems diVer from enzymes in Brassica species which are not P450-dependent and appear to be flavin monooxygenases or peroxidase-type enzymes.Two distinct P450 independent monooxygenases in Brassica napus leaves had been shown to convert homophenylalanine 228 or dihomomethionine 229 into the corresponding aldoximes by NADPH- and O2-dependent oxidative decarboxylation. In further studies on these systems,335 the latter enzyme was shown to have no activity towards methionine, homomethionine, phenylalanine, tyrosine or tryptophan.Immature seeds of rape (Brassica napus) have been shown to contain enzymes capable of converting 35S-labelled desulfoglucosinolates 225, e.g. desulfoindol-3-ylmethylglucosinolate 230, into the corresponding glucosinolates.336 Glucosinolates undergo hydrolysis by the action of thioglucoside glucohydrolases (myrosinases), giving (initially) a thiohydroximate O-sulfonate 231 which is normally followed by a Lossen-type rearrangement to give isothiocyanates 232 (Scheme 58).Other types of product, e.g. nitriles and thiocyanates, may be formed, depending on substrate, pH, and other factors. Myrosinase from Sinapis alba is normally involved with the hydrolysis of sinigrin 233, but actually has better aYnity for glucotropaeolin 234, and the aromatic substrate often features in mechanistic studies of the enzyme.The stereochemistry of hydrolysis has been examined by NMR spectrometry, and shown to involve retention of configuration at the anomeric centre, suggesting a mechanism similar to related O-glucosidase hydrolysis [Scheme 59(a)].337 However, the ready inactivation of myrosinase with 2-deoxy-2- fluoroglucotropaeolin via accumulation of a long-life glucosylenzyme intermediate indicates that there is probably only one catalytic residue, a nucleophilic glutamate, whilst the acidic catalyst residue found in the corresponding O-glucosidase is missing [Scheme 59(b)].Using a variety of synthetic deoxyglucotropaeolins, the role of the individual hydroxy groups during catalysis has been probed.338 Loss of a hydroxy group from any position produced a strong decline in reaction rate, with the 2-hydroxy being especially important. It is suggested that the 2-hydroxy forms hydrogen bonds with the essential carboxylate (Glu or Asp) involved in the hydrolytic reaction. Myrosinases appear to be encoded by gene families according to species.Gene families MA and MB in Brassica napus contain about four and ten genes, respectively, and a third family MC containing three or four genes has now also been isolated.339 Three myrosinase genes in Arabidopsis thaliana show little similarity to the MA and MB gene families in the Brassicaceae and cannot be grouped with either family.340 Two isozymes of myrosinase from Raphanus sativus have been isolated and purified.341 The biosynthesis of glucosinolates in Brassicas,342 and the organization and biochemistry of the myrosinase– glucosinolate system,343 are the subjects of recent reviews.CN GlcGlcO H CN GlcO H CN HO H CHO 223 Mandelonitrile R + HCN 222 Amygdalin Prunasin i iii ii R Scheme 56 Enzymes: i, amygdalin hydrolase; ii, prunasin hydrolase; iii, mandelonitrile lyase R CO2 H NH2 R CO2 H HN OH R N OH R N OH SH R N OH SGlc R N OSO3 – SGlc O2 Thiohydroximic acid 224 225 Desulfoglucosinolate UDPGlc 226 Glucosinolate –SR¢ PAPS NADPH Scheme 57 CO2H NH2 MeS CO2H NH2 N OH SGlc NH N OSO3 – SGlc HO 228 Homophenylalanine 229 Dihomomethionine 227 4-Hydroxybenzylglucosinolate 230 R N OSO3 – SGlc R N OSO3 – SH R NCS 226 Glucosinolate 231 232 i Scheme 58 Enzyme: myrosinase N OSO3 – SGlc N OSO3 – SGlc 233 Sinigrin 234 Glucotropaeolin 48 Natural Product Reports, 199813C-Isotopic enrichments at various atomic positions in aromatic glucosinolates and cyanogenic glycosides can be correlated with the known biosynthetic origins of the individual carbons.344 These values may be used to distinguish between natural and synthetic materials.The cruciferous phytoalexins such as brassinin 235 and cyclobrassinin 236 found in pathogen-inoculated Japanese radish (Raphanus sativus var. hortensis) roots, are synthesized by a pathway closely related to glucosinolate biosynthesis (Scheme 60). Feeding experiments have demonstrated the conversion of brassinin 235 into cyclobrassinin 236, but not into methoxybrassinin 237, and 237 was not incorporated into 236.345 Camalexin 238 is the major phytoalexin of Arabidopsis thaliana, but previous studies (see ref. 1) have suggested tryptophan does not appear to be a precursor. Accumulation of camalexin is accompanied by induction of mRNAs and proteins for all the tryptophan pathway enzymes tested for, suggesting this pathway and phytoalexin synthesis are coordinately regulated.However, camalexin biosynthesis probably branches away from the main tryptophan pathway, e.g. at indole-3-glycerolphosphate.346 11 Miscellaneous shikimate metabolites 11.1 Sphagnum acid Results of feeding experiments with labelled L-phenylalanine, cinnamic acid and 4-coumaric acid have demonstrated their incorporation into trans-sphagnum acid 240 in cultures of the peat moss Sphagnum fallax.347 It is suggested that sphagnum acid thus arises from 4-coumaric acid 239 by further incorporation of a C2 fragment from acetate (Scheme 61).Other typical phenolics found in peat mosses, e.g. the hydroxybutenolide 241, 4-hydroxyacetophenone 242 and 4-hydroxybenzoic acid 79, may also be formed via sphagnum acid (Scheme 61). Light-grown cultures of S. fallax incorporate 4-coumaric acid into a glucosyl conjugate 244 of cis-4-coumaric acid 243 as well as into sphagnum acid.348 In the dark, this material is not formed, indicating light-dependent trans–cis isomerization of 4-coumaric acid.A 4-coumaric acid O-glucosyltransferase with strict specificity towards the cis isomer was subsequently isolated from a cytosolic fraction. O O R HO HO OH OH HO O –O O O HO HO OH OH –O O O O H O H O OH HO HO OH OH HO O –O O O S R HO HO OH OH –O O O HO HO OH OH O O H O H O OH HO HO OH OH –O O (a) b-Glucosidase ( b) Myrosinase Scheme 59 N OSO3 – SGlc NH NCS NH NH NH SMe S NH S N SMe 235 Brassinin 236 Cyclobrassinin L-Met Scheme 60 NH N SMe S OMe NH 237 Methoxybrassinin 238 Camalexin S N CO2H HO CO2H HO O HO O HO O CO2H HO HO CO2H GlcO CO2H CO2H HO Acetate 240 Sphagnum acid 242 79 241 243 UDPGlc 244 hn 239 Scheme 61 Dewick: The biosynthesis of shikimate metabolites 4911.2 4-(4-Hydroxyphenyl)butan-2-one The aroma of ripe raspberry (Rubus idaeus) fruits is due to raspberry ketone, 4-(4-hydroxyphenyl)butan-2-one 246.This compound is known to arise from condensation of 4-coumaroyl CoA 126 with malonyl CoA, giving the benzalacetone 4-(4-hydroxyphenyl)but-3-en-2-one 245, which is then reduced to 246 (Scheme 62).The condensation resembles the chalcone synthase and stilbene synthase reactions, but stops after the addition of the first malonyl unit, and the product is decarboxylated before release from the enzyme. A benzalacetone synthase enzyme preparation converting 4-coumaroyl CoA and malonyl CoA into 245 has now been obtained from ripe raspberry fruits.349 During purification, the enzyme preparation became preferentially enriched in benzalacetone synthase activity over chalcone synthase activity, but whether this was due to contamination with chalcone synthase, or whether benzalacetone synthase may possess a level of dual activity has not been clarified. 11.3 Phenylacetylenes from Asparagus Cultured cells of Asparagus oYcinalis produce 4-[5-(4- methoxyphenoxy)pent-3-en-1-ynyl]phenol 248. Preliminary feeding experiments using [1-13C]- and [U-13C]-glucose precursors gave labelling patterns in 248 which indicated that both aromatic rings were derived from shikimate.350 Labelling in the C5 chain suggested it might have its origins in a C6C2 intermediate plus a C3 glycolysis metabolite; [1,2-13C2]acetate was not incorporated.In further studies with 2H- and 13Clabelled phenylanine substrates,351 it was shown that all 17 skeletal carbons were derived from phenylalanine. One phenylalanine molecule provided the phenylacetylenic C6C2 unit, and the second the phenoxypropenyl unit.It was also shown that the sequence of labels in the three carbons C-9 to C-11 was reversed when compared to the side-chain of its phenylalanine precursor. To accommodate these findings, an intermediate 247 of the spirotetrahydrofuran type has been proposed (Scheme 63). 11.4 Diarylheptanoids and phenylphenalenones 9-Phenyl-1H-phenalenones are a group of plant pigments for which there is limited evidence that the ring system is derived from phenylalanine/tyrosine and that two C6C3 units may be incorporated via a diarylheptanoid.The simplest member of the series is anigorufone 250, which occurs in the roots of the Australian plant kangaroo paws (Anigozanthos preissii). To investigate its biosynthesis, [2-13C]cinnamic acid was fed to root cultures of the plant and shown to yield anigorufone labelled at C-5 and C-8 (Scheme 64).352 Further, 13C-labelled heptadienone 249 was well incorporated. Cyclization of 249 to the phenylphenalenone skeleton resembles an intramolecular [4+2] cycloaddition of the Diels–Alder type.Indeed, periodate oxidation of 249 is known to generate a 1,2-quinone which spontaneously cyclizes to lachnanthocarpone 252, and this, by reduction, may be the precursor of anigorufone. In further studies,353 the origin of all the carbon atoms in hydroxyanigorufone 251 was established using 13C-labelled precursors. In particular, the high incorporation of C-2 of acetate proved that the central carbon (C-6a) was derived from this precursor.The chemistry of natural diarylheptanoids, including their biosynthesis, has been reviewed.354 11.5 Antibiotic LL-C10037· Antibiotic LL-C10037· 255 is known to be formed in a species of Streptomyces from 3-hydroxyanthranilic acid 54 (see Section 2.12), and a pathway via dihydroxyacetanilide 253 (Scheme 65) has been established in earlier work. A COSCoA HO HO O HO O 126 245 246 i ii NADPH Malonyl-CoA CO2, HSCoA Scheme 62 Enzymes: i, benzalactone synthase; ii, benzalactone reductase HO O OMe HO2C NH2 O O O OH Phe C6C2 Phe 248 247 Ñ · * Ñ Ñ * * · · 7 8 9 10 11 Scheme 63 CO2H OH HO O O OH O O O Acetate O OH 252 Lachnanthocarpone 249 OH 250 Anigorufone R = H 251 Hydroxyanigorufone R = OH • = 13C 5 6a 8 O R Scheme 64 50 Natural Product Reports, 1998dihydroxyacetanilide epoxidase from Streptomyces oxidizes 253 to the epoxy quinone 254 utilizing molecular oxygen but without a requirement for any other cofactor.An NADPHdependent oxidoreductase then produces LL-C10037·. It has been shown that a full equivalent of 18O from 18O2 is incorporated at the epoxide.355 However, control reactions revealed a rapid exchange of oxygen with H2 18O at the C-4 carbonyl. By coupling the dihydroxyacetanilide epoxidase reaction with the epoxyquinone oxidoreductase from the same organism, LL-C10037· was produced with about 20% incorporation of a second 18O atom at the C-4 alcohol.This suggests that dihydroxyacetanilide epoxidase is a dioxygenase with an epoxidation mechanism (Scheme 66) which is essentially the same as that observed for dihydrovitamin K epoxidation during the mammalian vitamin K-dependent glutamate carboxylase reaction (see ref. 2). 11.6 Brominated tyrosine metabolites from sponges Feeding experiments have demonstrated low but significant incorporations of label from L-tyrosine, L-3-bromotyrosine 256 and L-3,5-dibromotyrosine 257 into dibromoverongiaquinol 261 and aeroplysinin-I 259 in the sponge Aplysina fistularis.356 [methyl-14C]Methionine was specifically incorporated into the O-methyl group of aeroplysinin-I, but no incorporations from the O-methyl ethers of tyrosine or 3,5-dibromotyrosine were recorded.A tentative pathway to 261 via successive bromination of tyrosine, and oxime 258 and phenylacetonitrile 260 intermediates has been proposed (Scheme 67). Unfortunately, mono- and di-brominated (4-hydroxyphenyl)acetonitriles, although present in A.fistularis, were not incorporated in feeding experiments, though this may be a consequence of the experimental diYculties encountered in using sponges for biosynthetic studies. The O-methylation required for production of 259 is thought to occur before formation of the nitrile, but potential brominated (methoxyphenyl)acetonitrile intermediates were not transformed. 11.7 Cyclohexanecarboxylic acid Cyclohexanecarboxylic acid is a component unit in several secondary metabolites, and it is derived from shikimic acid by a surprisingly complicated series of reactions, a combination of dehydrations and double bond reductions arranged so that the ring system is never prone to aromatization (Scheme 68) (see ref. 2). The gene encoding cyclohex-1-enylcarbonyl CoA reductase, the reductase catalyzing the final stage (264]265) in the formation of the cyclohexyl moiety of ansatrienein A 266 in Streptomyces collinus, has been cloned and characterized.357 The deduced amino acid sequence for the protein showed high similarity with members of short-chain alcohol dehydrogenase enzymes.The gene was overexpressed in E. coli to give a protein with comparable properties to the enzyme from S. collinus. However, it was able to catalyze in vitro three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. In addition to the reduction of 264 into 265, this enzyme would also reduce 262 and 263 (Scheme 68).A mutant organism in which this gene had been disrupted lost this enzyme activity and the ability to synthesize cyclohexanecarboxylic acid and ansatrienin A. Since the three reductive steps all involve reduction of an ·,‚-conjugated double bond with the same stereochemical characteristics (shown in previous experiments to be anti, see ref. 2), it is possible that the single enzyme is responsible for catalyzing all three steps in the pathway. OH NH2 CO2H OH NH2 OH NH2 OH OH NHAc OH O NHAc O O O NHAc OH O – CO2 O2 NADPH 255 LL-C10037a 54 253 4 254 Scheme 65 OH NHAc OH O O Enz O OH O NHAc –O O– OH O NHAc O O NHAc O OH HO O NHAc O O O NHAc O O – H2O – H2O Scheme 66 CO2H NH2 HO Br CO2H NH2 HO Br Br CO2H NOH HO Br Br CN HO Br Br HO Br Br O NH2 O Br Br O NH2 OH CN MeO Br Br OH OH 259 Aeroplysinin-I 257 260 261 Dibromoverongiaquinol 256 L-Tyr 258 Scheme 67 Dewick: The biosynthesis of shikimate metabolites 51The potent immunosuppressants ascomycin (FK520) 270, FK506 269 and rapamycin 272 are macrocyclic polyketides that all contain a unit probably derived from 3,4-dihydroxycyclohexanecarboxylic acid.The biosynthetic pathway to this acid, while sharing features with that to cyclohexanecarboxylic acid, is known to diverge at the first intermediate beyond shikimate, namely the dihydroxy diene 262. The production of 4-hydroxy-3-methoxycyclohexanecarboxylic acid involves 3-O-methylation after assembly of the polyketide.The gene encoding 31-O-demethyl-FK506 methyltransferase has been isolated from two strains of Streptomyces species (FK506 producers) and S. hygroscopicus subsp. ascomyceticus (FK520 producer), sequenced and expressed in S. lividus.358 Disruption of the gene in one of the Streptomyces species yielded a mutant organism that produced 31-O-demethyl-FK506 267. The protein was established to methylate both 31-O-demethyl- FK506 267 and 31-O-demethyl-FK520 268.A second gene from these organisms was found to encode a protein with a strong sequence similarity to P450 hydroxylases. Disruption of this gene resulted in formation of 9-deoxo-31-O-demethyl- FK506 271, confirming that the gene indeed encoded a cytochrome P450 hydroxylase which was responsible for the 9-hydroxylation in FK506 and FK520. Clustered polyketide synthase genes in S. hygroscopicus responsible for the biosynthesis of rapamycin 272 have been characterized.359 A total of 70 constituent active sites makes this the most complex multienzyme system identified so far.The biosynthesis of the cyclohexanecarboxylic acidcontaining antibiotic asukamycin and other miscellaneous shikimate-derived antibiotics, e.g. reductiomycin, is discussed in a recent review.360 11.8 3-Amino-5-hydroxybenzoic acid and mC7N units The meta-C7N aminobenzoate units of several ansamycin antibiotics are provided by 3-amino-5-hydroxybenzoic acid 276 which is derived from a modification of the shikimate pathway.A DAHP synthase-like enzyme synthesizes amino- DAHP 273 via the introduction of nitrogen from glutamine, then the amino group is retained in what appears to be a normal pathway (Scheme 69) (see ref. 1). Cell-free extracts of the rifamycin B 277 producer Amycolatopsis mediterranei have been shown to transform the various intermediates in the pathway into 3-amino-5-hydroxybenzoic acid.361 Thus, amino- DAHP 273, aminoDHQ 274 and aminoDHS 275 were eY- ciently modified, but DAHP was not.The low, but significant, conversions of PEP and erythrose 4-phosphate into amino- DAHP and into 3-amino-5-hydroxybenzoic acid were also COSCoA HO OH OH COSCoA OH OH COSCoA OH OH COSCoA OH COSCoA OH COSCoA OH COSCoA COSCoA COSCoA O O NH OMe HO O O O NH O Shikimoyl-CoA 266 Ansatrienin A 265 262 Although all intermediates are shown as coenzyme A thioesters, the exact point of esterification is not known 264 263 Scheme 68 HO R1O O HO O R2 OMe O OMe OH O O N O HO HO O HO O OMe O OMe OH O N O HO MeO O O OMe OH O N 267 R1 = H; R2 = CH2CH=CH2 268 R1 = H; R2 = Et 269 FK506, R1 = Me; R2 = CH2CH=CH2 270 FK520, R1 = Me; R2 = Et O O O OMe OH O MeO 271 9 31 272 Rapamycin 52 Natural Product Reports, 1998noted.Cell-free extracts of the ansatrienin A 266 producer Streptomyces collinus would also occasionally catalyze transformations, but much less eYciently than A. mediterranei. Although a modified DAHP synthase is required for amino- DAHP synthesis, the next two reactions in the pathway could be normal pathway enzymes.Thus, 3-dehydroquinate synthase and shikimate dehydrogenase from E. coli can both catalyze transformation of the amino analogues, but 3-dehydroquinase (a type II enzyme, see Section 2.3) from A. mediterranei does not accept aminoDHQ. 11.9 3-Amino-4-hydroxybenzoic acid 3-Amino-4-hydroxybenzoic acid, a biosynthetic precursor of 4-hydroxy-3-nitrosobenzamide in Streptomyces murayamaensis is unusual in being the only aminohydroxybenzoic acid derivative so far reported which is not derived via shikimate.It has been established to arise from a C4 unit from the tricarboxylic acid cycle and a C3 unit, possibly PEP.362 11.10 Betalains Betalains are yellow to violet water-soluble nitrogenous pigments restricted to plants of the order Centrospermae, but found also in the caps of some larger fungi such as Amanita and Hygrocybe.Some structures, e.g. the betacyanin betanin 280 are derived from two molecules of DOPA 34 via cycloDOPA 36 (see Scheme 11) and the ring-cleaved betalamic acid 22, whilst others contain only the betalamic acid portion that is DOPA-derived. A tyrosinase enzyme (see Section 2.11) isolated from the betalain-producing coloured parts of the fly agaric (Amanita muscaria) is probably involved in betalain biosynthesis.363 This enzyme converted tyrosine into DOPA, but was not specific for tyrosine.In addition, it would oxidize a number of diphenols to o-quinones (diphenolase activity) and may therefore contribute towards melanin biosynthesis. Unusually for tyrosinases, the enzyme was a heterodimer. Betanidin O-glucosyltransferase activities have been isolated from cell suspension cultures of Dorotheanthus bellidiformis and shown to catalyze 5-O-glucosylation giving betanin 280 and 6-O-glucosylation giving gomphrenin I 281 (Scheme 70).364 Three isoforms of the 5-O-glucosyltransferase were identified.Kinetic data for the enzymes suggest that at low substrate concentrations, betanin formation would be favoured, whilst at high substrate concentration, gomphrenin I would be the preferred product. 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ISSN:0265-0568    

DOI:10.1039/a815017y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Application of electrospray mass spectrometry in biology Andrew R. Pitt Department of Pure and Applied Chemistry, Strathclyde University, Thomas Graham Building, 295 Cathedral Street, Glasgow, UK G1 1XL Covering: January 1984 to December 1996 1 Introduction 2 Electrospray mass spectrometry 2.1 Ion generation and mass analysis 2.2 Secondary ion generation 2.3 Protein and peptide analysis 2.4 Interfacing to separation techniques 2.4.1 High performance liquid chromatography 2.4.2 Capillary electrophoresis 3 Oligosaccharide analysis 4 Proteins 4.1 Covalent modification 4.1.1 Non-specific covalent adduct formation 4.1.2 Chemical modification 4.1.2.1 Post-translational modification 4.1.2.2 Active site directed irreversible inhibitors 4.2 Covalent enzyme–ligand complexes 4.3 Non-covalent enzyme–inhibitor complexes 4.4 Protein folding 5 DNA 6 Summary 7 References 1 Introduction Since electrospray ionisation (ESI) and matrix assisted laser desorption/ionisation–time of flight (MALDI–TOF) mass spectrometry (MS) became readily available at a reasonable cost in the late 1980s,1,2 mass spectrometry has become an ever more important tool for the biological scientist, both for polar small molecules that are diYcult to analyse using the traditional ionisation techniques and for the analysis of biological macromolecules. ESI and MALDI are both ‘soft’ ionisation techniques capable of producing ions of low energy, and hence have the power to generate ions from biological macromolecules of molecular masses in excess of 100 000, and in many cases these masses can be measured with accuracies better than 0.01%.Mass spectrometry relies on two key processes, the generation of gas phase ions of the compound of interest, a process that occurs within the ‘source’ area of the mass spectrometer, and the analysis of the mass to charge ratio of these ions using a ‘mass analyser’. Many combinations of sources and analysers have been used but this review will concentrate on the most common, the use of electrospray ionisation in conjunction with a quadrupole mass analyser to analyse larger biomolecules. A number of reviews have been published comparing ESI-MS with MALDI–TOF,3–8 and it is worth bearing in mind that they are, on the whole, complementary techniques. Comparisons to thermospray have been made,9 and the limitations of the use of electrospray in conjunction with sector mass analysers, which require a very stable ion beam and hence reasonably strong samples and good ionisation, have also been reviewed.10 As with any rapidly growing area of science new techniques with even higher performance are continuously being developed.The major advances have been in the design of the analyser. Fourier transform ion cyclotron resonance (FTICR) mass spectrometers,11–13 which have actually been interfaced to electrospray ionisation sources for many years,14 with mass accuracies of <1 ppm and exceptional sensitivity (theoretically a few thousand molecules would be enough) and resolution (the theoretical mass at which two peaks one Dalton apart would be resolved to half their heights) of 200 000 or better,15 are now commercially available and the use of quadrupole ion traps is becoming more popular.16,17 These exciting new techniques will only be mentioned in passing as they have not yet found widespread use but they are, without doubt, set to become the standards of the future. 2 Electrospray mass spectrometry 2.1 Ion generation and mass analysis A wide range of mass spectrometers are commercially available with electrospray sources18 and ESI-MS has now become a routine technique in many laboratories. Electrospray mass spectrometry has been around for a number of years, but some of the processes involved in the formation of the ions are still controversial. This section will attempt to deal briefly with the processes thought to be involved in the formation of the gas phase ions, the key process in any mass spectrometry.The process is outlined in Fig. 1. Electrospray ionisation is an atmospheric pressure ionisation technique, where ions are continuously formed in the source region from a solution of the analyte at, or near, atmospheric pressure. The pressure is then reduced in stages through the source by high capacity Sample + solvent ++++++ ++++ Electrospray capillary 2–3 kV relative to skimmer Ion beam Nebulising gas Drying gas Vacuum pumps Quadrupole analyser Ion beam focussing Ion source Electrospray capillary Sampling skimmer cone Fine spray of charged droplets formed due to high potential (+ nebulising gas) Drying gas ( a) ( b) Fig. 1 Schematic diagram of (a) one design of an electrospray source showing the general layout and (b) an expanded diagram of the area in which the spray and ions are formed Pitt: Application of electrospray mass spectrometry in biology 59vacuum pumps, to reach the 10"5 Torr or less needed in the quadrupole analyser.A continuous flow of solvent (at rates which can range from 10 nl min"1 up to 1 ml min"1) is passed into the source through a fine capillary needle which is held at a high potential (2–4 kV) relative to an adjacent sampling plate, resulting in a fine spray of droplets. The most widely accepted model for the formation of gas phase molecular or pseudomolecular (multiply charged) ions take place in four steps19,20 (Fig. 2): (i) the formation of a fine spray of droplets with relatively high surface charge densities due to the high potential on the capillary needle; (ii) evaporation of the carrier solvent molecules from the droplets, causing the droplets to shrink and the charge density on the surface to increase; (iii) explosive fragmentation of the droplets as the charge density on the droplet reaches a critical limit (the Raleigh limit); and (iv) the desolvation process continuing until the eventual formation of gas phase ions of individual molecules.The exact process for the formation of gas phase ions is still controversial, and other models have also been proposed.21,22 The formation of a stable spray of fine droplets is one of the critical processes for optimum performance of the electrospray source and can be aided at higher flow rates of solvent by the use of a sheath of N2 gas around the electrospray needle to promote nebulisation (pneumatically assisted electrospray) or by replacing the capillary needle with an ultrasonic nebuliser.The evaporation of solvent from the droplets is usually promoted by heating the source (from 40–150 )C), and in a number of designs by the use of a flow of nitrogen ‘drying’ gas through the source, usually across or against the flow of droplets, to help to remove solvent vapour. Ions are formed by the addition or removal of protons from the analyte and hence the formation of charged ions can be assisted by the addition of volatile acids or bases to the solvent.In many cases only a small proportion of the analyte produces ions and the sampling of the ions is relatively ineYcient, hence electrospray sources have limited sensitivity, especially at higher solvent flow rates. Electrospray sources can generate and analyse either positive or negative ions23 allowing the analysis of biomolecules with a wide range of pKa values. Quadrupole analysers have a reasonable mass to charge range (up to 4000) and are robust, easy to tune and do not require a very high vacuum, hence are ideal in the general laboratory environment.The electrospray source generally produces ions from biomolecules carrying a number of charges such that the mass to charge ratio (m/z) of the ions, which is the parameter that is measured, often lies in the region of 500–2000 (although under some conditions this may increase to >4000); this is ideal for detection by a quadrupole analyser.Proteins with masses up to 150 000 can be observed using a simple quadrupole analyser, although some care is needed in optimising the conditions.24 The importance of electrospray mass spectrometry in the analysis of biomolecules is now widely accepted and has been reviewed a number of times over the past years.25–34 The great advantage of FTICR as a mass analyser is its extreme sensitivity and resolving power; for example a resolving power of 63 000 has been reported for equine cytochrome c (12 352 Da).35 However, the authors also highlight some of the diYculties that need to be addressed when using FTICR, and for most applications this technique is still in its infancy. The extreme sensitivity of FTICR as an analyser for ESI-MS was demonstrated by the determination of the molecular weight of carbonic anhydrase (28 780) to 1 Da at a resolving power of about 60 000, using 29#10"18 moles of the enzyme from a crude preparation of red blood cells.36 This represents about 1% by weight of the protein in one red blood cell.Not only were the authors able to measure the mass to a high degree of accuracy, but they were also able to gain enough information from sequence specific fragment ions from the same sample to confirm unequivocally the identity of the protein from the protein database. Figure 3 shows a typical electrospray mass spectrum for a protein. It is usual to see a range of charged states adopting a Gaussian distribution (known as the charge state distribution), each of the adjacent peaks in the spectrum diVering by one charge caused by the addition (positive ion electrospray) or removal (negative ion electrospray) of a proton.The tops of these peaks represent the average isotopic mass of the peaks, as the quadrupole analyser is unable to resolve the individual isotope peaks in the multiply charged ions which will only be separated by 1/(the charge on the peak) Da.The conversion of the series of multiply charged peaks into a real mass spectrum is referred to as deconvolution (Fig. 3). The majority of operating systems for mass spectrometers are supplied with a computational package that will perform this operation, and it is relatively simple when a series of peaks from a single component can be readily identified. Under ideal conditions it should be possible to resolve species diVering by 1 Da in 10 000 with a quadrupole analyser, and 1 Da in 25 000 with a double focusing sector analyser.A mass accuracy of 1 Da in 10 000 has been demonstrated with a quadrupole analyser for compounds with molecular masses up to 80 000.37 A problem with ESI-MS is that complex mixtures can be diYcult to analyse due to the confusion of peaks that all appear at around the same mass. One of the most eVective methods of deconvoluting complex spectra to give a true mass output is the use of maximum entropy algorithms,38–41 which can be used to Evaporation of solvent Explosive fragmentation Process continues until formation of pseudomolecular ions + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Fig. 2 Schematic diagram of pseudomolecular ion formation Fig. 3 Electrospray mass spectrum of horse heart myoglobin (Sigma, Poole, Dorset) obtained on a VG Analytical Platform mass spectrometer (Altrincham, Cheshire) showing the Gaussian distribution of charge states. Numbers above peaks correspond to the number of charges on the ion 60 Natural Product Reports, 1998identify the components present from the raw data without the need for any preprocessing, and can give exceptionally good resolution if applied judiciously42 (see Fig. 6). This is supplied commercially as MaxEnt by Micromass, Altrincham, Cheshire. The problem of the determination of the number of charges on individual peaks in complex spectra (charge state assignment), which simplifies the assignment of a series of peaks in the raw data to a single component in the mixture, can also be simplified by utilising the 1:1 stoichiometry of the non-covalent adduct formation between peptides or proteins and 18-crown-6.43 The remarkable resolution of FTICR allows the resolution of the individual isotope peaks within any one multiply charged ion peak, obviating the need for any complex deconvolution routine. 2.2 Secondary ion generation A technique that has been used to great eVect for sequencing peptides is the fragmentation of the molecular ion (parent ion) into smaller units (daughter ions), the fragmentation usually taking place around the amide bonds generating a series of ions (Fig. 4). These daughter ions can then be analysed and the sequence of the peptide inferred.44,45 For a simple sample containing a single component the fragmentation can be induced in the electrospray source region by increasing the voltages on the skimmer cones, which results in the ions gaining kinetic energy and colliding with enough force to cause them to fragment.This is referred to as collisionally induced decomposition (CID) or cone voltage fragmentation (CVF). For more complex samples the spectra become too complicated and it is necessary to resort to tandem mass spectrometry. Tandem mass spectrometry, often referred to as MS/MS or MS2, uses two (or more) analysers in tandem, with an area between them where fragmentation is induced (Fig. 5). The first analyser (MS1) is used to select the ion of interest, referred to as the ‘parent’ ion.This is then passed into a collision cell, where fragmentation is usually induced by introducing a low pressure of gas. The ions leaving the first analyser have suYcient energy to cause fragmentation when they collide with the gas molecules in the collision cell, and the resulting ‘daughter’ fragment ions are then analysed in a second mass analyser (MS2). This is a particularly powerful technique for sequencing small peptides, and for identification of sites of modification, but will work with most compounds.A number of combinations of analysers have been used. The most common arrangement is two quadrupole analysers, but the development of QTof (Micromass, Altrincham, Cheshire), which is the combination of a quadrupole (MS1) with TOF (MS2), is showing great promise for simplicity and sensitivity. FTICR and quadrupole ion trap mass spectrometers have the capability to extend this technique to the selection of one of the initially formed fragment ions and further fragmentation of this ion, and MS4 is possible under ideal conditions. 2.3 Protein and peptide analysis There have been a number of reviews published in recent years in this area dealing with the simple characterisation of peptides from complex mixtures,27,46–50 protein analysis,51,52 on the application of mass spectrometry to the study of biological molecules and in analytical biochemistry.50,53–55 An example of the eVectiveness of the maximum entropy algorithm for the deconvolution of a complex spectrum is demonstrated by the analysis by allelic fingerprinting of major urinary proteins (18 600–19 000), showing the allelic diVerences between diVerent strains.56 The misincorporation of amino acids during the over expression of proteins57 and the incorporation of non-natural amino acids can also be followed by ESI-MS.58 The analysis can be extended to glycoproteins,59 although there can be some problems if there is a large contribution to the mass from the saccharide (see Section 4.1.2.1). 2.4 Interfacing to separation techniques One of the major advantages of the electrospray source is that it can be directly interfaced to a number of high performance separation techniques in order to eVect separation prior to mass analysis, the result often being referred to as ‘hyphenated methods’,60 thus allowing the on-line monitoring of separated materials directly.This has proved to be a powerful technique for the identification of components of complex mixtures, especially in the use of proteolytic digests for the location of modifications (see Section 4.1) and for variant protein analysis.61 O HN R O NH R OHN O NH O NH R R R O HN R O NH R OHN O NH O NH R R R CO2H R NH2 O NH R OHN O NH O NH R R R CO2H HN R CO2H HN O NH R R CO2H O NH R OHN HN O NH R R O HN RO NH R NH2 O HN R O NH R OHN O NH O NH R R R O HN R O NH R OHN O NH O R R R NH2 NH HN H2N R1 O R2 O R3 NH CO2H O R4 Collision cell Fragmentation of parent ion Measure mass of daughter ions MS1 MS2 z3 m/ z y1 m/ z y3 M x1 z2 x3 M y2 z1 x2 a1 b1 Parent ion spectrum b2 b3 c1 Daughter ion spectrum c2 c3 SEQUENCE a2 a3 a3 b3 a2 b2 y1 a1 b1 A+ + B+ + C+ + ......( b) P+ ( a) ( c) Daughter ions Select ion of interest Parent ion Fig. 4 MS/MS of peptides showing (a) the formation of daughter ions from a parent peptide ion, (b) the expected backbone fragmentation pattern of a peptide and the annotation used to denote the ions formed, and (c) a representation of a secondary ion spectrum MS1 MS2 Ion source Ion detection Deflector Collision cell Fig. 5 Schematic of a tandem mass spectrometer Pitt: Application of electrospray mass spectrometry in biology 612.4.1 High performance liquid chromatography High performance liquid chromatography (HPLC) has been routinely used for the separation of biological samples for many years, and its use in conjunction with ESI-MS is now widespread.62–65 The problems associated with the coupling of HPLC to the mass spectrometer are mainly associated with the high solvent flow rates and the use of ion pairing reagents.The flow rate problem has been overcome by the use of splitters to direct only a portion of the eluent into the mass spectrometer, along with the use of large vacuum pumps, high source temperatures and a high flow rate of drying gas to remove rapidly the larger volumes of solvent vapour, or the use of microbore HPLC.The use of ion pairing reagents, for example trifluoroacetic acid, which are commonly added to polar HPLC eluents to improve the resolution of the technique for a range of polar biomolecules, can significantly reduce the sensitivity of ESI-MS, especially for DNA samples. This can be overcome by careful choice of the reagent, or by the addition, just at the point of formation of the spray, of an additional reagent.66,67 HPLC is particularly useful for proteolytic mapping, for example in glycopeptide and glycoprotein analysis.68,69 2.4.2 Capillary electrophoresis The high resolving power of capillary electrophoresis (CE) and the low flow rates involved make it an ideal technique to interface with ESI-MS; this was first demonstrated for proteins in 1989.70 There are a number of problems associated with the very low flow rates and high buVer concentrations required for CE in some instances, and also in raising the potential on the capillary to a suitable voltage to generate the electrospray.These have been overcome by a number of methods68 including the addition of a second solvent at the probe tip via a larger coaxial capillary, or coating the capillary in gold to make the contact. It is a powerful analytical tool due to its impressive resolving power, and has found use for the analysis of proteins70–73 and complex mixtures of peptides, especially proteolytic digests.65,74–81 3 Oligosaccharide analysis Electrospray mass spectrometry has been less widely applied to oligosaccharide analysis, although there have been some notable successes.Fragmentation by CID or tandem mass spectrometry will generate daughter ions that can be used to assign both the sequence and the position of the linkages. Hydrophobic oligosaccharides without easily ionisable groups can be problematic to analyse, and the formation of adducts with sodium in place of a proton is common.Carbohydrate sequence analysis by ESI-MS has been recently reviewed,82 and the linkage position and anomeric configurations of underivatised glucopyranosyl disaccharides have been determined using ESI-MS.83 4 Proteins Electrospray mass spectrometry has been extensively used to study covalent modifications in proteins. These studies can be roughly divided into four areas; non-specific covalent adduct formation, specific chemical modification, post-translational modification and irreversible inhibitors. 4.1 Covalent modification 4.1.1 Non-specific covalent adduct formation ESI-MS has been shown to be useful in identifying covalent non-specific chemical modifications of proteins. However, some care needs to be taken in assuming that an observed mass change is due to an expected modification. A disulfide link between 2-mercaptoethanol (present in the buVer during puri- fication) and a surface cysteine of the class II fructose-1,6- bisphosphate aldolase from E.coli (expected mass change of 76 amu) was shown to be responsible for an apparent mass change of 80 amu, which could have easily been assigned to a phosphate group.84 Although this modification was benign, it would have gone unnoticed without the use of ESI-MS in the analysis of the protein. The adducts formed with histidines on treatment of myoglobin with 4-hydroxy-non-2-enal, a byproduct of lipid peroxidation, are easily distinguished by ESI-MS85 and can be further characterised by tryptic digest and tandem MS.86 ESI-MS has also proved useful for the monitoring of the reactions of the biologically important compound nitric oxide with peptides and proteins.87 Cysteine residues were shown to be rapidly nitrosated, with an increase in mass of 29 Da.being observed for each NO addition. The further oxidation of nitroso thiols to sulfonic acids and nitrosation of tyrosine were also noted. 4.1.2 Chemical modification ESI-MS can be used to characterise rapidly a range of chemical modifications of proteins.83 ESI-MS alone, and in conjunction with liquid chromatography techniques, has become a powerful tool for the identification of active site residues using chemical modification.It has been extensively used to characterise the adduct between the arginine specific reagent phenylglyoxal and type I and type II dehydroquinases,88–91 and to relate this to the activity of the enzyme. It was possible to characterise the phenylglyoxal modified protein and show that there may be 1:1 or 1:2 stoichiometries with this reagent which may cause misinterpretation of radioactive labelling results.Using proteolytic digestion and LC-MS the authors were able to locate the position of the hyper-reactive arginine as ESI-MS is soft enough to observe the highly labile modified peptides (Fig. 6). Boots et al.92 used the active site directed inhibitor 1-bromooctan-2-one to label specifically and to identify the active site histidine of Staphylococcus hyicus lipase as His 600, and the active site cysteine of thiaminase I from Bacillus thiaminolyticus was identified using ES–FTICR of the 4-amino-2-methyl-6-chloropyrimidine inactivated enzyme and identification of the modified peptide generated from fragmentation of the protein.93 The ability of iron to generate free radical species has been used to locate the metal binding site of actin.94 Treatment of iron bound actin with oxygen and either dithiothreitol or ascorbate as the reductant caused cleavage of the protein at sites adjacent to the iron binding site, and the resultant fragments were analysed using ESI-MS to locate the fragmentation sites. 4.1.2.1 Post-translational modification ESI-MS has been used to study a range of post-translational modifications, but has been particularly eVective for studying sites of phosphorylation and glycosylation patterns. Full characterisation of the modifications may require the combination of a number of techniques, including enzymatic digestion, chemical modification and accurate mass analysis, as well as ESI-MS,95 and care needs to be taken in assigning an apparent mass shift to a particular modification without additional evidence.84 The expected mass changes for a range of post translational modifications are given in Table 1.ESI-MS was used to demonstrate that the post-translational modification of barley ·-amylase expressed in yeast involved the removal of the C-terminal Arg–Ser dipeptide, glutathionylation and O-glycosylation.96 The complex glycosylation pattern of human lecithin:cholesterol acyltransferase was determined by microbore reverse-phase HPLC–ESI-MS;97 a combination of enzymatic digestion of the protein and sequential glycosidase digestion followed by HPLC–ESI-MS using a method of monitoring carbohydrate specific fragment ions was able to locate four N-linked glycosylation sites containing sialylated bi- and tri-antennary complexes, along with two O-linked glycosylation sites.A range of post translational 62 Natural Product Reports, 1998modifications of a the humanised monoclonal antibody Campath-1H were also identified by ESI-MS in combination with capillary chromatography following disulfide reduction and trypsin digestion.98 Two diantennary carbohydrate moieties and conversion of the terminal glutamate into pyroglutamic acid were observed, and partial removal of the C-terminal lysine was confirmed by MS/MS sequencing.The combination of ESI-MS and FAB mass spectrometry were used to identify the two sites and the nature of the glycosylation of the potent immunosuppressant glycodelin.99 Multiple isozymes often reflect diVerential post-translational modification. The catalytic subunit of mouse cAMP-kinase expressed in E. coli was shown to have a number of isozymes reflecting diVerent levels of phosphorylation using ESI-MS to measure the mass of the protein.100 The phosphorylation was not due to endogenous E. coli enzymes but was due to autocatalysis, giving a maximum of four, but predominantly three, phosphates.ESI-MS was also used to show that the two isozymes of the same enzyme isolated from porcine heart had the same mass, corresponding to bis-phosphorylation and N-terminal myristoylation. The in vivo phosphorylation sites of protein kinase C ‚-2 were identified by ESI-MS using collisionally induced dissociation, and this technique gave more detailed results than had been obtained in the previous in vitro studies.101 The four tyrosines that are the sites of phosphorylation of the intra- and extra-cellular domain of the heparin-binding fibroblast growth-receptor tyrosine kinase (FGF-R1) were identified using trypsin digestion followed by Edman degradation and tandem mass spectrometry,102 and ESI-MS was subsequently used extensively to identify the sites Table 1 Common post-translational modifications (excluding glycosylations) Modification Mass change Modification Mass change Pyroglutamic acid from glutamine "17.0306 Phosphorylation 79.9799 Disulphide bond formationa "2.0159 Sulfonation 80.0642 C-Terminal amide from glycine "0.9847 Cysteinylation 119.1442 Deamidation of glutamine or asparagine 0.9847 Incomplete N-terminal methionine removala 131.1986 Methylation 14.0269 Farnesylation 204.3556 Hydroxylation 15.9994 Myristoylation 210.3598 Oxidation 15.9994 Biotinylation 226.2994 Oxidation of methionine: sulfoxide sulfone 15.9994 31.9988 Pyridoxal phosphate (SchiV base to lysine) 231.1449 Formylation 28.0104 Glutathionylation 305.3117 Acetylationa 42.0373 5*-Adenosylation 329.2091 Carboxylation of asparagine or glutamine 44.0098 4*-Phosphopantotheine 339.3294 aIncomplete post-translational processing of overexpressed proteins is quite a common occurrence and will result in mixtures of proteins being observed, separated by the indicated masses. O O +N H H2N NH2 Arg HN NH OH HO +N H Arg O O O O HN NH HN NH +N H Arg +N H Arg Phenylglyoxal Singly modified (+116 amu) Doubly modified Takahashi adduct (+250 amu) – H2O – H2O ( a) ( b) Fig. 6 Chemical modification of arginines using phenylglyoxal. (a) Scheme showing the chemical formation of 1:1 (+116 amu) and 2:1 (+250 amu) adducts, and (b) MaxEnt (Micromass, Altrincham, Cheshire) deconvoluted mass spectrum of Streptomyces coelicolor dehydroquinase 95% inactivated with phenylglyoxal showing the formation of singly (+116 amu for each addition) and doubly (+250 amu for each modification) modified arginines.Pitt: Application of electrospray mass spectrometry in biology 63of the phosphorylations, usually using LC-MS to separate peptic digest fragments103 or tandem mass spectrometry.104 This technique is also useful for the study of covalently attached cofactors. Morris et al. were able to use ESI-MS to study the loading of Saccharopolyspora erythrea acyl carrier protein overexpressed in E.coli with 4*-phosphopantothene.105 The crude material isolated from the cells consisted of apoenzyme, holoenzyme dimer and holoenzyme glutathione adduct. The same group were able to show that the C-terminal acyl carrier protein thioesterase domain of the multifunctional 6-deoxyerythronolide B synthase from S. erythrea overexpressed in E. coli was not loaded with the 4*-phosphopantothene group, but that the cysteine predicted to lie in the active site could be selectively labelled with phenylmethylsulfonyl fluoride.106 They also used ESI-MS to study a number of other features of the acyl carrier protein.107 ESI-MS has been used to show that a subunit of NADH:ubiquinone reductase from bovine heart mitochondria, that has a closely related sequence to the acyl carrier proteins, actually caries a 4*-phosphopantothene group,108 although the role of this group was unknown at the time.The modification was identified by treatment of the subunit under alkaline conditions resulting in a mass loss of 339 expected for that of the 4*-phosphopantothene group.The flavinylation of trimethylamine dehydrogenase from Methylophilus methylotrophus and the wild type and mutant enzymes expressed in E. coli have shown that the enzyme is expressed almost exclusively in the holoenzyme form in the natural host, whereas the recombinant and mutant forms studied are not correctly posttranslationally modified, and therefore are prevented from undergoing flavinylation to the same extent.109 This may be due to the higher levels of expression in E.coli. 4.1.2.2 Active site directed irreversible inhibitors The ability of ESI-MS to characterise rapidly the complexes between proteins and covalent irreversible inhibitors, and in many cases to extend this to the identification of the specific site of the modification using proteolytic digests in conjunction with HPLC–ESI-MS, has played a major role in the increasing popularity of the technique.The high degree of sensitivity, and the ability to compare directly and rapidly the extent of modification with the remaining activity of the enzyme makes it the method of choice in these studies. The formation of acyl–enzyme inhibitor complexes between proteins and inhibitors has been a fertile area for ESI-MS studies. Farmer et al. have studied the inhibition of a range of ‚-lactamases with the penem BRL-42715 1.110 They used ESI-MS to show rapid and stoichiometric binding, and the observed mass diVerence between inhibited and uninhibited complexes confirmed the formation of an acyl-enzyme species with no further fragmentation of the inhibitor.Aplin et al. have also used ESI-MS to study the inhibition of ‚-lactamases. 6-‚-Halogenated penicillanic acids were seen to react with class A and C ‚-lactamases to give a mass consistent with the previously proposed enzyme bound dihydrothiazine derivative (Scheme 1).111 They were also able to observe the ‚-lactamase acyl-enzyme intermediate with a class C ‚-lactamase from Enterobacter cloacae P99 and four poor substrates that behave as inhibitors.112 A series of phosphonamidate peptide analogues were found to be inhibitors of the serine ‚-lactamase from the same organism,113 and ESI-MS was used to show that the reaction of the majority of these inhibitors with the enzyme resulted in apparent phosphonylation of the active site serine.The adjacent variable amino acid which forms the phosphonamidate bond acts as the leaving group in this case. The complex mechanisms of the inactivation of the TEM-2 ‚-lactamase from E. coli by clavulanic acid were unravelled by the use of ESI-MS.114 Four diVerent modified forms were observed, consisting of a serine O-linked decarboxylated form, a vinyl ether cross linked form, a hydrated aldehyde and a ‚-linked acrylate (Scheme 2). The sites of the modifications were then localised by proteolysis and HPLC–ESI-MS.These types of study are not limited to ‚-lactamases. The inhibition of porcine pancreatic elastase by two cephalosporins, L-647957 2 and L-658758 3, has also been studied by Aplin et al. using ESI-MS.115 The mass shifts that they observed were consistent with initial formation of an acylenzyme intermediate, followed by expulsion of the acetoxy group from the 3*-methylene position. Further elimination of HCl was observed for L-647957 2 (Scheme 3).The same research group was also able to use ESI-MS to study the inhibition of this enzyme by a number of chloro methyl ketones and a range of other alkylating inhibitors.116 A more detailed investigation of the inhibition of human leukocyte elastase and porcine pancreatic elastase by ‚-lactams using ESI-MS and NMR was able to determine that the mechanism of the chemical reaction was dependent on the structure of the ‚-lactam, and that the stability of the final complex is controlled by its molecular structure and conformation, which is also dependent on the initial ‚-lactam.117 Depending on the stereochemistry and constitution of the ‚-lactam, the inhibitor could be observed covalently attached to the enzyme intact, or after fragmentation, or after trapping of the fragmentation product by water to give a hydroxy amine.Other inhibitor complexes have also been seen in the case of proteases. For example (2R,3S)-2-benzyl-3,4-epoxybutanoic acid methyl N N N N S O CO2 – H 1 X N S O CO2 – S NH CO2 – O O Ser X = I, Br Scheme 1 O HN O O O O O HN O OH N O HN O OH O Ser 70 Ser 70 Ser 70 Asp 130 Ser 70 Scheme 2 64 Natural Product Reports, 1998ester was shown using ESI-MS and HPLC–ESI-MS to inhibit ·-chymotrypsin by covalently modifying the active site serine.118 An electrophilic alkylating analogue of ATP, 4 was used by McKay et al.to study the aminoglycoside antibiotic phosphotransferase responsible for the resistance to these drugs in many pathogenic bacteria.119 They were able to observe the inhibited complex, and using a combination of peptide mapping, Edman degradation and ESI-MS, were able to locate the modified sites in the protein as Lys33 and Lys44, indicating that the ATP binding site lies towards the N-terminus.The 2*,3*-dialdehyde (2*,3*-oxidised) analogue of ATP has been used to study the binding of ATP to GroEL.120 ESI-MS was used to show that the stoichiometry was approximately 1:1, and the use of peptide mapping with ESI-MS indicated that the covalent modification probably occurs at one of two cysteines, although the lability of the adduct made a definite identification diYcult.Ovine ceroid lipofuscinosis protein was shown by ESI-MS to have an identical mass to the F0 subunit c of bovine ATP synthase, and even has the same +42 mass units post-translational modification. Its reaction with a series of ATP synthase inhibitors was then studied by ESI-MS121 and shown to be similar to that of the F0 subunit and tandem mass spectrometric analysis allowed the site of modification to be determined.The identification of the active site nucleophile in the reaction of yeast ·-glucosidase was investigated using fluorinated inhibitors.122 A glucosyl-enzyme species was observed which was shown by tandem mass spectrometry and daughter ion analysis to be bound to Asp-214, one of the three conserved aspartates in the active site.The suicide inhibition of yeast cytochrome c peroxidase by resorcinol has been studied by ESI-MS.123 It was possible to confirm the previous findings that the resorcinol mainly became bound to the peptide and also to show that there were concomitantly two oxidations of the protein, probably on methionine, tyrosine or tryptophan residues and they were also able to use ESI-MS to demonstrate that the haem unit remained substantially unaltered, although a small amount of resorcinol modified haem could be identified.The substrate analogues 5-chlorolaevulinic acid 5 and 5-amino-3-thialaevulinic acid 6 have been shown by ESI-MS to be a non-specific alkylator and potent mechanism based inhibitor of B. subtilis 5-aminolaevulinic acid dehydratase, respectively, using ESI-MS (Scheme 4).124 The residue modified by the aYnity label N-bromoacetyl cellobiosylamine 7, which inhibits a Cellulomonas fimi exoglucanase, was unequivocally determined as Glu-127 by a combination of the ESI-MS analysis of tryptic digests and tandem mass spectrometry on the modified peptide.125 This confirmed the previous mutagenesis studies that suggested that this was the acid–base catalytic residue.ESI-MS was used to confirm that acetelynic GABA 8 binds covalently to GSH aminotransferase, with an observed mass increase equivalent to C6H6O3. This can be assigned to a species generated via attack of lysine on the conjugated acetylene, which is only formed slowly after tautomerisation of the initially formed imine, followed by hydrolysis (Scheme 5).126 The imine formed between 8 and GSH aminotransferase was too labile to be observed directly by ESI-MS, but on sodium borohydride reduction the resultant amine was observed.Mass spectrometry has been used very eVectively to study the mechanism of inhibition of the cysteine protease papain by the hydroxylamine derivative [Bz-Phe-Gly-NH-O-CO-(2,4,6-trimethylphenyl)].127 ESI-MS was used to identify the oxidised form of papain resulting from treatment with the peptidyl hydroxamate in the absence of a reducing agent as a sulfinic acid, along with a sulfenamide covalent adduct between the inhibitor and papain.The catalytic aspartate of soluble epoxide hydrolase was determined using ESI-MS to identify a number of radiolabelled peptides formed on incubation with the inhibitor 4-fluorochalcone oxide. The four peptides identified were overlapping and had only Asp-333 in common.128 The same group went on to use an elegant ESI-MS experiment to demonstrate the formation of a covalent intermediate and to con- firm the site of attachment of the inhibitor to soluble epoxide hydrolase.129 Using 18O-labelled water in single turnover experiments they were able to identify a tryptic peptide that N S O O OAc O OBut O Cl N S O O OAc O N O MeO N S O O O OBut –O2C O Cl O Ser 2 –HCl 3 Scheme 3 S F O O O O O N N N N NH2 OH OH 4 5 6 S CO2H H2N O CO2H Cl O S CO2H H2N O SH CO2H O NH H2N H2N Enz Enz Scheme 4 O O HO HO OH O OH OH OH HN O O O Br O O– HO 7 Glu 127 Pitt: Application of electrospray mass spectrometry in biology 65was labelled with 18O, and eventually to identify 18O incorporation into Asp-333 in this peptide, suggesting the intermediacy of an ·-hydroxyacyl-enzyme intermediate (Scheme 6). 4.2 Covalent enzyme–ligand complexes The study of covalent modification of proteins has been very eVectively extended to look at covalent interactions with substrates.Covalent enzyme–intermediate complexes can be seen when the breakdown of the intermediate complex is the rate-limiting step of the reaction. For example an acyl-protein intermediate has been observed in an antibody catalysed hydrolysis of a p-nitrophenyl ester;130 the covalent species, which showed an increase in mass consistent with the acyl portion of the substrate, accounted for about 8% of the total Fv concentration, and is not observed in the presence of the hapten, showing active site specificity.Interestingly, no covalent modification is observed on switching to the p-chlorophenyl ester, indicating that the nature of the leaving group is important in determining the rate limiting step. The E166Y mutant of TEM-1 ‚-lactamase shows the build up of an acyl-enzyme intermediate when treated with penicillin G, indicating that breakdown of this complex has become the rate limiting step.131 Acyl-enzyme intermediates have also been observed for the reaction of three serine proteases with cinnamoyl imidazole and indoleacryloyl imidazole.132 The mild nature of electrospray also makes it possible to observe other relatively labile enzyme–substrate complexes and a number of other types of enzyme–substrate and enzyme– product complexes have been observed by ESI-MS.Enzyme bound intermediates in the assembly of the tetrapyrrole unit that is the precursor to the tetrapyrrole pigments can be observed by ESI-MS.133 A series of intermediates with one to four of the monomeric porphobilinogen units that make up the final tetrapyrrole attached to the enzyme can be observed under substrate limited conditions (Fig. 7). Mutation of an essential histidine in the active site of E. coli type I dehydroquinase results in stalled catalysis with preferential formation of the SchiV base intermediate, as shown by isoelectric focusing and ESI-MS.134 This finding of a dual role for the histidine in catalysis and product release could explain the unexpected syn stereochemistry of the elimination process.Borthwick et al. used ESI-MS to observe the formation of the imine between E. coli dihydrodipicolinate synthase and pyruvate.135 They were also able to see the formation of the imine with the substrate analogues 3-bromopyruvate and 3-fluoropyruvate. The formation of an imine intermediate has also been detected between dehydroquinase and its substrate quinate using ESI-MS, con- firming the proposed mechanism.136 The requirement for the 4- and 5-hydroxy groups of dehydroquinic acid in the recognition of the substrate by dehydroquinase was studied using 5-deoxydehydro- and 4,5-dideoxydehydro-quinic acid and looking for the formation of the intermediate imine complex with the enzyme by ESI-MS.137 Distinct diVerences between type I and type II dehydroquinases were observed.The imine formed between pyridoxal phosphate and glutamate-1- semialdehyde aminotransferase is too labile to be observed by ESI-MS, but reduction of the imine with sodium borohydride gave a peak in the mass spectrum corresponding to covalently bound pyridoxal phosphate.126 The rates of phosphorylation and dephosphorylation of phosphoglycerate mutase from Saccharomyces cerevisiae and the fusion yeast Schizosaccharomyces pombe, which lacks the C-terminal section of the protein, were studied by ESI-MS.138 It was possible to determine the approximate rates of the addition and removal of the phosphate for the two enzymes, and also to observe the increase in the rate of dephosphorylation of the S.cerevisiae enzyme in the presence of the substrate analogue 2-phosphoglycolate. The straight forward measurement of rates of phosphorylation and dephosphorylation could be useful in the study of this important modification. 4.3 Non-covalent enzyme–inhibitor complexes ESI-MS has been shown to be an eVective method for the identification and characterisation of a wide range of noncovalent complexes, including protein–substrate, protein– inhibitor, protein–DNA, protein–protein and DNA–drug interactions.Low skimmer cone voltages are needed to avoid collisional activation from causing some dissociation, and careful choice of buVers and pH is essential. There have been a number of reviews in this area covering general eVects,139 the preservation of non-covalent associations, such as higher order protein structure, enzyme complexes and multimeric peptide association into the gas phase,140 and the evaluation of the performance of several designs of electrospray source for the detection of non-covalent complexes between ribonuclease A and cytidylic acids.141 One productive area has been the study of the binding of metal ions to proteins.The choice of conditions can play a major role in the successful detection of bound metal ions.NH2 NaBH4 NH2 CO2H N CO2H N NH CO2H N NH CO2H O NH2 NH2 N CO2H NH2 HN CO2H Lys PYP Lys PYP Lys PYP Lys PYP Lys PYP Lys PYP H+ 8 Scheme 5 H R R N NH O– O N NH O O N NH O– O OH O H O H H R = 18O * : * * Scheme 6 66 Natural Product Reports, 1998Studies on a number of metal substituted rubredoxins demonstrated that the apoenzyme form tended to predominate at acidic pHs using positive ion detection, and was the only form observed when the metal was zinc, whereas at neutral pH and using negative ion detection the predominant form was the holoenzyme.142,143 Jaquinod et al.144 have observed iron bound to a synthetic siderophore analogue and two iron– sulfur proteins using ESI-MS, and the mass spectrum of isopenicillin-N synthase also shows a species that appears to have the catalytic iron bound to the protein.145 The study of metal binding has been eVectively extended to zinc finger proteins.The interaction of zinc and copper with a 71 residue peptide containing two four-cysteine clusters was used to develop the methodology which was then exploited to study why copper might inhibit the function of this type of zinc finger.Species with up to two zinc or four copper atoms coordinated were observed, and a charge state shift (see Section 4.4) indicated a change in conformation on binding Cu in place of Zn.146–148 Electrospray data were obtained even with 100 micromolar zinc present. The calcium binding properties of calmodulin (up to 4 calcium ions)149 and a comparison of calmodulin and parvalbumen (up to 2 calcium ions)150 have also been determined by ESI-MS, with strong cooperativity being observed for the binding of the second calcium to parvalbumen and the fourth calcium to calmodulin, closely mimicking the observed solution behaviour of the proteins.The observation of bound cofactors is not limited to metal ions. Jaquinod et al.151 have observed the non-covalent complex between pig lens aldose reductase and NADP+ using positive ion ESI-MS.They were also able to see the covalent adduct formed with 3-chloroacetyldihydropyridineadeninedinucleotidephosphate 9, an alkylating analogue of NADP+. Drummond et al.152 isolated the proposed 28 kDa cobalmin binding domain from the 136 kDa methionine synthase and used ESI-MS to show that the isolated domain does indeed bind cobalamin, and they were also able to resolve some anomalies in the previously reported sequences.The eVect of A P A P A A P A P P A P A P A P A P ES3 ES4 ES2 NH NH S NH NH NH S NH ( a) ( b) H2N A P NH NH S A P A P A P HMBS holoenzyme NH3 NH A P A P A P PBG A HN HN A P ES1 NH3 A P NH P A P NH NH S NH HN HN NH NH S NH HN Porphobilinogen (PBG) A P PBG NH3 PBG NH3 Fig. 7 Observation of intermediate complexes for hydroxymethylbilane synthase. (a) Formation of intermediate complexes by the addition of 1 (denoted ES1) to possibly 4 (ES2, ES3 and ES4) substrate units to the dipyrromethane cofactor, and (b) MaxEnt (Micromass, Altrincham, Cheshire) deconvoluted spectrum of substrate limited experiment showing formation of the intermediate complexes (A.R. Pitt and A. R. Battersby, unpublished data). Pitt: Application of electrospray mass spectrometry in biology 67pH and solvent composition were shown to have significant eVects on the observation of non-covalent complexes between the ras protein and GDP and GTP.153 Apoenzyme, GDP and GTP bound enzyme were all observed in diVering amounts depending on the composition and pH of the solvent used in the ESI-MS.The factors that influence the stability of non-covalent complexes in the gas phase have been studied by using ESI-MS to measure the binding of acyl CoA to acyl CoA binding protein and the diVerences caused by a range of modifications of both enzyme and cofactor.154 Changes in chain length of the acyl portion had little eVect on the gas phase binding, although they did have a substantial eVect on the solution state binding, whereas removal of three tyrosine residues involved in key contacts with the ligand by mutagenesis gave substantially lower binding in the gas phase.The complex of bovine pancreatic trypsin inhibitor and soya bean trypsin inhibitor with trypsin and a K15V mutant, and a modified inhibitor used as a control, can all readily be observed by ESI-MS.155 There are changes in the charge state distribution of the components on binding which may give some indication of the contact surfaces between the protein and peptide (see Section 4.4).ESI-MS analysis of the binding of peptides to carbonic anhydrase has been used to screen two peptide combinatorial libraries consisting of 289 and 256 compounds for tight binding inhibitors.156 The ability of ESI-MS to look at comparative binding energies simultaneously for a complex mixture of compounds is a powerful technique in the analysis of libraries, and a compound with a binding constant of 1.4#10"8 M"1 was identified using this technique.The non-covalent binding of metal ions and two inhibitors to matrylysin (a matrix-metalloprotein) was studied at a range of pH values using ESI-MS.157 Below pH 2.2 the enzyme adopted a denatured conformation and no binding was observed. As the pH was raised above 4.5, binding of both metal ions and inhibitor was observed, and the stoichiometry appeared to be close to that observed at the optimum pH of 7.0 for the activity of the enzyme. The observed stoichiometry was for one inhibitor molecule, 2 calcium and 2 zinc ions.The intensity of the signal due to non-covalently bound ligand in the gas phase appeared to correlate with the known solution binding behaviour. Non-covalent adducts could be observed between the antibiotics vancomycin, ristocetin A and teicoplanin and two short peptides acting as analogues of the bacterial cell wall.158 However, the observed complexes ranged from simple homodimers of the antibiotics to complex associations of a number of antibiotic and peptide molecules, and relating these to the actual situation was not straightforward. It should be emphasised at this point that great care needs to be taken in assigning observed non-covalent complexes to specific binding, as there is ample evidence that some interactions are non-specific.For example, a non-specific interaction of cytidylic acids with ribonuclease A has been observed if an excess of the compound is added,159 but reliable results have been obtained provided suYcient care is taken to avoid non-specific eVects.160,161 Observations of non-covalent complexes are not limited to small molecule–large molecule interactions.The geneV protein is observed as a dimer by ESI-MS using ammonium acetate solution as the carrier solvent, and the addition of a 16mer oligonucleotide results in a 1:1 protein dimer–oligonucleotide complex.162 Furthermore, ESI-MS could be used to measure stoichiometries and relative binding constants for a range of oligonucleotides to the geneV dimer.A dimer was also observed in the ESI-MS of the oestrogen receptor ligand binding domain.163 The binding of biotin to the complete streptavidin tetramer was be observed by ESI-MS, and the streptavidin tetramer appeared to be particularly stable in the gas phase.164 A similar study used an extended mass range quadrupole analyser to study the binding of biotin (Kd, ca. 10"15 M) and iminobiotin (Kd, ca. 10"7 M) to the streptavidin tetramer.165 With biotin four molecules were observed bound to the tetrameric complex without any appearance of random aggregation. Under the same conditions full loading with iminobiotin was not observed. The thermally induced dissociation of the complex of biotin or iminobiotin with streptavidin in the mass spectrometer caused by applying softer or harsher conditions in the source appeared qualitatively to follow their solution binding behaviours.Some care needs to be taken in setting up the mass spectrometer for studies on tertiary association of proteins, as some observations of multimeric proteins again appear to be non-specific.166 4.4 Protein folding The ability of electrospray mass spectrometry to measure the average mass of a protein, and the apparent relationship between the most intense peaks observed in the charge state distribution and the folded state of the protein, makes ESI-MS a potent tool for the study of conformation, folding and dynamics in protein structures167–171 and cooperativity between the folding of elements of the structure.172 The simplest experiments involve the interpretation of a shift in the maximum intensity of the envelope of charged states towards higher numbers of charges (lower mass to charge ratios) to be due to a larger number of polar residues becoming exposed to the solvent as the protein changes conformation or unfolds.167 An alternative and more sensitive technique that is in many ways complementary to the charge state shift is to use ESI-MS to measure deuterium incorporation or washout from a protein.There appears to be a good correlation between the exchangeable protons and the folding or conformational changes of the protein, for example the number of protons that could be exchanged in tuna cytochrome c was in good agreement with that calculated using a computational method that determined the solvent accessible surface.173 ESI-MS has the advantage over NMR for measuring hydrogen–deuterium exchange in that it is not limited to small proteins that are soluble at mM concentrations.However, care needs to be taken as in some experiments it appears that diVerent protein conformations may have the same charge state distribution, but have diVerent exchange rates, and that the rapid interconversion of alternative conformations can lead to the same exchange rate being observed for alternative conformations showing diVerent charge states.174 The advantage of charge state distribution studies is that they are relatively rapid and simple to perform.There is some debate as to whether the solution (normal biochemical) and gas phase (in ESI-MS) behaviour of proteins is the same, but studies have indicated that the gas phase distribution of ions is in reasonable agreement with the calculated solution behaviour of the proteins.175 A number of observations of the changes in charge state distribution have already been mentioned.147–149,155,167 The addition of a small quantity of organic solvent to an aqueous solution of ubiquitin resulted in N Cl O O O OH OH O P O O– O P O– O O O N N N N NH2 OH OH 9 68 Natural Product Reports, 1998the observation of a bimodal charge–state distribution.176 The high mass to charge (low charge–state) distribution represents the protein in its native fully folded state, whereas the distribution with a higher charge state suggests a more extended conformation.Addition of more than 20% acetonitrile or 40% methanol completely eliminated the higher mass distribution by unfolding all of the protein. The refolding of acid denatured myoglobin could also be followed by this method, indicating that a compact native like structure forms initially without the assistance of the haem, which then associates with the newly formed binding site.177 In studies on proton–deuterium exchange of proteins, the rate of proton or deuterium exchange gives information on the conformational stability of proteins, whereas the total number of exchanged protons gives information on the folding of the protein.The study of the eVect of conformational changes on the extent of deuterium incorporation goes back many years to some of the first studies which were on the solvent dependent denaturing of ubiquitin.178 The same group went on to follow the eVect of reduction of the disulfide bonds of hen egg lysozyme on unfolding using the same techniques.179 A shift in the charge state distribution to a higher mass to charge ratio of both components on the binding of soya bean trypsin inhibitor to trypsin and a trypsin mutant have been used to suggest that less surface is available in the complex.155 The slower rate of exchange of protons in ·-helices was neatly demonstrated using mutagenesis on the 32-residue growth hormone releasing factor;180 introduction of helixpromoting or helix-disrupting residues resulted in slower or faster rates of exchange, respectively, and this correlated well with CD measurements of helical content.Addition of an organic solvent, which induces helix formation, also slowed the rate of exchange. Further studies of helical unfolding have been reported.181 Addition of a tight binding phosphopeptide ligand to a cloned Src homology 2 domain slowed proton incorporation into the protein dramatically.As NMR and X-ray crystallographic data show no significant conformational changes, the authors put the change in exchange rate down to an increase in conformational stability.182 Studies performed on apo-and holo-myoglobin see similar eVects; Johnson and Walsh183 showed that 47% of the amide hydrogens in apomyoglobin, but only 12% in holomyoglobin, exchange in 30 seconds. The authors went on to analyse individual peptides that had been generated by rapid peptic digests of the exchanged protein using tandem mass spectrometry, and were able to identify peptides where the exchange had been slowed highlighting the stabilised and destabilised regions of the protein.A combination of charge– state distribution, isotope exchange and CD analysis at a range of pHs was able to show the conformational instability of apomyoglobin and was able to identify two distinct charge states that probably represent diVerent, rapidly interconverting conformations.184,185 The folding of a 4-helix bundle has also been studied using rapid isotope exchange experiments.186 This technique was also applied to the determination of the changes in stability in Desulfovibrio vulgaris Hildenborough cytochrome c on mutation of a key tyrosine residue to a number of diVerent amino acids,187 which induced changes ranging from a slight increase in stability (Y64F) to significant destabilisation (Y64S).The eVect on the stability and conformation of the protein of mutagenesis of key residues of Rhodobacter capsulatus ferrocytochrome c could also be followed using the same hydrogen–deuterium exchange techniques.188,189 The use of exchange experiments monitored by ESI-MS in conjunction with complementary NMR studies has proved to be a powerful technique for unravelling diVerent pathways of protein folding.Miranker et al.190 have shown that ESI-MS gives the distribution of masses within a population, whereas NMR measures occupancy of individual sites; these two techniques can therefore be used together to provide information on the populations of transient folding intermediates.The rate of deuterium exchange and the higher number of protons protected from exchange for RTEM ‚-lactamase bound to GroEL at 48 )C, compared to the uncomplexed protein is good evidence that there is a significant amount of native structure remaining in the GroEL ‚-lactamase complex at these temperatures. 191 The protection of hydrogen exchange on ligand binding has been observed for the binding of acyl CoA to acyl CoA binding protein.153 In studies comparing the gas phase and solution phase behaviour of the complexes the rate of exchange was used to determine the extent of association and conformation stability of the complex under these conditions. 5 DNA ESI-MS can be used to observe short sections of single stranded DNA, including modified DNA for use as antisense compounds,192,193 duplex DNA194–198 and even quadruplex DNA.199 In its simplest application ESI-MS can be used to confirm the sequence of the DNA and, coupled to HPLC, to analyse more complex mixtures.200 One of the major problems with DNA is its aYnity for sodium ions, which can complicate the spectra to such an extent that it can be diYcult to make any interpretation.On-line microdialysis has been used to clean up DNA samples by removing sodium from the solution as it is introduced into the mass spectrometer, and by using ammonium acetate as the buVer the DNA remains in the duplex form.201 The addition of modifiers such as acetic or formic acid with imidazole or piperidine can also simplify assignment by shifting the signals to a higher mass to charge ratio, and can also suppress sodium ion adduct formation. 202,203 A micro electrospray source has been developed that has proved particularly useful for the analysis of duplex DNA and duplexes consisting of one strand of DNA and one of peptide nucleic acid.204 DNA duplex could be observed between the self complementary 12-mer (5*-dCGCAAATTT GCG-3*), and the non-covalent complex of this duplex DNA with the minor groove binding drugs distamycin, pentamidine and Hoechst 33258 could also be observed and the stoichiometry of binding was determined.205 The sensitivity of ESI-MS in detecting DNA modification was shown by the study of the formation of endogenous adducts with malondialdehyde in the liver.206 6 Summary The wide range of experiments using ESI-MS described in this review, most of which can be performed with the relatively simple quadrupole analyser, pay tribute not only to the hard work of scientists throughout the world, but also the flexibility, utility and simplicity of the technique.To say that ESI-MS has become routine in many laboratories is not understating the facts. 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Mass Spectrom., 1995, 30, 1157. 72 Natural Product Reports, 1998

ISSN:0265-0568    

DOI:10.1039/a815059y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Natural sesquiterpenoids Braulio M. Fraga Instituto de Productos Naturales y Agrobiología, CSIC, 38206-La Laguna, Tenerife, Canary Islands, Spain Covering: 1996 Previous review: 1997, 14, 145 1 Farnesane 2 Monocyclofarnesane 3 Bicyclofarnesane 4 Nanaimoane, acanthodorane and isoacanthodorane 5 Bisabolane 6 ·-Santalane and ‚-santalane 7 Trichothecane, cuparane, herbertane, laurane and gymnomitrane 8 Chamigrane 9 Carotane, acorane, cedrane, duprezianane, italicane and anisatin group 10 Cadinane, cubebane, copaane, oploponane, copabornane and picrotoxane 11 Himachalane, longipinane and longibornane 12 Caryophyllane, botrydiane and quadrane 13 Humulane, alliacane, pentalenane, hirsutane, lactarane, marasmane, precapnellane, capnellane, illudane and africanane 14 Germacrane 15 Elemane 16 Eudesmane, valerane, oppositane and manicolane 17 Vetispirane and nudenane 18 Eremophilane, chiloscyphane and bakkane 19 Guaiane, xanthane, pseudoguaiane, rotundane and patchoulane 20 Aromadendrane and bicyclogermacrane 21 Pinguisane 22 Miscellaneous sesquiterpenoids 23 References 1 Farnesane The structure of the hydrocarbon caparratriene has been determined as 1.This compound has been isolated from Ocotea caparrapi and possesses significant growth inhibitory activity against leukemia cells.1 The sesquiterpenes 5-hydroxy- 12-oxo-farnesol 2 and 5-acetoxy-12-hydroxyfarnesol 3 have been found in an extract of the aerial parts of Inula salsoloides. 2 A novel furanosesquiterpene 4 has been obtained from the soft coral Lobophytum catalai, which has been collected in the Andaman and Nicobar Islands.3 The structures of ‘primitive’ membranes have been shown to be formed by single-chain polyprenyl phosphates.4 Feeding experiments with labelled acetates have been used to show that the dorid nudibranches, such as Archidoris odhneri and Archidoris montereyensis, can biosynthesize terpenoid acid glycerides de novo.5 The biotransformation of the acyclic terpenoid (2E,6E)-farnesol by the plant pathogenic fungus Glomerella cingulata has been studied.6 The absolute configuration of (3S)-nerolidol, the precursor of the acyclic homoterpene 4,8-dimethylnona-1,3,7- triene, has been determined by GC–MS analysis of the homoterpene produced after feeding to various plants a mixture of equal amounts of labelled (3S)- and (3R)-nerolidol derivatives. 7 Two new farnesyl protein transferase inhibitors have been isolated from a Streptomyces strain.8 The stereochemical course of human protein-farnesyl transferase has been shown to be similar to that of FPP synthase.9,10 New farnesyl phosphonate derivatives of phenylalanine have been prepared and used as inhibitors of farnesyl protein transferase.11 The full paper on the chemistry and biology of cylindrols, novel inhibitors of Ras farnesyl protein transferase, has appeared.12 Syntheses of oxocrinol,13 ·-farnesene hydroperoxides14 and furoic acid15 have been reported, whilst chiral syntheses of (+)-ipomeamarone16 and of several hydroxylated farnesane sesquiterpenes17 have been described.The synthesis and biological evaluation of several farnesyldiphosphate derivatives have been carried out.18,19,20 The electron-transfer photoreaction between (E,E)-farnesol and 1,4-dicyanobenzene has been studied.21 (E)-Nerolidol has been employed as starting material in the preparation of racemic ambrox,22 whilst farnesyl acetate has been used in a chiral synthesis of the same compound, in which a lipase catalysed resolution of racemic drimane-8,11-diol is involved.23 2 Monocyclofarnesane Two novel cyclonerolidol derivatives 5 and 6 have been isolated from the liverwort Porella subobtusa.24 The new sesquiterpenes 7–9 have been found in an extract of the aerial parts of Artemisia chamaemelifolia.25 The new norisoprenoids 10–13 have been obtained from the leaves of Apollonias barbujana.26,27 Another compound of this latter type 14 has been isolated from Viburnum dilatatum.28 The structure of 10-normegastigmane glycoric acid has been determined as 15.This compound has been found in Glycosmis arborea.29,30 Several megastigmane glycosides have been isolated from Alangium premnifolium,31 Bunias orientalis,32 Cydonia vulgaris, 33 Pistia stratiotes34 and Vitis vinifera.35 The absolute configurations of rehmaionosides A–C, three ionone glycosides from Rehmannia glutinosa, have been determined by chemical and physicochemical methods.36 O O O O O O O O O O O R1 OH OR2 O O O H Ang = ; Epang = ; Meacr = Epmeacr = ; Mebu = ; Vali = Sen = ; (CO)Pri = ; Tig = 1 4 2 R1 = O; R2 = H 3 R1 = OH,H; R2 = Ac Fraga: Natural sesquiterpenoids 73The structure of an antibacterial sesquiterpene, previously obtained from Premna oligotricha, has been revised to 16 on the basis of its chemical synthesis.37 The total synthesis and the absolute configuration of riccardiphenols A and B have been reported.38 Syntheses of theaspirone and vitispirane have been devised.39 Two independent syntheses of the potent antiulcerogenic compound (+)-cassiol40,41 and its enantiomer (")- cassiol42 have been accomplished this year.The first enantioselective syntheses of (")-pallescensone and (")-ancistrodial have been reported.43 A rapid synthesis of an intermediate 17 for the preparation of monocyclofarnesyl derivatives has been described.44 The natural occurrence of abscisic acid in Portuguese heather honey has been determined by HPLC analysis.45 A conformational analysis of ABA analogues, produced by Cercospora cruenta, has been carried out.46 The role of the hydroxy group in the activity of abscisic acid has been evaluated using derivatives, with a methyl ether at C-1, in the bioassays.47 Optically active forms of oxygenated analogues of ABA have been tested to evaluate its biological activity.48 3 Bicyclofarnesane The isolation of isodrimenediol 18, a possible intermediate in the biosynthesis of drimane sesquiterpenes from Polyporus arcularius, has been described.49 The two novel endothelin type B receptor antagonists RES-1149-1 19, RES-1149-2 20,50 6-epi-albrassitrol 21 and 12-hydroxy-6-epi-albrassitrol 2251 have been obtained from Aspergillus species.Compound 20 has also been isolated together with 23 from another species of this genus, Aspergillus ustus.52 A revision of the absolute configuration of the drimane sesquiterpene 24 has been reported.This compound had been obtained from Aspergillus oryzae.53 The new sesquiterpene 2·-hydroxyisodrimeninol 25 has been found in cultures of a fungus of Pestalotiopsis genus, which is associated with species of Taxus.54 The epicuticular wax of the fern Nephrolepis biserrata contains the three drimane sesquiterpenes 26–28.55 A compound Sch-65676 29, which shows inhibitory activity against the cytomegalovirus protease, has been obtained from the fermentation broth of a fungal culture,56 whilst the substance BE-40644 30, a new inhibitor of the human thioredoxin system, has been found in cultures of an Actinoplanes species.57 The genus Stachybotrys is also a good source of novel endothelin receptor antagonists, such as the lactam 31, stachybocin A 32 and other analogues. 58,59,60 Another fungus Memnoniella echinata contains the new IMPase inhibitors ATCC 20998A 33 and ATCC 20998C.61 The levels of the sesquiterpenes polygodial and 9-deoxymuzigadial in the foliage of several New Zealand populations of Pseudowintera colorata have been determined using HPLC and NMR methods.62 Known drimane, bisabolane and pinguisane sesquiterpenes have been isolated from a cell suspension culture of the liverwort Porella vernicosa.63 The brown alga Dictyopteris undulata contains a new sesquiterpene-substituted benzoquinone with antifeedant properties, which has been named cyclozonarone 34.64 The novel sesquiterpenes deoxyspongiaquinone 35, (E)- chlorodeoxyspongiaquinone 36, spongiaquinol 37 and (E)- chlorodeoxyspongiaquinol 38 have been isolated from a marine sponge of the Euryspongia genus, collected at the Great Australian Bight.65 The five new drimane derivatives 39–43 have been obtained from the sponge Dysidea fusca,66 whilst the two sesquiterpenes 44 and 45 have been found in Dysidea fragilis, collected in the lagoon of Venice.67 The absolute OMe OH OH AcO CO2H OH OH OH O O HO OH HO OH AcO HO HO OH OH OH OH OH HO OH OH HO 7 8 14 5 6 7 D7(8) 8 D7(14) 9 10 11 12 13 14 15 O O C6H5(CO)O OH OH 16 17 O O HO CHO O O HO HO OH OH HO OH OH CHO O O O R1 R2 OH 18 19 20 24 25 21 R1 = b-OH,H; R2 = H 22 R1 = b-OH,H; R2 = OH 23 R1 = O; R2 = H 74 Natural Product Reports, 1998stereochemistry of puupehenone and related metabolites has been determined.68 A cytotoxic red dimer of this sesquiterpene, dipuupehedione 46, has been isolated from a New Caledonian marine sponge of the genus Hyrtios.69 A general strategy for the synthesis of drimane sesquiterpenes, exemplified by the preparation of siccanin, has been developed.70 A racemic synthesis of cinnamolide and methylenolactocin has been described,71 whilst the enantioselective synthesis of (+)-avarol and (+)-avarone,72,73 and that of their enantiomers,73 have been reported. A synthesis of various model compounds for the central tricyclic ring system of popolophuanone E has been described.74 4 Nanaimoane, acanthodorane and isoacanthodorane The biosynthesis of nanaimoal, acanthodoral and isoacanthodoral in the dorid nudibranch Acanthodoris nanaimoensis has been investigated.75 Studies of the synthesis of these sesquiterpenes have also been reported.76 5 Bisabolane Nidulal 47 is a new bisabolane sesquiterpene with biological activity, which has been found in an extract of the basidiomycete Nidula candida.77 Four new sesquiterpene polyol esters 48–51 have been isolated from Cremanthodium ellisi.78 Seven new sesquiterpenes 52–58 have been obtained from the aerial O O O O O O O CHO CHO HO OH HO HO HN O N N O OH O O O R1 AcO HO HO HO OH OH AcO R2 CHO CHO HO HO CO2H 29 30 31 32 33 26 R1 = R2 = H 27 R1 = OAc; R2 = H 28 R1 = H; R2 = OAc O O OMe O O OH HO OMe MeO OMe O O MeO OMe OH HO Cl Cl 34 35 36 37 38 O O O O O O O OAc OAc O O O O O O O O OH HO CO2H CHO HO HO OH 39 40 41 42 43 44 45 46 OHC O O O O OH R3 R4O H OH OAng R2O OH OR1 O(CO)Pri 47 48 R1 = R3 = R4 = H; R2 = (CO)Pri 49 R1 = Ac; R2 = R3 = H; R4 = (CO)Pri 50 R1 = Ac; R2 = (CO)Pri; R3 = R4 = H 51 R1 = R4 = H; R2 = (CO)Pri; R3 = OH Fraga: Natural sesquiterpenoids 75parts of Achillea cretica.79 The bisabolane derivative 59 has been found in an extract of the foliage of Fitzroya cupressoides, 80 whilst the bisabolane endoperoxides 60 and 61 have been isolated from the aerial parts of Eupatorium rufescens.These metabolites showed schizonticidal activity against Plasmodium falciparum.81 The gum exudates of Commiphora kua contain the new sesquiterpene 62.82 The isolation and structural determination of the bisabolane derivative 63 has been reported.This compound has been obtained from the heartwood of Juniperus formosana.83 The known antifouling sesquiterpene 3-isocyanotheotheonellin 64 has been found in extracts of four nudibranches of the family Phyllidiidae.84 The synthesis of turmeronol B has been accomplished.85 The ultraviolet irradiation of isoperezone acetate has been studied.86 6 ·-Santalane and ‚-santalane Two new sesquiterpenes ·-santaldiol 65 and ‚-santaldiol 66 have been obtained from the heartwood of Santalum insulare.87 A new enantiospecific synthesis of ·-santalane derivatives has been described.88 7 Trichothecane, cuparane, herbertane, laurane and gymnomitrane Trichothecinols A, B and C 67–69 are potent antitumor promoting sesquiterpenoids, which have been isolated from the fungus Trichothecium roseum.89 A study of tricoverroid stereoisomers, produced by another fungus Myrothecium verrucaria, has been carried out.90 The biosynthesis of the trichothecene 3-acetyldeoxynivalenol has been investigated.91 Enzymatic formation of multiple sesquiterpene skeletons, by genetic alteration of the trichodiene synthase active site, has been reported.92 15-Hydroxytrichodiene 70, produced by hydroxylation of trichodiene, has been obtained in transformed Nicotiana tabaccum cell suspension cultures, expressing a trichodiene synthase gene from Fusarium sporotrichioides.93 The results of these two last studies demonstrate that the alteration or introduction of a sesquiterpene gene can result in the formation of new sesquiterpene metabolites. The sesquiterpene hydrocarbons ‚-bazzanene, ·-barbatene and ‚-barbatene, which are characteristic constituents of liverworts, have been identified for the first time as components of a higher plant, Meum athamanticum.94 An enantioselective synthesis of (")-cuparene and (")-‰- cuparenol has been described.95 Two new herbertane sesquiterpenes 71 and 72 have been found in an extract of the liverwort Herbertus aduncus, whilst the known sesquiterpenes herbertene and ·-herbertenol have been obtained from Herbertus borealis.96 Total syntheses of herbertenediol,97 herbertenolide, ·-herbertenol, ‚-herbertenol,98 tochuinyl acetate and dihydrotochuinyl acetate have been reported.99 An enantiocontrolled100 and a racemic101 synthesis of filiformin have been described.A new gymnomitrane 73 and a novel norgymnomitrane derivative 74 have been isolated from the liverwort Jungermannia truncata, collected in Malaysia.102 CO2H CO2H O OH O O OH O O O OH O OH OH H HO H HO H AcO H HO H HO H AcO HO HO R 52 53 54 55 56 57 R = a-OH 58 R = b-OH OH H O O O O H H H H H H OH OH O OH H CN 59 60 61 62 63 64 OH OH OH OH 65 66 O R1 O O O OH R2 70 67 R1 = O; R2 = OH 68 R1 = a-OH,H; R2 = H 69 R1 = a-OH,H; R2 = OH OH OH R OH O H H 71 R = CHO 72 R = CO2Me 73 74 76 Natural Product Reports, 19988 Chamigrane A new approach to the synthesis of racemic ·-chamigrene has been reported.103 9 Carotane, acorane, cedrane, duprezianane, italicane and anisatin group The structure and stereochemistry of fersorin 75 and fersoridin 76 have been reported.These new carotane sesquiterpenes were obtained from the roots of giant fennel, a Ferula species.104 Another two compounds of this type, isoferuone and 2,3-epoxyakichenol, have been isolated from Ferula jaeschkeana.105 The chromatography of a methanolic extract of Acorus calamus gave six novel acorane derivatives 77–82, which were shown to inhibit the germination of lettuce seeds.106 Another acorane sesquiterpene 15-hydroxyacora-4(14),8-diene 83 has been obtained from the heartwood of Juniperus chinensis.107 This plant also contains a cedrane sesquiterpene, cedr-3-en- 15-ol 84 and a duprezianane derivative, junipercedrol 85.The latter possesses a new skeleton.108 Another species of this genus, Juniperus thurifera, produces the sesquiterpenes ·-duprezianene 86, ‚-duprezianene 87 and sesquithuriferol 88. This last compound was transformed by solvolytic rearrangement into 86 and 87, confirming the structure of the duprezianane skeleton by chemical methods.109 The biotransformation of cedrol and related compounds by Mucor plumbeus has been investigated.110 Racemic syntheses of ·-biotol, ‚-biotol111 and ƒ2-cedrene112 have been described.The oxidation of the hydrocarbon italicene has been studied.113 A novel sesquiterpene lactone, 3-benzoylpseudoanisatin 89, has been found in the pericarps of Illicium dunnianum.114 Another species of this genus, Illicium verum, contains the neurotropic sesquiterpenoids veranisatin A 90, veranisatin B and veranisatin C.115 10 Cadinane, cubebane, copaane, oplopanane, copabornane and picrotoxane The structure of 10-isocyano-4-cadinene has been determined as 91.This sesquiterpene has been found in extracts of nudibranches of the family Phyllidiidae and showed antifouling activity against larvae of Balanus amphitrite.84 Three new cadinane sesquiterpenes 92–94 have been identified as components of species of the genus Baccharis.116 Other compounds of this type, ·-hinokienol 95 and ‚-hinokienol 96, and the cubebane derivative 97 have been isolated from the leaf oil of Chamaecyparis obtusa.In this study, the absolute configurations of 95 and 96 were established by synthesis from (")- menthone.117 The sesquiterpene (+)-10·-hydroxy-4-muurolen- 3-one 98, a new inhibitor of leukotriene biosynthesis, has 75 R = H 76 R = OAng HO R O O OH R 81 R = a-H 82 R = b-OH 83 77 R = H 78 R = Ac O OR O O R 79 R = H 80 R = OH OH H H OH H OH H 8 9 15 84 86 D8(9) 87 D8(15) 85 88 O O O O OMe C6H5(CO)O O O O HO OH OH HO HO 89 90 H H H HO H OH H H OH H H OH NC O 91 92 93 94 HO H R O H OH 97 98 95 R = a-OH 96 R = b-OH Fraga: Natural sesquiterpenoids 77been obtained from fermentations of a Favolaschia species.118 Other inhibitors of this type have been found in Leutinellus cochleatus.119 The cadinane derivatives 99 and 100–102 have been isolated from Artemisia chamaemelifolia25 and Fitzroya cupressoides,80 respectively.The heartwood of Juniperus formosana contains (")-15-hydroxycalamenene 103.83 The sesquiterpene o-naphthoquinones 104–107 have been isolated from the root bark of Ulmus davidiana.Their antioxidative activities were determined by a thiobarbituric acid method using rat liver microsomes.120,121 Other antioxidative sesquiterpenes, 7-hydroxy-3,4-dihydrocadalin and 7-hydroxycadalin, have been found in the dried flowers of Heterotheca inuloides. These compounds also showed cytotoxic activity.122 The novel furanosesquiterpene 108 has been obtained from Bursera leptophloeos.123 Several sesquiterpene glycosides, which have been named alangicadinosides A–E, have been isolated from Alangium premnifolium.124 The absolute stereostructure of (1S,4S)-cis-5-hydroxycalamenene 109 has been revised to (1R,4R)-cis-5-hydroxycalamenene utilizing X-ray analysis.This compound has now been isolated from the liverwort Bazzania trilobata.125 The complete 1H and 13C NMR spectra of ‰-cadinene have been assigned. This sesquiterpene hydrocarbon has been obtained from a Juniperus species.126 Purifi- cation of (+)-‰-cadinene synthase from bacteria-inoculated Gossypium hirsutum has been reported,127 whilst the cloning and heterologous expression of a second (+)-‰-cadinene synthase from Gossypium arboreum have been described.128 Syntheses of oxo-T-cadinol,129 halipanicine,130 hibiscoquin one131 C and 7-demethyl-2-methoxy-calamenene132 have been accomplished. An asymmetric synthesis of (+)-apogossypol hexamethyl ether has been devised.133 An enantiomerically pure form of an intermediate in the synthesis of (+)-heptelidic acid has been prepared.134 A review on the structure, biosynthesis and functions of artemisinin (qinghaosu) has appeared.135 A new artemisinic acid analogue has been obtained from the mature stems of Tithonia diversifolia.136 The assignment of the 1H NMR signals of artemisinic acid has been revised,137 whilst the 13C NMR spectra of ·- and ‚-dihydroartemisinin have been assigned.138 The cytotoxicity of several artemisinin derivatives has been evaluated.139 The production of methyl 3-oxoartemisinate by biotransformation of methyl artemisinate with suspension cell cultures of Mentha piperita has been studied.140 The isolation of clones of Artemisia annua, containing high amounts of artemisinin141 and artemisinic acid142 have been reported.An immunoquantitative analysis of artemisinin using polyclonal antibodies has been developed.143 Ferrous ion induced the cleavage of the peroxy bond in artemisinin and its derivatives.DNA damage due to this process has been observed and may be responsible for the antimalarial activity of these substances. 144 On the other hand, the rearrangements of artemisinin in the presence of heme and non-heme iron(II) and iron(III) have been investigated.145,146 The biotransformation of the semisynthetic sesquiterpene artemether, using Cunninghamella elegans and Streptomyces lavendulae, has been studied.147 Novel asymmetric total syntheses of (+)-artemisinin,148 (")- artemisinin D and (")-arteannuin D149 have been described. A radiolabelled synthesis of 14C-artemisinin has been reported.150 The preparation of a new artemisinin dimer has been achieved.151 Artemisinic acid has been converted into (")-fabianane in seven steps.152 An eYcient total synthesis of (")-10-desmethylarteannuin B has been described.153 The novel sesquiterpenes 4-hydroxycopa-2-ene 110 and 2·-hydroxycopa-3-ene 111 have been obtained from a petrol extract of Entandrophragma cylindricum.154 Two new bioactive oplopanane derivatives tussilagonone 112 and neotussilagolactone 113 have been isolated from Tussilago farfara.155 The stereoselective synthesis of copaborneol by an intramolecular double Michael reaction has been reported.156 Picrotoximaesin 114 is a new sesquiterpene, which has been isolated from the berries of Maesobotrya floribunda.157 11 Himachalane, longipinane and longibornane A homosesquiterpene 3-methyl-·-himachalane 115 has been characterized as the main sex pheromone of Lutzomya H H HO O OH OH H R 99 102 103 100 R = a-OH 101 R = b-OH O O O OR2 R1 OH O O 107 104 R1 = CH2OH; R2 = Me 105 R1 = Me; R2 = CO2Me 106 R1 = Me; R2 = CH(OMe)2 O O OH 109 108 O O MeCH O MeCH HO OH H OH H OMebu H OMebu H H O O HO 110 111 112 113 114 78 Natural Product Reports, 1998longipalpis, a diptera from Brazil.158 The himachalane derivative 116 has been isolated from the foliage of Fitzroya cupressoides. 80 The structure of the sesquiterpene 117, which had been obtained from Cedrus atlantica, has been resolved by X-ray analysis.159 The leaves of Artemisia argyi (Chinese moxa) contain the novel longipinane derivative moxartenone 118.160 Other sesquiterpenes with this carbon framework ·-longipin-2-en-3-one 119 and 12-hydroxy-·-longipinene 120 have been isolated from Achillea millefolium161 and Juniperus chinensis,107,108 respectively.The acid rearrangement of longipinane into arteaganane has been studied,162 whilst the photochemical rearrangement of a longipinane derivative into a vulgarone derivative and a compound with a new tricyclic skeleton, has been investigated.163 The structure of isoculmorin has been determined as 121 by X-ray analysis.This compound has been obtained from the marine fungus Kallichroma tethys.164 A stereoselective synthesis of longiborneol has been reported.156 12 Caryophyllane, botrydiane and quadrane The sesquiterpenes pestalotiopsin A 122 and pestalotiopsin B 123 have been isolated from a Pestalotiopsis species, a fungus associated with the bark and leaves of Taxus brevifolia.165 A new trypanocidal sesquiterpene, lychnophoic acid 124, has been found in a species of the genus Lychnophora.166 The rearrangements of caryophyllene oxide in acid medium167 and in tetracyanoethylene168 have been studied.The gastric cytoprotection of the antiinflammatory sesquiterpene ‚- caryophyllene in rats has been investigated.169 (")-Clovane-2·,9‚-diol 125 is one of the components obtained from the aerial parts of Baeckea frutescens.170 Five novel bioactive sesquiterpenes, botryenalol 126, botryendial 127, methyl acetyl botryenaloate 128, 10-epi-dihydrobotrydial 129 and 10-dehydroxydihydrobotrydialone 130, have been isolated from cultures of the fungus Botrytis cinerea.171 Another two sesquiterpenes, previously isolated from this fungus, botrydial and dihydrobotrydial, appear to be responsible for the phytotoxic activity of this microorganism.172 The presilphiperfolane sesquiterpene 131, compound 132, formed by oxidative cleavage, and four silphiperfolane acids 133–136 have been isolated from Artemisia chamaemelifolia.25 A formal asymmetric total synthesis of (")-isocomene has been achieved,173 whilst a racemic synthesis of presilphiperfolan- 9-ol has been carried out.174 An extract of the gorgonian Subergorgia suberosa contains a novel cytotoxic sesquiterpene suberosenone 137.This is the first quadrane derivative found in a marine species.175 The molecular structure of terrecyclodiol 138, a derivative of the antifungal metabolite terrecyclic acid, has been determined by X-ray analysis.176 13 Humulane, alliacane, pentalenane, hirsutane, lactarane, marasmane, precapnellane, capnellane, illudane and africanane A highly functionalized humulane derivative 139 has been isolated from an endophytic fungus of Taxus brevifolia.177 The O H H O O H H H HO OAc 115 116 117 118 O H H H H H H OH OH OH 119 120 121 O CO2H H OH OMe AcO OH AcO HO OMe HO H 122 123 124 O O CHO R OAc H OH OH OAc H OAc H OH H HO OH O 125 129 130 126 R = CH2OH 127 R = CHO 128 R = CO2Me H H H OH H HO2C HO2C O O R R 131 132 133 R = a-OMe 134 R = b-OMe 135 R = a-Me 136 R = b-Me Fraga: Natural sesquiterpenoids 79total synthesis of two alliacane derivatives of marine origin has been carried out.178 A synthesis of racemic pentalenene has been reported.179 Four hirsutane derivatives 140–143 with antibiotic properties have been obtained from cultures of the fungus Lentinus crinitus.180 Racemic syntheses of ceratopicanol have been described.181,182 The syntheses of the linear and angular triquinane skeletons have been reported.183,184 Subvellerolactone C 144 is a new lactarane sesquiterpene, which has been found in an extract from the fruit bodies of Lactarius subvellereous.185 The trans-fused lactarane sesquiterpene 3-O-ethyl-8-epi-9-epi-furandiol has been synthesized and its structure determined by X-ray analysis.186,187 A reinvestigation of an ethanolic extract of Lactarius vellereus aVorded four new lactones with the marasmane skeleton 145–148.188 Two dialdehydes with this skeleton, merulidial and isovelleral, react stereoselectively with the natural triketide triacetic acid lactone giving pentacyclic pyranone adducts.One of these adducts, that formed with merulidial, was identical with a compound previously isolated from cultures of Merulius tremellosus.189 A concise total synthesis of dactylol and 3·-epi-dactylol has been devised.190 Two asymmetric syntheses of natural (")- ƒ9(12)-capnellene have been described.191,192 The structure of lentinellone has been determined as 149.This protoilludane derivative has been obtained from submerged cultures of Lentinellus cochleatus.193 Two new active metabolites against bacteria and phytopathogenic fungi, illudin C2 150 and illudin C3 151, have been found in a culture filtrate of Coprinus atramentarius.194 The design, synthesis and antitumor activity of bicyclic and isomeric analogues of illudin M have been reported.195 Normal and reverse phase HPLC methods have been established for the isolation of illudin M and illudin S, from extracts of fungi of the genus Omphalotus.196 An illudin S derivative, (hydroxymethyl)acylfulvene, has been shown to have antitumor properties.197 Two independent syntheses of the fern sesquiterpene pterosin Z have been carried out.198,199 An extract of the Colombian liverwort Porella swartziana contains five africanane derivatives, caespitenone 152 and swartzianin A–D 153–156, two secoafricanane sesquiterpenes, secoswartzianin A 157 and secoswartzianin B 158, and one norsecoafricanane norsecoswartzianin 159.200 Another africanane derivative 160 has been isolated from Porella subobtusa.24 The known sesquiterpene ƒ9(15)-africanane has been obtained from the soft coral Sinularia hirta.201 Dermatolactone 161 is a cytotoxic sesquiterpene with a novel carbon skeleton, which has been found in an extract of an Ascomycete belonging to the family Dermateaceae.202 O OH H H HO 137 138 O HO O OH R1 O H H R1 139 140 R1 = H; R2 = O 141 R1 = OH; R2 = O 142 R1 = H; R2 = a-OH,H 143 R1 = OH; R2 = a-OH,H O O H H O O H H O O R1 R1 O O HO OH H H Et O OH 144 145 146 147 R1 = H; R2 = OH 148 R1 = OH; R2 = H R O OH O H H OH OH 149 150 R = a-CH2OH 151 R = b-CH2OH O O O R O O MeO2C O O O HO HO H HO H O 152 155 156 157 158 159 153 R = H2 154 R = O O O O OAc O O H H 160 161 80 Natural Product Reports, 199814 Germacrane The structure of 9-methylgermacrane B has been determined to be a novel homosesquiterpene, which has been obtained from the sex pheromone glands of Lutzomyia longipalpis.203 The minimum energy conformations of two sex pheromones, periplanone A and periplanone B, of the American cockroach, Periplaneta americana, and of eleven structural analogues have been calculated using molecular mechanics methods.204 The hydrocarbon germacrene C is the main constituent of the liverwort Preissia quadrata, collected in Germany.205 Another liverwort Porella swartziana contains the germacrane diketone 162.200 The relative and absolute configuration of (+)- allohedycaryol 163 have been determined by synthesis of its enantiomer.206 This compound had been isolated from Ferula communis.Another species of this genus Ferula leucographa contains the new sesquiterpene leucoferin 164.207 Two novel sesquiterpenes, parvigemone 165 and neolitrane 166, have been found in an extract of the stems of Neolitsea parvigemma.208 The hydrocarbon germacrene A has been proposed to be an intermediate in the biosynthetic conversion of FPP to (")- aristolochene. Thus, when (7R)-6,7-dihydrofarnesyl diphosphate was incubated with aristolochene synthase dihydrogermacrene was obtained.209 The biotransformation of allylically activated (E,E)-cyclodeca-1,6-dienols by Cichorium intybus has been studied.210 Many new germacrane lactones have been isolated from natural sources during 1996.The structures 167–190 represent the new germacranolides, whilst the structures 191–202 have been assigned to the heliangolides, 203–205 to the melampolides, and 206 to the cis,cis-germacranolides (Table 1). There are several points to note in relation to these lactones.Known germacranolides have been obtained from Cyrtocymura cincta.214 The metabolites, mainly sesquiterpenes and sesquiterpene lactones, isolated from species of the subtribe Gochnatiinae (tribe Mutisieae, family Compositae) have been reviewed.222 The antiplasmodial activity and the cytotoxic eVects of aqueous extracts and sesquiterpene lactones from Neurolaena lobata have been evaluated.223 The antibacterial activity of several sesquiterpene lactones has been reported.224 Spectral data of chemical modification products of costunolide have been described.225 However, costunolide has been shown to have DNA-damaging properties.226 The structure of 1(10)Z,4Z-hanphyllin has been determined using X-ray analysis. 227 The acid cyclisation of 5-oxo-germacren-6,12-olide has been investigated.228 15 Elemane An investigation of the aerial parts of Onopordon myriacanthum aVorded the elemane derivative 207 and the elemanolide 208.217 The lactone 209 has been found as a component of the aerial parts of Centaurea nicaensis.229 Another compound of this type 210 has been obtained from the roots of Neolitsea hiiranensis,230 whilst the novel lactam clavulinin 211 has been isolated from the soft coral Clavularia inflata.231 16 Eudesmane, valerane, oppositane and manicolane Four new eudesmane sesquiterpenes 212–215 have been obtained from the liverwort Lepidozia vitrea,232 whilst the furan–eusdemane 216 has been isolated from another liverwort, Lophocolea heterophylla.233 The bisesquiterpene biatractylode 217 has been found in an extract of the Chinese medicinal herb Atractylodes marocephala.234 A study of Acorus calamus led to the isolation of the two sesquiterpenes 218 and 219.106 The aerial parts of Artemisia eriopoda235 and Artemisia mongolica236 contain the novel eudesmane derivatives 220–223 and 224, respectively. The structures 225 and 226 have been assigned to two metabolites isolated from Eremophila spectabilis. 237 Four new eudesmane derivatives pterodontic acid 227, 1‚-hydroxypterodontic acid 228, 3‚-hydroxypterodontic acid 229 and 2·,3‚-dihydroxypterodontic acid 230 have been found in the medicinal plant Laggera pterodonta.238 Another study of this plant aVorded other eudesmanes and eudesmanoic glucosides. 239 Eudesm-4(14)-en-3·,11-diol 231 is a new sesquiterpenoid, which has been found in the heartwood of Neocallitropsis pancheri.240 The structure of machikusanol has been determined as 232.This compound has been isolated from the xylem of Persea japonica.241 The new sesquiterpene 233, which possesses antibacterial activity has been obtained from Epaltes mexicana.242 An investigation of a hexane extract of the aerial parts of Pluchea quitoc yielded four novel eudesmane derivatives 234–237.243 Other novel products of this type 238 and 239 have been obtained from Senecio flammeus,244 and Tanacetum 162 163 O O OH 164 165 166 O O O O OAc OH O CO OH O OH O OAng O Table 1 Sources of germacrane lactones Source Ref.Germacranolides Anvillae garcinii 168, 169 211 Artemisia pallens 167 212 Carpesium nepalense 187–190 213 Eirmocephala megaphylla 170 214 Elephantopus mollis 172 215 Inula salsoloides 179–181 2 Mikania mendocina 186 216 Onopordon myriacanthum 171 217 Stevia maimarensis 173–178 218 Stevia vaga 182–185 219 Heliangolides Bajaranoa sp. 191–197 220 Mikania mendocina 198–199 216 Stevia vaga 200–202 219 Melampolides Inula salsoloides 203, 204 2 Stevia vaga 205 219 cis,cis-Germacranolides Acanthospermuim australe 206 221 Fraga: Natural sesquiterpenoids 81O O O O O O O O O O OH O O O O O O O O O OH HO O O OH O HO O O OR1 O O OH O O R O O O O O O R2 OAc OR3 R2 O OMeacr OAc OH O O O O O O O OAc O O O Cl O O O O O R3 O O O O O R2 OTig OH R1 R1 R2 OH HO OR OTig OH OH OR1 HO HO OH O O O O OAc O OH O O O OAc O O O CHO O O O O O (CO)Pri O OAc O O HO OR OH O O O R1 O HO 199 200 205 206 201 R = H 202 R = OAc 203 R = H 204 R = Vali 167 168 Partenolid-9-one 169 170 3-Deacylglaucolide B 171 172 173 R1 = R2 = H; R3 = OTig 174 R1 = H; R2 = OH; R3 = Tig 175 R1 = H; R2 = OH; R3 = 4-OHTig 176 R1 = OH; R2 = OAc; R3 = Tig 177 R1 = Tig; R2 = OH; R3 = H 178 186 197 198 179 R = H 180 R = OH 181 R = OAc 182 R1 = OAc; R2 = OH 183 R1 = OAc; R2 = H 184 R1 = H; R2 = OH 185 R1 = H; R2 = OAc 187 R1 = Sen; R2 = CH2 188 R1 = Ang; R2 = CH2 189 R1 = Tig; R2 = CH2 190 R1 = H; R2 = b-Me,H 191 R1 = H; R2 = 5-AcOTig; R3 = 192 R1 = H; R2 = 5-AcOTig; R3 = a-OH, b-CH2Cl 193 R1 = OH; R2 = Tig; R3 = 194 R1 = OH; R2 = Tig; R3 = a-OH, b-CH2Cl 195 R = Ang 196 R = Tig OAc H H H H CO2Me O O OH O OH OH O CO2Me O O O O N O OH O OH OH OH O O OH O H OH 207 208 209 210 211 3 4 216 217 212 D4(15); R = H 213 D4(15); R = b-OH 214 D3(4); R = H 215 D3(4); R = a-OAc 218 R = H 219 R = OH 15 O O O O O O OH OH OH R Rpraeteritum,245 respectively.A new dinorsesquiterpene guayulone 240 with fungistatic properties has been isolated from the resin of Parthenium argentatum.246 This species also contains five novel sesquiterpenes 241–245.247 The phytotoxic metabolite zingibertriol 246 has been found in the fungus Pyricularia oryzae.248 The structure of 5-O-acetylcuauhtemonyl- 6-O-2*,3*-epoxy-2*-methylbutyrate, isolated from Pluchea carolinensis, has been determined by X-ray analysis.249 This technique has been also used in the structural determination of emmotin-2 247.250 An eYcient and stereoselective synthesis of (+)-·-cyperone has been devised.251 (&)-Dihydrocarvone has been used as starting material in the synthesis of (+)-12-hydroxy-·- cyperone, (+)-12-oxo-·-cyperone and (+)-3-oxo-eudesma- 4,11(13)-dien-12-oic acid,252 whilst santonin has been employed in the preparation of (+)-‚-cyperone, eudesma-3,5- diene,253 furanoeudesma-1,3-diene and tubipofurane.254 A modified synthesis of racemic occidentalol has been reported.255 A practical synthesis of enantiomerically pure (")-geosmin has been achieved.256 Several dihydroagafuran sesquiterpenes have been obtained from members of the Celastraceae family: Celastrus flagellaris, 257 Celastrus hindsii,258 Celastrus orbiculatus259 and Maytenus buchananii.260 New eudesmanolides have been obtained from diVerent species (Table 2), and their structures shown to be 248–261.The cytotoxic and antibacterial activities of the sesquiterpene lactones isolated from Tanacetum praeteritum have been evaluated. 267 The 1H and 13C NMR assignment of the alantolactone moiety of the adduct of this lactone with (Z)-L-Cys-Ala-OMe has been reported.268 A short synthesis of the sesquiterpene lactone 1-oxoeudesma-2,4-dien-11‚,12,6·-olide has been achieved.269 A new (salen)-manganese(III) complex bearing a sesquiterpene salicylaldehyde derivative has been used in the CO2H CO2 H HO R R 227 R = H 228 R = OH 229 R = H 230 R = OH CO2Me OH OH OH OH O OH R O OH OH HO OH OH HO 220 221 224 225 226 222 R = O 223 R = b-OH,H OH O OOH EpangO HO HO 231 D4(15) 232 D4(5) 233 4 5 15 CO2Me O OH O O OH O HO OH OH AcO AngO AngO AngO AcO OH HO OH R 236 237 238 239 234 R = a-OH 235 R = b-OAc O OMe O O R1 MeO R2 OH OH HO CHO OH OH HO 240 247 241 R1 = Me; R2 = H2 242 R1 = H; R2 = H2 243 R1 = CH2OH; R2 = H2 244 R1 = Me; R2 = O 245 R1 = H; R2 = O 246 Table 2 Sources of eudesmanolides Source Eudesmanolides Ref.Artemisia giraldii 248 261 Artemisia herba-alba 249 262 Artemisia lerchiana 250, 251 263 Artemisia pontica 252–254 264 Inula salsoloides 255 2 Onopordon myriacanthum 257 217 Stevia maimarensis 256 218 Sarcandra glabra 261 265 Tanacetum praeteritum 258 245 Wedelia prostrata 259, 260 266 Fraga: Natural sesquiterpenoids 83catalysed epoxidation of unfunctionalized olefins with iodosylbenzene and molecular oxygen–pivalaldehyde as terminal oxidant.270 A HPLC method for the analysis of valerenic acids in extracts of Valeriana oYcinalis has been described.271 Enantiospecific syntheses of (+)-valerane and (")-valeranone have been achieved,272,273 while a racemic synthesis of isovalerenol has been reported.274 The sesquiterpene 262 has been isolated from the marine sponge Acanthella cavernosa.This compound inhibits the metamorphosis of Balanus amphitrite.275 A racemic synthesis of axamide and axisonitrile has been described,276 whilst enantioselective synthesis of (")- homalomelol A and homalomenol B has been reported.277 An enantiospecific construction of the carbon skeleton associated with the antineoplastic sesquiterpene manicol 263 has been accomplished.278 Spectral data of several eudemanolides obtained by epoxidation and cyclization of costunolide have been described.225 17 Vetispirane and nudenane The new sesquiterpene ethers 264 and 265 have been found in the apolar part of Haitian vetiver oil.279 The eVects of agarospirol and jinkoheremol, obtained from agarwood, on the central nervous system in mice have been investigated.280 A racemic synthesis of the spirovetivane phytoalexin (&)- lubiminol has been achieved,281 whilst an enantiospecific synthesis of (")-solavetivone has been carried out.282 The Taiwanese liverwort Mylia nuda contains the sesquiterpene nudenoic acid 266, which possesses a new carbon skeleton named nudenane, probably derived from vetisperane.283 18 Eremophilane, chiloscyphane and bakkane An eremophilane derivative 267 has been found in an extract of Pedicularis striata,284 whilst the new sesquiterpene ethers 268 and 269 have been isolated from vetiver oil.279 The structure of ligulaverin A 270 has been determined by X-ray analysis.This compound, which has been isolated from Ligularia veitchiana, possesses a new carbon framework possibly derived from an intramolecular Diels–Alder reaction of a hydroxymethylacrylate ester of an eremophilane sesquiterpene. 285 Another species of this genus, Ligularia virgaurea, contains the novel sesquiterpenes virgaurin A 271, furanomexican- 9-en-8-one and 9‚,10‚-epoxyfuranomexicanan-8- one.286 The phytotoxicity and the electrochemical properties of the herbicide cacalol and its derivatives, isolated from the roots of Psacalium decompositum, have been investigated.287,288 Four new dinoreremophilane derivatives with a rare skeleton eremopetasinorone A 272, eremopetasinorone B 273, eremopetasinol 274 and epoxyeremopetasinorol 275, and four novel eremophilenolides eremosulfoxinolide A 276, eremosulfoxinolide B 277, 3‚,8·-dihydroxy-6‚-methoxyeremophil- 7(11)-en-12,8-olide 278 and 2‚-hydroxyeremophil-7(11)-en- 12,8‚-olide 279 have been obtained from the rhizomes of O O O O O O O O O O O O O O O O O R R2 O O OH O O O O O(CO)Pri OH OH OR1 OH OH OH OH AcO OH OH O OR OH OH OH OR HO H H CHO R 248 249 252 R = various 257 258 261 250 R = CH2 251 R = a-OH,Me 253 R = b-OH 254 R = a-OVali 255 R1 = H; R2 = a-Me,H 256 R1 = various; R2 = CH2 259 R = Ang 260 R = Tig O O SCN HO O OH 262 263 O R CO2H 264 R = CH2 265 R = OMe,H 266 O O OH O O O O O OH O OH OH H O OH 267 268 269 270 271 84 Natural Product Reports, 1998Petasites japonicus.289 Another four lactones 280–283 and two new secoeremophilane derivatives, 284 and 285, were also isolated from this species.290,291 The roots of Roldana sessilifolia292 contain three new eremophilanolides 286–288.Another two compounds of this type have been obtained from Ligularia intermedia.293 The eremophilane derivative 289 has been found in Senecio hualtaranensis, whilst a closely related species, Senecio fabrisii, does not contain eremophilane derivatives.294 The chiloscyphane derivative 290 has been isolated from the liverwort Jungermannia vulcanicola.295 A highly stereoselective synthesis of an A-ring functionalized bakkane has been achieved.296 The synthesis of spirolactones related to the bakkenolides has been accomplished.297 19 Guaiane, xanthane, pseudoguaiane, rotundane and patchoulane Guaiswartzianin A 291 and guaiswartzianin B 292 are two guaiane sesquiterpenes, which have been isolated from Porella swartziana.200 The essential oil from the heartwood of Thuja occidentalis298 contains the two guaiane derivatives 293 and 294. A 1,5,11-trihydroxyguaiane has been isolated from Caryodaphnosis tonkinensis,299 whilst the compounds 295–297 have been obtained from Viburnum awabuki.300 Other guaiane derivatives valeracetate 298 and pancherione 299 have been obtained from Valeriana oYcinalis301 and Neocallitropsis pancheri,240 respectively. The bis-sesquiterpene assafulvenal 300, which is formed from a guaiane and a patchoulane sesquiterpene, has been isolated from the root bark of Joannesia princeps, and its structure has been determined by X-ray analysis.302 A guaiazulene pigment 301 has been found in the gorgonian Calicogorgia granulosa.303 Known guaiane derivatives have been obtained from Lepechinia urbaniana304 and Rubus rosifolius.305 Another known sesquiterpene with cytotoxic properties, guaianediol 302, has been isolated from the Red Sea soft coral Sinularia gardineri.306 Koike et al.307 have reported the synthesis of natural dictamnol, but later De Groot et al.308 have prepared cis-dictamnol stating that dictamnol has a trans- and not a cis-fused hydroazulene system.Thus, the structure of dictamnol (Nat. Prod. Rep., 1995, 12, 313, structure 273) has been revised to 303.The synthesis of (")- clavukerin A and (")-11-hydroxyguaiene has been achieved, but the spectroscopic data of the latter did not match with O O O O O OAng RO H H HO H R S O O S O O O O HO OMe H O O H HO H OH : : 5 6 272 R = a-Me 273 R = b-Me 274 275 5,6-epoxy 276 R = 277 R = 278 279 O O OAng H O O OR2 H R1 O O OH H OH O OH OAng O O OH OAng O OH O O O O HO HO OH OMe Cl O HO 280 283 284 285 281 R1 = OH; R2 = 282 R1 = R2 = H O O OH O O OH R O O(CO)Et H O O OH OAng OH OH 288 289 290 286 R = H 287 R = OH O O OH H H H HO 291 R = D4(15) 292 R = D3(4) 293 294 OH OH OH O OAc O OH H HOO HOO H H H H H H 295 296 297 298 299 Fraga: Natural sesquiterpenoids 85those reported for the natural compound.309 An asymmetric synthesis of the perhydroazulene (")-isoclavukerin A has been achieved.310 The sources of the new guaianolides 304–320 that have been isolated from plant species during the period of coverage of this review are listed in Table 3.A reinvestigation of the Caribbean sea plume, Pseudopterogorgia americana, aVorded the novel lactones americanolide A 321, americanolide B 323 and americanolide C 322.316 The structures of the guaianolides, canin, tanaparthin ·-peroxide, secotanapartholide A, artecanin, tanaparthin ‚-peroxide and secopartholide B, present in the feverfew, Tanacetum parthenium, have been revised by X-ray analysis and chemical correlations. The activity of some of these compounds as inhibitors of human blood platelet function has been determined, and its relation to migraine prophylaxis by feverfew has been discussed.317 Known guaianolides have been obtained from Artemisia pedemontana.318 The production and characterization of polyclonal antibodies against the bitter sesquiterpene lactones of chicory, Cichorium intybus, has been described.319 The crystal structure of 11‚,13-dihydromicheliolide has been reported.320 The absolute configuration of chlorojanerin has been determined by X-ray analysis.321 The circular dichroism spectra of eight guaianolides, obtained from Centaurea scoparia, have been studied.322 The new xanthanolide glycosides 324, 325 and 326, 327 have been isolated from the flowers of Arnica amplexicaulis323 and the aerial parts of Xanthium spinosum,324 respectively.The structure of the pseudoguaianolide hymenograndin B has been determined as 328. This lactone has been found in Hymenoxys brachyactis.325 A biosystematic study of Argentinian species of Gaillardia has been carried out, using the pseudoguaianolides as chemotaxonomical markers.326 A species of this genus, Gaillardia grandiflora,327 contains the new lactones 329 and 330.The cytotoxicity and the NMR spectral assignments of ergolide and bigelovin have been described.328 The phytotoxicity of parthenin on aquatic weeds has been studied.329 A formal enantiospecific synthesis of (+)-carpesiolin, (+)- confertin, (")-damsin, (")-helenalin, (+)-bigelovin, (+)- mexicanin I and (+)-linifolin A has been reported.330 The lactones rotundopontilides A–F 331 possess the uncommon rotundane skeleton.They have been obtained from the OHC H OH HO H H HO H 300 301 302 303 O O O O O O O O O O O O O O O O O O O O O O O O O O O OH O O OR2 R OAc R OAng H H HOH HO H H OMeacr H H H H OH OH GlcO H H HO HO H H OH OH H H O O O O O H HO H R1 R HO HO HO R OH 304 Moxartenolide 305 R = a-Me 306 R = b-Me 307 Gnaphaloide 308 R1 = a-CH2OH; R2 = H 309 R1 = a-CH2OH; R2 = Ac 310 R1 = b-CH2OH; R2 = H 311 R1 = b-CH2OH; R2 = Ac 312 313 314 315 318 323 321 R = OH 322 R = H 316 R = CH2 317 R = a-Me,H 319 R = CH2 320 R = a-OH,Me, Table 3 Sources of guaianolides Source Guaianolides Ref.Arnica mollis 313 311 Artemisia argyi 304 161 Artemisia lerchiana 314, 315 263 Crepis rhoeadifolia 305, 306 312 Mikania mendocina 316–318 216 Picris radicata 319, 320 313 Ptilostemmon gnaphaloides 307 314 Stevia vaga 308–311 219 Tanacetum argenteum 312 315 86 Natural Product Reports, 1998aerial parts of Artemisia pontica.331 The stereoselective synthesis of (&)-isonorpatchoulenol has been achieved.332 The asymmetric synthesis of vulgarolide and deoxocrispolide has been accomplished.333 20 Aromadendrane and bicyclogermacrane The sesquiterpene 332 has been isolated from the marine sponge Acanthella cavernosa. This compound inhibits the metamorphosis of the barnacle Balanus amphitrite.275 The new aromadendrane derivative isoplagiochilide 333 and the 2,3- secoaromandendrane 334 have been found as a constituent of the liverworts Plagiochila elegans334 and Heteroscyphus coalitus, 335 respectively.Three ent-alloaromadendranes 335–337, two ent-2,3-secoaromadendrane 338 and 339, and one bicyclogermacrane 340 have been obtained from cultured cells of Heteroscyphus planus.336 The first natural occurrence of the (")-ledol has been reported.This metabolite has been obtained from another liverwort, Cephaloziella recurvifolia.337 In a study of the constituents of Dicranolejeunea yoshinagana it has been observed that (")-spathunelol is an artefact formed from (")-ent-bicyclogermacrane, when it is allowed to stand at room temperature or during the extraction of liverworts.338 A total synthesis of (+)-ledol has been described.339 Stereoselective total syntheses of (+)-aromadendrane and (")- alloaromadendrane have been achieved.340 The biomimetic synthesis and absolute configuration of (")-tanzanene have been reported.341 The ozonolysis of (+)-aromadendrane and other terpenoids have been investigated.342 The rearrangements of ledene and aromadendrene in superacidic media have been studied.343 On the other hand, the rearrangement of (+)-ledene epoxide in acid medium has been described.344 21 Pinguisane Two novel pinguisane derivatives 341 and 342 have been obtained from the liverwort Dicranolejeunea yoshinagana.345 Another three new sesquiterpenes of this type 343–345 have been isolated from axenic cultures of Aneura pinguis.346 The structure and biosynthesis of several pinguisane sesquiterpenes have been reported.347 A novel pinguisanoic acid 346 has been found in an extract of the liverwort Porella platyphylla.348 In this work the stereochemistry of ‚-pinguisenediol has been revised to 347.Known pinguisane sesquiterpenes have been O O GlcO R O O R2O O O OR1 OAc H AcO AcO 326 R1 = H; R2 = Glc 327 R1 = Glc; R2 = H 328 324 R = a-Me 325 R = b-Me O O OH H HO RO OAc 329 R = H 330 R = Ang HO OR H O O 331 R = various O HO O O H H NCS H 332 333 334 335 R1 = R2 = H 336 R1 = H; R2 = Ac 337 R1 = R2 = Ac 338 339 340 H R1O AcO H O AcO H AcO H H HO AcO HO O OH R2O O R O O O O O O OR 341 R = CHO 342 R = CH2OAc 343 R = H 344 R = Me 345 O MeO2C OH OH OH 346 347 Fraga: Natural sesquiterpenoids 87obtained from another species of this genus, Porella vernicosa. 63 A racemic synthesis of ‚-pinguisene and pinguisenol has been described.349 22 Miscellaneous sesquiterpenoids The myltaylane derivative 348 has been found in the liverwort Bazzania trilobata.125 Benkarlaol 349 is a new sesquiterpene with a new skeleton, which has been isolated from a Chinese red alga Laurencia karlae.350 Rarisetenolide 350, epoxyrarisetenolide 351 and epirarisetenolide 352 are new sesquiterpene lactones with a new carbon framework, which have been obtained from the marine ciliated morphospecies Euplotes rariseta.These compounds are used by this organism as defensive agents.351 The novel antifouling sesquiterpenes isocyanotrachyopsane 353 and 10-epi-axoisonitride 354 have been found in a nudibranch of the family Phyllidiidae.84 The antiinflammatory and antipyretic activities of the sesquiterpene spartidienedione have been studied.352 Dimeric sesquiterpene thioalkaloids have been shown to have potent immunosuppressive properties.353 An HPLC method for the separation of bilobalide and ginkgolides has been described.354 (")-Furodysinin has been synthesized in enantiomeric355 and racemic356 forms.Syntheses of ent-herbasolide357 (&)-2- pupukeanone358 and tavacpallescencin359 have been reported. 23 References 1 E. 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ISSN:0265-0568    

DOI:10.1039/a815073y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Diterpenoids James R. Hanson School of Chemistry, Physics and Environmental Science, University of Sussex, Brighton, Sussex, UK BN1 9QJ Covering: 1996 Previous review: 1997, 14, 245 1 Introduction 2 Acyclic and related diterpenoids 3 Bicyclic diterpenoids 3.1 Labdanes 3.2 Clerodanes 4 Tricyclic diterpenoids 4.1 Pimaranes 4.2 Abietanes and related diterpenoids 5 Tetracyclic diterpenoids 5.1 Kaurenes 5.2 Beyerenes, atiserenes and aphidicolanes 5.3 Gibberellins 6 Macrocyclic diterpenoids and their cyclization products 6.1 Taxanes 6.2 Cembranes and other cyclization products 7 Miscellaneous diterpenoids 8 References 1 Introduction This review continues the pattern of coverage of its predecessors. 1 The period under review has again been dominated by publications on the taxanes and their biological activity.The number of known gibberellin plant hormones now exceeds 100. Diterpenoids continue to be associated with the biological activity of many plant extracts. 2 Acyclic and related diterpenoids Analysis of the labial gland secretion of the cuckoo-bumblebee (Psithyrus vestalis) males revealed2 that geranylcitronellyl acetate was a major component. These secretions play an important role as territorial marking pheromones. The unusual geometry of the aldehydic dicarboxylic acid 1 obtained from an Eremophila species, was established3 by X-ray crystallography of its semicarbazone. Some glycosides based on 20-hydroxygeranyllinalool have been found4 in Nicotiana tabacum. The epimeric viridiols A and B 2 were isolated5 from the marine red alga Laurencia viridis. 3 Bicyclic diterpenoids 3.1 Labdanes Copaiba oil, which is a mixture of oleoresins extracted from Copaifera species and used for medicinal and cosmetic purposes, has been shown6 to contain the norlabdane 3 and a clerodane 7·-acetoxybacchotricuneatin D 4. Extraction of the leaves of Guarea trichilioides (Meliaceae) gave7 the labdane 5 and the simple clerodane 6, whilst 7 together with ent-13- epimanool have been obtained8 from the liverwort Jungermannia vulcanicola.The seeds of Alpinia zerumbet (Zingiberaceae), which are used in Chinese medicine for stomach problems, have been shown9 to contain zerumin A 8. Investigation into the larvicidal activity of the diterpenes produced by the marine pulmonate Trimusculus reticulatus have shown10 that 6‚-isovaleroxylabda-8,13-dien-7·,15-diol was responsible for the biological activity. Examination of the bark of Juniperus formosana (Cupressaceae), indigenous to Taiwan, revealed11 the presence of the known dicarboxylic acid, ent-oliveric acid, and (13S)-15-hydroxylabd-8(17)-en-19-oic acid 9 together with some 15-esters. The oxidative degradation of the labdane side chain to provide ambergris odorants and starting materials for diterpenoid partial synthesis, has continued to attract attention with HO2C H O CO2H OH OH 1 2 O HO H H HO H H OH OH H HO2C H H CH2OH CHO H H CO2H H HOCH2 OAc H OH 3 4 5 6 7 8 9 O Hanson: Diterpenoids 93work being reported on larixol 10,12,13 including some microbiological studies, and on sclareol14 and copalic acid.15 Some relatives of andrographolide including 11 (wightional) have been obtained16 from Andrographis wightiana (Acanthaceae), a medicinal herb found in the Annamalais Hills of India.Leopersin C 12 was among17 the furanoid and secolabdanoid diterpenes of Leonurus persicus (Labiatae). The insect antifeedant properties of compounds of the grindelic acid series has stimulated interest in the biotransformation of grindelic acid 13.18 Microbiological hydroxylation by Cunninghamella echinulata occurred at the 2‚,3‚ and 6· positions.The X-ray crystal structure of tarapacol 15-acetate 14, from Grindelia tarapacana (Asteraceae), has been published. 19 The resinous wood of Excoecaria agallocha (Euphorbiaceae) has been used as incense (Okinawa-jinko). The constituents of the wood include20,21 ent-13-epimanoyl oxide and its 3·-alcohol (ribenol), and 3-ketone (ribenone), the 11·-hydroxy derivative of ribenone and some ent-8·,13:14,15- diepoxides known as the excoecarins A–C and exemplified by 15.Forskolin activates adenylyl cyclase and although it is widely used as a biochemical probe for characterizing adenylyl cyclase coupled biological responses, therapeutic application is limited due to its poor solubility. A series of carbamate esters have been examined22 in an eVort to overcome these problems.The known labdanes, vulgarol and marrubiin have been found23 in Marrubium anisodon (Labiatae) whilst an X-ray structure has been published24 of leonotinin 16 which was obtained from Leonotis nepetaefolia (Labiatae), a plant used for treating skin infections. Extraction of the Egyptian herb Leucas neufliseana (Labiatae) gave25 3-oxomarrubiin and the prefuran 17. Continuing investigations of diVerent chemotypes of Halimium viscosum have given26 some isofregenedane diterpenoids including the diol 18.This skeleton may be obtained27 by iodine catalysed rearrangement of labdane diterpenes, e.g. 20]19. The unusual structure of saudinolide 21, obtained from Cluytia richardiana, may be derived28 by cleavage of the 6,7-bond of a labdane and recyclization. A norfriedo-labdane 22 has been isolated29 from a Brazilian collection of Vellozia stipitata. 3.2 Clerodanes The triol 23 has been isolated30 from two varieties of Viguiera tucumanensis (Asteraceae) in the course of a chemotaxonomic study of this genus.Soulidiol 24 was obtained31 from Aster souliei, whilst the lactone 25 and a perhydroazulenoid rearrangement product 26 of a clerodane were isolated32 from a Chilean collection of Baccharis linearis (Asteraceae), a medicinal herb known locally as romerillo and used for the treatment of rheumatism. The clerodane 27 has been isolated33 from the toxic bark of the Madagascan tree Croton hovarum (Euphorbiaceae).The glucoside, amarisolide 28, was obtained34 from a Mexican collection of Salvia amarissima (Labiatae). The aglycone was recovered by incubation of the glucoside with the fungus Fusarium monoliforme. The absolute stereochemistry of tanabalin 29, an insect antifeedant from the bitter tasting Brazilian plant Tanacetum balsamita (Compositae), catinga-de-mulata, was established35 by a combination of X-ray crystallography and a modified Mosher’s method.Extraction of Teucrium sandrasicum (Labiatae), a plant used in folk medicine in Turkey in the treatment of diabetes, gave36 the clerodane, sandrasin A 30, whilst teucrasiatin 31 was obtained37 from a Spanish collection of Teucrium asiaticum. Several studies have been reported on Ajuga species, including A. decumbens,38 A. lupulina, which aVorded lupulin A 32,39 and A. parviflora.40 The latter, which occurs in the H H OH OH CHO H H HOCH2 CH2OH OH H O O 10 11 12 O O OH O H O CO2H O O O OAc OH H H O H H H 13 R 14 15 CO O H CO O H O O O OH O O O O OR OAc O O O O O OH OAc H H RO AcO H OH 16 17 18 R = H 19 R = Ac HO2C CO2H H 20 21 22 94 Natural Product Reports, 1998Himalayas, aVorded two groups of clerodanes exemplified by deoxyajugarin 1 33 and 3‚-acetoxyclerodinin C 34.The neoclerodane diterpenoids which have been isolated from Scutellaria (Labiatae) species, are of interest because of their biological activity as insect antifeedants and antifungal agents.Two separate studies41,42 on S. altissima have aVorded the lactol, scutaltisin 36, whilst S. albida gave scutalbin 37.42 The dibenzoate ester 38 was obtained from S. balcalensis,43 whilst the nicotinic acid derivative, scutebarbatine A 39, was isolated44 from S. barbata. Other new compounds that have been isolated include scutecyprol A 35 from S. cypria,45 the 15-ethoxyclerodin 34 and some relatives of jodrellin and scutaltisin from the Nepalese drug S. discolor,46 scutegalin C 40 from S.galericulata47 and scutorientalin A 41 from S. orientalis.48 8-Hydroxysalviarin 42 was obtained49 from Salvia reflexa whilst the unusual 5,10-secoclerodane structure 43 was assigned50 to tonalensin, from S. tonalensis, on the basis of a HO OH O O O O HO2C HO HO OH O O O O O R2 CHO HO CHO HO HO H OH H H H HO H R1 H 25 23 24 26 27 28 R1 = OGlu; R2 = H 29 R 1 = H; R2 = OAc O OAc O O O O O OAc O O O O CH2 OAc O O MeO OAc HO OH H OAc HO OH H H EtCH Me O C 30 31 32 OAc OAc H O OH OAc H O OAc OAc H O O OAc H O O O O O O O O OH O O H R H H H H H H H H H C O 33 36 37 34 R = OEt 35 R = OH CHEt Me O H OR OR O H OTig OAc O O O O O O OCOCHMe2 OAc O O O O O O O O O O H H H OH OH H OH OTig O O CC6H5 O CC5H4N 38 R = 39 R = 40 41 42 43 Hanson: Diterpenoids 95crystallographic study.The eVect of the clerodanes isolated from Salvia species on the feeding behaviour of Spodoptera littoralis has been examined.51 Chemosystematic studies on liverworts have continued to reveal the presence of clerodanes. Heteroscypholide A 44 was obtained52 from cell cultures of Heteroscyphus planus, whilst the 3,4-secohalimane 45 was isolated53 from H.coalitus. Some chemical transformations of the clerodanes have been reported including the cleavage of ring A of eriocephalin and capitatin,54 and the selective reduction55 and the basecatalysed rearrangement56 of teucvidin 46 to the ketone 47. The latter has potent insect antifeedant activity against the larvae of Leucania separata. The X-ray crystal structure of the 18-chloro-4·-hydroxy derivative 48 of 19-acetylteupolin has been reported.57 A number of A/B cis-clerodanes have been examined.The synthesis of the cis-clerodane acid 49 has been reported58 but the product diVered from the natural product suggesting that the structure of the latter requires revision. Nakamurol A 50 has been obtained59 from the Okinawan sponge Agelas nakamurai. The glucoside, rumphioside 51 has been isolated60 from the Philippine medicinal plant Tinospora rumphii (Menispermaceae), whilst the amphiacrolides, e.g. 52, are lactones which were extracted61,62 from Amphiachyris dracunuloides. Extraction of the bark of Casearia tremula (Flacourtiaceae) gave63 the cis-clerodane 53, whilst the relative 54 was obtained64 from Laetia procera (Flacourtiaceae). Both plants came from Costa Rica. 4 Tricyclic diterpenoids 4.1 Pimaranes A phytotoxic pimarane, sphaeropsidin A 55, has been isolated65 from the cypress canker fungus Sphaeropsis sapinea var.cupressi. The structure is the same as that previously assigned to an Aspergillus metabolite. The zythiostromic acids, e.g. 56, are antifungal metabolites of a Zythiostroma species associated with the aspen tree.66 The plant growth regulatory O O OAc OAc O H MeO2C O C O O O H 44 45 O O O O O H HO2C O O O O H H H H O O O H HO Cl OAc H O OAc 46 47 48 O CO2H OH MeO2C O O OGlu O O O O O C O EtCH Me O O CH2OH H H HO H EtO OH H OR2 AcO OAc HO H AcO OAc OR1 OMe O H 49 50 51 52 53 54 CO O OH H OH HO OH H H H OH CO2H O MeO2C OH H H O OH H H O OH HO AcO AcO 55 56 57 58 96 Natural Product Reports, 1998activity of the vouacapanes of Pterodon polygalaeflorus has been examined.67 Extraction of the Indonesian medicinal plant Caesalpinia major (Fabaceae), known as ‘dekar’ and used as an anthelmintic, has aVorded68 the caesaldekarins, e.g. 57, whilst the cassane derivative 58 was obtained69 from C.bonduc. The betonicosides A–D, e.g. 59, are similar compounds which have been isolated70 from the roots of Stachys oYcinalis (Labiatae). The swartziarboreols are cassane derivatives which have been isolated71 from Swartzia arborescens. The X-ray crystal structure of trinervinol 60 has been published.72 During studies on the partial synthesis of gibberellin analogues, the base-catalysed cyclization of 61 has been shown73 to give the unexpected spiro-diketone 62.The cytotoxic spongian diterpene 63 has been isolated74 from the nudibranch Chromodoris obsoleta. 4.2 Abietanes and related diterpenoids 19-Hydroxy-abieta-8(9),15-diene 64 has been isolated75 from Vellozia flavicans (Velloziaceae) in a continuation of studies on this species. Extraction of the bark of Juniperus formosana var. concolor (Cupressaceae) gave76 totarol and a number of abietanes including dehydrosugiol 65. 6‚-Hydroxyferruginol 66 together with some 6·,7‚-dimeric ethers have been obtained77 from the heartwood of J.formosana. In this work it has been suggested that the 6‚-hydroxyferruginol which had been previously isolated78 from Cryptomeria japonica, may in fact be the 6·-isomer. The ortho-quinone 67, obtained79 from the bark of J. procera, has strong antibacterial action. Evidence has been presented80 indicating that the yellow colour of buddlejone 68, obtained from the roots of Buddleja albiflora (Loganiaceae), arises from the presence of the enol tautomer 69.The widely distributed Japanese cedar, sugi, Cryptomeria japonica (Taxodiaceae), has been the source of many diterpenoids. Recent isolates included81 the ketol 70. The quinonemethide 72 and the phenol 73 have been isolated82 as antimicrobial constituents from Plectranthus elegans (Labiatae). The diketone forskalinone 71, which possessed antimicrobial activity, was isolated83 from the roots of Salvia forskohlei. Euphorbia calyptrata (Euphorbiaceae) is a poisonous shrub growing in the Sahara which produces a series of lactones, the helioscopinolides that have CNS activity.Cell culture lines have been established84 to produce quantities of these lactones including some novel members of the series such as helioscopinolide F 74.85 The rearranged abietane 75, possessing moderate antibacterial activity, has been isolated86 from Plectranthus hereroensis (Labiatae), whilst examination of Caryopteris incana gave87 incanone 76 and Coleus scutellarioides yielded88 scutequinone 77.Some ring A cleavage products, including limbinal 78 and a compound with the rather surprising structure 79, have been isolated89,90 from Salvia limbata. Salvibretol 80 from S. montbretii, represents91 a further unusual modification of the basic abietane skeleton. The roots of S. miltiorrhiza (Tan-shen) have continued92 to yield novel diterpenoids including tanshinketolactone 81. A survey of the tanshinone diterpenoids from the roots of some Salvia species have been reported.93 The Californian white sage, S.apiana, has been used as a medicinal herb. Examination of this species has yielded two unusual O O O O HO CH2OH OH OH OH OH CO2Me MeO2C O O AcO H H H CO2Me O H H HO 59 60 61 62 63 HO OH O OH O O H H H OH OH 64 65 66 67 OH O O OH H H 68 69 O R2 O R1 HO OMe O HO HO OMe O O H H H OH H H H H 70 R1 = H; R2 = a-OH, b-H 71 R1 = OH; R2 = O 72 73 74 Hanson: Diterpenoids 97families of C23 terpenoids, known as the apiananes94 and hassananes95 and exemplified by 82 and 83, respectively.The rearrangement of ring B of abietanes has been observed96 in the structure 84 of taiwaniaquinone D, which was obtained from the leaves of Taiwania cryptomerioides (Taxodiaceae). [4+2] Cycloaddition products of the quinone with ‚-myrcene and with trans-ozic were also isolated. The chemistry of the readily available tricyclic diterpenoids has continued to attract interest. Studies have been reported97 on the photosensitized oxidation of diterpenoids.The partial synthesis of umbrosone 85 from dehydroabietic acid has been reported.98 The conversion of abietic acid into methyl (13S)- 13-hydroxy isoatisiren-18-oate 87 via the unsaturated ketone 86 has been described.99 This also represented a formal synthesis of methyl trachyloban-18-oate. Methyl 11,12-di-Omethyl- 6,7-didehydrocarnosate 88, obtained from carnosol, has been shown100 to undergo an interesting rearrangement when treated with potassium tert-butoxide in dimethyl sulfoxide involving rearrangement of the C-20 carboxy group to C-7 resulting in the formation of 89.This rearrangement facilitated a partial synthesis of the potent benzodiazepine agonist, miltirone 90. NMR methods have been used101 to examine the temperature-dependence of conformational changes involving hydrogen bonding to the carboxamide group in 91. The organometallic chemistry of the ring C aromatic diterpenoids has continued to be explored102 with reports on their ruthenium catalysed alkylation. Dimers of podocarpic acid derivatives have been produced103 by thallium(III) trifluoroacetate oxidation. 5 Tetracyclic diterpenoids 5.1 Kaurenes In further studies on Yugoslavian species of Achillea (Asteraceae), examination of A. clypeolata gave104 3·-acetoxyent- kauran-16,17-epoxide 92. 17-O-‚-D-Glucopyranosyl-16‚- H-ent-kauran-19-oic acid 93 has been isolated105 from Inula britannica (Compositae), a Chinese medicinal plant used in the treatment of inflammation. A series of ent-kaur-16-en-15-ones, O O OH OH OH HO OH O OH O O OH CHO O O OH HO HO O OH H OH H OMe HO AcO OH 75 76 77 78 79 80 O O O O O O O O HO O O CO O CHO O O OH H OMe OH OMe H H 81 82 83 84 O O OH O HO MeO2C H H MeO2C H H 85 86 87 MeO OMe MeO OMe O O H MeO2C 88 89 90 CO2H KOBut OMe CONEt2 OH H MeOCH2 91 98 Natural Product Reports, 1998exemplified by 94, have been found106 in the liverwort Jungermannia truncata.The acid 95 was obtained107 from Adenostemma brasilianum (Compositae). ent-16‚,17- Dihydroxykauran-19-oic acid was identified108 as an anti-HIV principle in Annona squamosa whilst annosquamosin A was shown to be 96. Some further bioactive kaurenes have been detected109 in A. senegalensis. ent-3‚,7·,18-Triacetoxykaur-16- ene (triacetoxyfoliol) has been found110 in Turkish Sideritis huber-morathic. More highly oxygenated kaurenes, such as exsertifolin B 97 and some 7–11 dimers based on the seco ring B ent-kaurene 98, have been isolated111 from the liverwort Jungermannia exsertifolia subsp.cordifolia. Some glycosides closely related to atractyloside have been isolated from the toxic plant Xanthium spinosum (Compositae).112 Examination of Isodon ternifolius aVorded113 the isodoternifolins A 99 and B. The ease with which the ·,‚-unsaturated ketone of the enmein and oridonin series reacts with methanethiol has been noted.114 The 8,9-secokaurene 100 has been identified115 as a cytotoxic constituent of the New Zealand liverwort Lepidolaena taylorii.The partial purification of the enzyme system from Stevia rebaudiana which mediates the conversion of ent-kauren-19-oic acid to steviol has been reported.116 The microbiological hydroxylation of isosteviol at C-7· and C-12‚ by Fusarium verticolloides has been described.117 The formation of ent-11·,16·-epoxykaurane from kauranol by Gibberella fujikuroi has been reported.118 The biotransformation of 3,15- oxygenated kauranes by G.fujikuroi has also been shown119 to lead to the formation of ent-11·,16·-ethers exemplified by the conversion of 101 to 102. The stereospecificity of the microbiological reduction of ·,‚-unsaturated ketones by G. fujikuroi has been examined.120 The 13C NMR spectra of some phyllocladene diterpenoids have been assigned,121 whilst the unusual phyllocladene (13‚- kaurene) stereochemistry has been assigned to 103 which was obtained122 from Plectranthus ambiguus. 5.2 Beyerenes, atiserenes and aphidicolanes The ruthenium-catalysed rearrangement of ent-14- (benzoyloxy)-15,16-epoxybeyerane to compounds of the kaurene series has been explored.123 The atiserene skeleton has been assigned124 to the lactone spiramilactone 104, obtained from Spirea japonica. The production of scopadulcic acid 105 by tissue cultures of Scoparia dulcis (Scrophulariaceae) and its inhibitory eVect on bone resorption as well as its antiviral and antitumour activity have been examined.125,126 5.3 Gibberellins Over 100 gibberellin plant hormones are now known and those reported during 1996 are mentioned here.GA95 106 was detected127 in Prunus cereus (sour cherry) seed and the 19–2 isolactone was also found. GA96 107 was identified128 as a minor antheridiogen in the ferns Lygodium circinnatum and L. flexuosum. GA97 108, GA98 109 and GA99 110 were obtained129 from spinach Spinacia oleracea.Authentic samples were synthesized130 from gibberellic acid. Gibberellins A100– A102 111–113 were isolated131 from Helianthus annuus (sunflower). The structure elucidation and synthesis of these three 13,15‚-dihydroxygibberellins which diVer in the oxidation level of C-20, involved the conversion of gibberellic acid to gibberellin A19 and thence to the new gibberellins. The gibberellins that are present in the Japanese cherry Prunus spachiana, have been identified132 by GC–MS.A major review of gibberellin biosynthesis has appeared133 covering not only O AcO CO2H CH2OGlu H H H 92 93 H H H CO2H H H CHO H H H H OH O O O O O O AcO OH AcO O OH HO OH OH H H CH2OAc OAc OAc H O OH O O OR 94 95 96 97 98 99 100 H H H H O HO HO OH OH 101 102 H H C O OH Me2C CH AcO O OH 103 H H O OH OH O O H OBz HO2C H 104 105 Hanson: Diterpenoids 99the formation of the carbon skeleton but also the relationship between the variously hydroxylated gibberellins found in higher plants and recent studies on the enzymology of the gibberellins.The partial synthesis of GA32 114, one of the rare but biologically potent gibberellins, from gibberellic acid has been reported.134 An interesting aspect of this work was the utilization of the 7-carboxy group to assist the reduction of the C-15 ketone. GA53 115 and its 17-2H2-labelled derivative have also been synthesized135 from gibberellic acid for metabolic studies. An unexpected C-arylation at C-10 has been observed136 in the course of a thionocarbonate radical deoxygenation.Thus treatment of the phenylthionocarbonate 116 with tributyltin hydride and AIBN in benzene, gave the aryl derivative 117. The synthesis of the glucosyl conjugates of [17-2H2]GA34 118 for metabolic studies has been described.137 Further studies have been reported138 on the decomposition of gibberellic acid in aqueous solution. 6 Macrocyclic diterpenoids and their cyclization products 6.1 Taxanes A review of the naturally occurring oxetane group of taxanes has appeared.139 A particularly diYcult step in the isolation of paclitaxel (Taxol>) from Taxus brevifolia is its separation from the closely related cephalomannine.Improved methods have been described140,141 for achieving this. A number of new taxanes have been described. Immunological methods have been used142 in the detection of a new taxoid from Taxus baccata stem bark whilst some new Taxus alkaloids have been obtained143 from the needles.Teixidol 119 is an abeotaxane which was obtained144 from this source whilst 120 was obtained145 as a minor product during the large scale extraction of paclitaxel from T. brevifolia. The production of paclitaxel and cephalomannine in cell cultures of T. brevifolia has been investigated146 using very sensitive HPLC and tandem mass spectrometric methods of separation and detection. Two new abeotaxanes, the taxchinins L 122 and M 123, have been isolated147 from T.chinensis var. mairei. Further investigations148–151 of the Japanese yew T. cuspidata have led to the isolation of a series of taxanes and abeotaxanes known as the taxuspines K–W and exemplified by taxuspine K 124, the seco derivative U 125 and the 2(3–20)-abeotaxane W 126. Examination of the Taiwanese species, T. mairei, has aVorded152 the taxane 127 and the taxumairols, e.g. 128,153 whilst the abeotaxane 121 was obtained154,155 from the Himalayan yew T.wallichiana. The C-14 oxygenated taxane 129 was isolated156 from T. yunnanensis cell culture. The same species has yielded157 an 11,12-epoxide 131 and some 11(15–1)- abeotaxanes158 whilst a 2(3–20)-abeotaxane identical to taxuspine W 126, has also been found159 in the needles of a Taxus x media cultivar. The biotransformation of the taxane 130, the parent alcohol of which was obtained in high yield from cell cultures of T. yunnanensis, using the fungi Cunninghamella elegans and C.echinulata has been described.160 The mechanism of action CO2H O CO2H CO2H R CO H CO2H O CO H HO2C R OH OH HO2C OH H H H H HO OH H OH 106 107 108 R = Me 109 R = CH2OH (19®20-lactone) 110 R = CHO 111 R = Me 112 R = CH2OH (19®20-lactone) 113 R = CHO HO2C O CO H CO2H OH OH OH HO2C H H OH H HO 114 115 H H ArOC S C O O O O O Ar 116 117 O CO CO2H O CO HO GluO 118 119 120 R = H 121 R = Ac AcO OAc H OAc AcO OH HO OAc H OH BzO OH OR O OAc OH 122 R = H 123 R = Ac 124 125 126 RO OAc H OBz HO OH OAc O OAc OAc AcO O AcO H OAc OAc OH HO O HO AcO AcO H CH2OH OAc OAc OH OAc OH OAc OH OAc 100 Natural Product Reports, 1998of taxadiene synthase, a diterpene cyclase which catalyses the initial stages in Taxol biosynthesis from geranylgeranyl pyrophosphate, has been examined,161 Structure–activity studies have shown that the 2*-hydroxy group in the side chain is very important for biological activity.Combined NMR and molecular modelling studies of paclitaxel have revealed two predominant conformations – one in nonpolar and the other in polar solution.Taxol 2*-acetate displays no significant in vitro microtubule polymerization activity. NMR studies have shown162 that the presence of the acetate does not modify the side chain conformation implying that the hydroxy group may be interacting directly with a protein residue in the Taxol–microtubule complex possibly as a hydrogen bond donor. The X-ray crystal structure of the 7-methanesulfonate of paclitaxel has revealed163 a further side chain conformation whilst the ‘hydrophobic collapse’ conformation has been observed164 in the crystal structure of 10-deacetyl-7-epitaxol.The taxane skeleton undergoes a number of rearrangements. The conditions have been explored for the formation of 11(15–1)-abeotaxanes. Toluene-p-sulfonic acid catalysis brings165 about the rearrangement of 132 to 133, whilst 9,10-dioxotaxanes in the presence of trichloroacetic acid also undergo166 a ring-contraction reaction (134 to 135).The taxanes form167 an unusually stable enol 136 on reduction of the unsaturated ketone with zinc and acetic acid. However 11,12-dihydro derivatives are biologically inactive168 and 2-epipaclitaxel also shows a significantly reduced biological activity.169 The synthesis of azetidine taxanes in which the oxetane ring has been replaced by a nitrogen ring, and the synthesis of 7,9-pyrazoline adducts, have been reported.170,171 The partial synthesis of some 7-deoxypaclitaxel analogues has also been described.172 A new method based on using thioesters has been developed173 for introducing C-13 side chain.The synthesis, conformational analysis and biological evaluation of paclitaxel analogues containing heteroaromatic rings on the side chain has been reported.174 These include derivatives which show a biological activity comparable to paclitaxel. 2*-Phosphonoxy methyl ethers have been prepared175 as water soluble paclitaxel pro-drugs. The preparation of phenolic paclitaxel metabolites has been reported.176 6.2 Cembranes and other cyclization products Marine organisms have continued to provide the source of cembrane and related diterpenoids.The cembranolide, sartol A 137 has been found177 as an ichthyotoxic metabolite of a Sarcophyton species. A number of cytotoxic cembranoids such as 138 have been isolated178 from the soft corals Sinularia gibberosa and Sarcophyton trocheliophorum, whilst 3,4- epoxysarcophytonin 139 was obtained179 from an Okinawan Sarcophyton species. Some norcembranoids were obtained180 from the Red Sea soft coral Sinularia gardineri.Various collections of the gorgonian octocoral Briareum asbestinum have yielded novel briareolides such as 140,181 141182 and 142.183 The carbon skeleton of sarcoglane 143 isolated184 from the soft coral Sarcophyton glaucum, may arise via a cembrane 127 128 129 R = H 130 R = Ac 131 OAc AcO OH CH2OBz AcO H OH OAc OAc H OH AcO OAc H OAc O C O MeCH Et RO OR H OAc RO OAc AcO OAc H OH H H OAc O O MeO OBz H OH HO OH OH OH HO OH AcO OBz AcO H OAc OH O HO OH 132 133 HO HO C OH OAc HO O O OBz HO H OAc OH O HO OBz O O OH 134 135 O AcO OBz HO H OAc OTES O HO 136 O O O OH O O OH OMe 137 138 139 Hanson: Diterpenoids 101or a xeniaphyllane.The labiatamides A and B, e.g. 144, are eunicellane diterpenoids which have been obtained185 from a deep water gorgonian Eunicella labiata.Members of the Euphorbiaceae produce a number of groups of toxic and irritant diterpenoids. A new lathyrane 145 has been obtained186 from Euphorbia portulacoides whilst 19-acetoxyingenol was found187 in the latex of E. poisonii. The latter was also the source of a new tigliane 146 possessing a 9,10-methyleneundecanoate ester.188 The aleppicatines A 147 and B were isolated189 from the Turkish shrub E. aleppica. Two unusual bishomoditerpenes, the terracinolides A 148 and B, have been obtained190 from E.terracina. A number of phorbol analogues which bind to protein kinase C have been prepared.191 7 Miscellaneous diterpenoids The pseudopteradienoic acid 149 has been isolated192 from the Caribbean sea plume Pseudopterogorgia acerosa. The dimethylamino derivatives, aceropterine 150193 and its relative alanolide,194 were obtained from the same species. Sinulariadiolide 151 is a norditerpenoid which has been found195 in an Okinawan Sinularia species of soft coral.The Xenia species of soft corals have been a rich source of diterpenoids containing the nine-membered xenicane ring and exemplified in the current report by the azamilides, e.g. 152,196–198 whch are esterified with long chain fatty acids. Examination of Mulinum crassifolium (Umbelliferae) which is a Chilean plant used in folk medicine for the treatment of diabetes and bronchial disorders, has aVorded199 mulinolic acid 153, whilst other mulinanes were obtained200 from M.spinosum. The erinacines are a group of diterpenoid xylosides possessing a cyathan skeleton which have been isolated from the mycelium of Hericium erinaceum. They have attracted interest as stimulators of nerve growth factor synthesis with potential O O O O MeO2C O O PrC O O O O N Ac Me O H H HO AcO H AcO AcO Cl AcO AcO H AcO OAc OH Cl H OH OAc H H AcO H H AcO OAc OAc O H 140 141 142 143 144 O CPr O H AcO H OAc OAc O AcO O CH2OH C(CH2)7 O AcO H O OAc OH O H TigO AcO H H AcO HO H CH CHCH3 CH2 O O OCOCHMe2 O H AcO AcO OAc AcO OAc OAc 145 146 147 148 O O O CO2H O O CO2Me O HO Me2N O H O O OH O MeO2C O O C17H35C O OH AcO H OH H H H OH 149 150 151 152 CO2H H OH 153 102 Natural Product Reports, 1998application in the treatment of degenerative neuronal disorders such as Alzheimer’s disease.Recent examples that have been described201,202 include the erinacines D–G, exemplified by 154. The Basidiomycete Lepista sordida, has aVorded lepistal 155 which has been found203 to induce the diVerentiation of human leukaemic cells. Chemotaxonomic studies of the liverworts have yielded204 some 13-epineoverrucosane diterpenoids, e.g. 156, which was isolated from a New Zealand collection of Jamesoniella tasmanica. The sphenolobane 157 was isolated205 from Anastrophyllum donianum, the sacculatane 158206 from Porella platyphylla and infuscatrienol 159207 from Jungermannia infusca. The lobane derivative 160 has been obtained208 from the soft coral, Lobophytum microlobulatum whilst the 2-epicedrene isoprenologue 161 was isolated209 from the resin of the desert adapted species Eremophila pungens (Myoporaceae).The kalihinane derivative 162, extracted from the marine sponge Acanthella cavernosa, has been shown210 to have antifouling activity against barnacle larvae. A futher secotrinervitane 163 has been isolated211 from soldiers of the Madagascan termite Nasutitermes canaliculatus. The amphilectane and isocycloamphilectane isonitrile and isocyanate derivatives, e.g. 164, which have been found212,213 in the tropical marine sponge Cymbastela hooperi, have attracted interest because they show significant in vitro antimalarial activity. Leaf extracts of Ginko biloba (the maiden-hair tree) are widely used as a medicinal drug. The ginkolides are potent platelet-activating factor antagonists and their stereochemistry at C-1 has now been confirmed by an NMR study.214 Ryanodine is a potent regulator of the calcium release channel of mammalian muscle and a series of structure:activity studies have been reported.215,216 The neovibsanines A 165 and B have been found217 in Viburnum awabuki, a poisonous plant used for catching fish.Compounds of this structural type were obtained by the photochemical rearrangement of vibsanine B 166 which had been obtained previously from this plant. 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Hammink, S. J. E. Mulders, F. M. H. de Groot, H. L. M. van Rozendaal and H. W. Scheeren, J. Org. Chem., 1996, 61, 7092. Hanson: Diterpenoids 105173 C. Gennari, A. Vulpetti, M. Donghi, N. Mongelli and E. Vanotti, Angew. Chem., Int. Ed. Engl., 1996, 35, 1723. 174 G. I. Gunda, G. C. B. Harriman, M. Hepperle, J. S. Clowers, D. G. van der Velde and R. H. Himes, J. Org. Chem., 1996, 61, 2664. 175 J. Golik, H. S. L. Wong, S. H. Chen, T. W. Doyle, J. Wright, J. Knipe, W. C. Rose, A. M. Casazza and D. M. Vyas, Bioorg. Med. Chem. Lett., 1996, 6, 1837. 176 H. Park, M. Hepperle, T. C. Boge, R. H. Himes and G. I. Georg, J. Med. Chem., 1996, 39, 2705. 177 T. Iwagawa, S. Nakamura, H. Okamura and M. Nakatani, Bull. Chem. Soc. Jpn., 1996, 69, 3543. 178 C.Y. Duh and R. S. Hou, J. Nat. Prod., 1996, 59, 595. 179 H. Miyaoka, S. Taira, H. Mitome, K. Iguchi, K. Matsumoto, C. Yokoo and Y. Yamada, Chem. Lett., 1996, 239. 180 K. A. El Sayad and M. T. Hamann, J. Nat. Prod., 1996, 59, 687. 181 B. S. Mootoo, R. Ramsewak, R. Sharma, W. F. Tinto, A. J. Lough, S. McLean, J. P. Yang and M. Yu, Tetrahedron, 1996, 52, 9953. 182 A. D. Rodriguez, C. Ramirez and O. M. Cobar, J. Nat. Prod., 1996, 59, 15. 183 M. T. Hamann, K. N. Harrison, A.R. Carroll and P. J. Scheuer, Heterocycles, 1996, 42, 325. 184 E. Fridlovsky, A. Rudi, Y. Benayahu, Y. Kashman and M. Schleyer, Tetrahedron Lett., 1996, 37, 6909. 185 V. Roussis, W. Fenical, C. Vagias, J. M. Kornprobst and J. Miralles, Tetrahedron, 1996, 52, 2735. 186 T. Morgenstern, M. Bittner, M. Silva, P. Aqueveque and J. Jakupovic, Phytochemistry, 1996, 41, 1149. 187 M. O. Fatope, L. Zeng, J. E. Ohayaga and J. L. McLaughlin, Bioorg. Med. Chem., 1996, 4, 1679. 188 M.O. Fatope, L. Zeng, J. E. Ohayaga, G. Shi and J. L. McLaughlin, J. Med. Chem., 1996, 39, 1005. 189 S. Oksuz, F. Gurek, L. Lin, R. R. Gil, J. M. Pezzuto and G. A. Cordell, Phytochemistry, 1996, 42, 473. 190 J. A. Marco, J. F. Sanz-Cervera, A. Yuste, J. Jakupovic and J. Lex, J. Org. Chem., 1996, 61, 1707. 191 K. Sugita, D. Sawada, M. Sodeoka, H. Sasai and M. Shibasaki, Chem. Pharm. Bull., 1996, 44, 463. 192 A. D. Rodriguez and J. J. Soto, Chem. Pharm. Bull., 1996, 44, 91. 193 A. D. Rodriguez and J. J. Soto, Tetrahedron Lett., 1996, 37, 2687. 194 A. D. Rodriguez and J. J. Soto, J. Org. Chem., 1996, 61, 4487. 195 K. Iguchi, K. Kajiyama, H. Miyaoka and Y. Yamada, J. Org. Chem., 1996, 61, 5998. 196 T. Iwagawa, Y. Amano, M. Nakatani and T. Hase, Bull. Chem. Soc. Jpn., 1996, 69, 1309. 197 T. Iwagawa, Y. Amano, H. Okamura, M. Nakatani and T. Hase, Heterocycles, 1996, 43, 1271. 198 T. Iwagawa, T. Matsuda, H. Okamura and M. Nakatani, Tetrahedron, 1996, 52, 13 121. 199 L. A. Loyola, J. Borquez, G. Morales and A. San Martin, Phytochemistry, 1996, 43, 165. 200 M. Nicoletti, A. Di Fabio, A. D. Andrea, G. Salvatore, C. van Baren and J. D. Coussio, Phytochemistry, 1996, 43, 1065. 201 H. Kawagishi, A. Simada, K. Shizuki, H. Mori, K. Okamoto, H. Sakamoto and S. Furukawa, Heterocycl. Commun., 1996, 2, 51 (Chem. Abstr., 1996, 125, 190 112). 202 H. Kawagishi, A. Shimada, S. Hosokawa, H. Mori, H. Sakamoto, Y. Ishiguro, S. Sakemi, J. Bordner, N. Kojima and S. Furukawa, Tetrahedron Lett., 1996, 37, 7399. 203 X. Mazur, U. Becker, T. Anke and O. Sterner, Phytochemistry, 1996, 43, 405. 204 M. Toyota, E. Nakaishi and Y. Asakawa, Phytochemistry, 1996, 43, 1057. 205 M. S. Buchanan, J. D. Connolly and D. S. Rycroft, Phytochemistry, 1996, 43, 1297. 206 M. S. Buchanan, J. D. Connolly and D. S. Rycroft, Phytochemistry, 1996, 43, 1249. 207 F. Nagashima, A. Tamada and Y. Asakawa, Chem. Pharm. Bull., 1996, 44, 1628. 208 A. S. R. Anjaneyulu and K. V. S. Raju, Indian J. Chem. Sect. B., 1996, 35, 45. 209 Y. M. Syah and E. L. Ghisalberti, Phytochemistry, 1996, 41, 859. 210 H. Hirota, Y. Tomono and N. Fusetani, Tetrahedron, 1996, 52, 2359. 211 Y. Ranarivelo, M. Andriantsiferana, F. Tillequin, J. V. Silverton, H. M. GarraVo, T. F. Spande, H. J. C. Yeh and J. W. Daly, J. Nat. Prod., 1996, 59, 883. 212 G. M. Koenig, A. D. Wright and C. K. Angerhofer, J. Org. Chem., 1996, 61, 3259. 213 A. Linden, G. M. Koenig and A. D. Wright, Acta Crystallogr., Sect. C: Cryst. Struct. Commun., 1996, 52, 2601. 214 T. A. van Beek and P. P. Lankhorst, Tetrahedron, 1996, 52, 4505. 215 P. R. JeVeries, T. A. Blumenkopf, P. J. Gengon, L. C. Cole and J. E. Casida, J. Med. Chem., 1996, 39, 2331. 216 P. R. JeVeries, T. A. Blumenkopf, M. J. Watson and J. E. Casida, J. Med. Chem., 1996, 39, 2339. 217 Y. Fukuyama, H. Minami, K. Takeuchi, M. Kodama and K. Kawazu, Tetrahedron Lett., 1996, 37, 6767. 218 G. Guella, F. Dini and F. Pietra, Helv. Chim. Acta, 1996, 79, 439. 106 Natural Product Reports, 1998

ISSN:0265-0568    

DOI:10.1039/a815093y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Amaryllidaceae and Sceletium alkaloids John R. Lewis Department of Chemistry, Aberdeen University, Old Aberdeen, UK AB24 3UE Covering: 1996 Previous review: 1997, 14, 303 1 Introduction 2 Occurrence and structural studies 3 Synthetic studies 4 References 1 Introduction In this annual review seven new alkaloids have been described, while new analytical techniques have shown that additional alkaloids are present in previously investigated plant extracts. Interest in galanthamine 1 continues, due to its proposed role in the treatment of Alzheimer’s disease.1 Although viable yields have been achieved in the synthesis of this alkaloid,1 commercial production via plant culture methodology remains a goal yet to be reached.A study of in vitro culturing of Narcissus confusus by the ‘shoot clump’ procedure has achieved an alkaloid yield of 2.50 mg l"1 of culture; most of the alkaloid (1.97 mg) being found in the medium. Interestingly the addition of trans-cinnamic acid, a known precursor in the biosynthesis of galanthamine 1, inhibited the production of this alkaloid while the concentration of its congener, N-formylgalanthamine 2, was enhanced.1 A report on the analgesic properties of the four alkaloids lycorine 3, haemanthidine 4, haemanthamine 5 and tazettine 6, isolated from Sternbergia clusiana found in Turkey, showed that lycorine 3 and haemanthidine 4 possessed greater activity than that of aspirin.2 Using circular dichroism (CD) measurements on eight alkaloids obtained from the plant Hippeastrum equestre it was possible to determine, relatively easily, their basic ring systems and the stereochemistry of their ring junctions.Six other alkaloids were also obtained from other plant sources so as to complement the analyses. In all, this method provides a useful addition to the armoury for structure/stereochemical assignments.3 2 Occurrence and structural studies A reinvestigation of the alkaloid content of Amaryllis belladonna, using a multidimensional screening system involving liquid chromatography following by UV and MS detection, has led to the identification of nine additional alkaloids: anhydrolycorin-7-one 7, 6·-hydroxybuphanisine 8, R5O O N R1 R3 R4 R2 a 1 Galanthamine R1 = lone pair; R2 = R5 = Me; R3 = H; R4 = OH; ab unsat. 2 N-Formylgalanthamine R1 = lone pair; R2 = CHO; R3 = H; R4 = OH; R5 = Me; ab unsat. 35 Norgalanthamine R1 = lone pair; R2 = R3 = H; R4 = OH; R5 = Me; ab unsat. 45 Lycoramine R1 = lone pair; R2 = R5 = Me; R3 = H; R4 = OH; ab sat. b N R1O R2 R4 H R3 H b a 3 Lycorine R1R2 = CH2O; R3 = R4 = OH; ab unsat. 11 Galanthine R1 = R2 = Me; R3 = b-OMe; R4 = a-OH; ab sat. 48 Pseudolycorine R1 = H; R2 = R4 = OH; R3 = OMe; ab sat. N R3 O O R1 R2 R4 4 Haemanthidine R1 = H; R2 = R4 = b-OH; R3 = a-OMe 5 Haemanthamine R1 = R2 = H; R3 = b-OH; R4 = a-OH 8 6a-Hydroxybuphanisine R1 = R4 = H; R2 = a-OH; R3 = a-OMe 9 6-Hydroxycrinine R1 = R4 = H; R2 = R3 = a-OMe 10 Crinine R1 = R2 = R 4 = H; R3 = a-OH 16 Buphanisine R1 = R2 = R4 = H; R3 = b-OMe 17 Buphanidrine R1 = OMe; R2 = R4 = H; R3 = a-OMe 18 Epibuphanisine R1 = R2 = R4 = H; R3 = a-OMe 30 Vittatine R1 = R2 = R4 = H; R3 = b-OH 34 6b-Hydroxybuphanisine R1 = R4 = H; R2 = b-OH; R3 = a-OMe O O O R4 NMe R3 R2 R1 6 Tazettine R1 = b-OMe; R2 = R4 = H; R3 = a-OH 26 Criwelline R1 = a-OMe; R2 = R4 = H; R3 = a-OH 32 Littoraline R1 = b-OH; R2 = OH; R3 = b-H; R4 = H 43 Pretazettine R1 = Me; R2 = R3 = b-H; R4 = O 44 Macronine R1 = R2 = H; R3 = a-OH; R4 = O N O O O 7 Lewis: Amaryllidaceae and Sceletium alkaloids 1076-hydroxycrinine 9, crinine 10, galanthine 11, hippadine 12, ismine 13, pratorimine 14 and pratosine 15.All these compounds are new additions to the alkaloid content of this plant.4 In the bulbs of Brunsvigia orientalis, 12 alkaloids were found,5 namely lycorine 3, crinine 10, buphanisine 16, buphanidrine 17, epibuphanisine 18, undulatine 19, crinamidine 20, crinamine 21, 6-hydroxycrinamine 22 and the new alkaloids 1-epibowdensine 23, 1-epidemethoxybowdensine 24 and 1-epideacetylbowdensine 25.Crinum firmifolium var hygrophilium whole plant extracts6 contain eight alkaloids, lycorine 3, ismine 13, crinamine 21, 6-hydroxycrinamine 22, hamayne 27, criwelline 26, trisphaeridine 28 and the novel alkaloid 3-hydroxy-8,9- methylenedioxyphenanthridine 29. Dried plant and bulbs obtained from Hippeastrum solandri- florum have been shown to contain five alkaloids.7 They are the cytotoxic lycorine 3, ismine 13, hamayne 27, vittatine 30, and ungeremine 31.Because extracts of Hymenocallis littoralis showed in vitro cytotoxic activity, a reinvestigation of this plant’s alkaloidal content identified 14 alkaloids. One of these, littoraline 32, is new,8 and the others are quoted in Table 1. A new crinane type alkaloid cantabricine 33 has been found in extracts of the whole plant Narcissus cantabricus.9 Also present in the extract were tazettine 6, crinamine 21, vittatine 30, 6·-hydroxybuphanisine 8, and 6‚-hydroxybuphanisine 34.The cultivated daVodil Narcissus CV salome has now been investigated for its alkaloidal content.10 The bulbs contained six known alkaloids, namely crinamine 21, norgalanthamine 35, hippeastrine 38, tortuosine 39, vasconine 40, pseudolycorine 48, and the hitherto unreported alkaloid 2·-hydroxy- 6-O-methyloduline 41. 3 Synthetic studies In the first part of a study on the synthesis of crinum type alkaloids it is now possible11 to generate tetracyclic N O R1O R2O 12 Hippadine R1R2 = CH2 14 Pratorimine R1 = H; R2 = Me 15 Pratosine R1 = R2 = Me CH2OH MeNH O O 13 Ismine N O O R5 R3 R2 R1 R4 19 Undulatine R1R2 = b-O; R3 = a-OMe; R4 = H; R5 = OMe 20 Crinamidine R1R2 = b-O; R3 = a-OH; R4 = H; R5 = OMe 23 1-Epibowdensine R1 = b-OMe; R2 = b-OAc; R3 = R4 = H; R5 = OMe 24 1-Epidemethoxybowdensine R1 = R2 = b-OAc; R3 = R4 = R5 = H 25 1-Epideacetylbowdensine R 1 = R2 = b-OH; R3 = R4 = H; R5 = OMe R3 N R1O R2O R5 R4 a b 21 Crinamine R1R2 = CH2; R3 = b-OH; R4 = a-OMe; R5 = H; ab unsat. 22 6-Hydroxycrinamine R1R2 = CH2; R3 = b-OH; R4 = a-OMe; R5 = OH; ab unsat. 27 Hamayne R1R2 = CH2; R3 = b-OH; R4 = a-OH; R5 = H; ab unsat. 33 Cantabricine R1 = R3 = R5 = H; R2 = Me; R4 = a-OAc; ab sat. 46 Demethylmaritidine R1 = Me; R2 = R3 = R5 = H; R4 = b-OH; ab unsat. N R O O 28 Trisphaeridine R = H 29 3-Hydroxy-8,9-methylenedioxyphenanthridine R = OH + 31 N O– O 36 Homolycorine R1 = R2 = Me; R3 = O; R4 = H 37 Lycorenine R1 = R2 = R4 = H; R3 = b-OH 38 Hippeastrine R1R2 = CH2; R3 = O; R4 = a-OH 41 2a-Hydroxy-6- O-methyloduline R1R2 = CH2; R3 = a-OMe; R4 = a-OH 42 O-Methyllycorenine R1 = R2 = R4 = H; R3 = b-OMe O R1O R2O R3 MeN R4 H N MeO R1O R2 39 Tortuosine R1 = Me; R2 = OMe 40 Vasconine R1 = Me; R2 = H 108 Natural Product Reports, 1998anhydrolycorin-7-one 7 by a palladium acetate catalysed cyclisation of the bromo dihydroindole 49.An eYcient synthesis of (&)-crinane 60 in eight steps with an overall yield of 23% has been achieved starting from 3,4-methylenedioxyphenylbromide 50, with the key step being a pyrolytic decomposition of azide 57 to imine 58 and hence to crinane (Scheme 1).12 Firstly compound 50 was condensed with cyclohexenone 51 to give 52 which upon reduction gave enol 53 and thence its acetate 54. Treatment of this acetate with tert-butyldimethylchlorosilane (TBSCl) according to the method of Keck aVorded the carboxylic acid 55 which could be reduced to alcohol 56 with LiAlH4.A ‘one pot’ transformation of this alcohol to azide 57 was accomplished under Mitsunobu conditions. Thermolysis of this azide in refluxing toluene under nitrogen, gave imine 58 which upon reduction and reaction with Eschenmoser’s salt for 48 h, gave (&)-crinane 60. A new synthesis of the membrine ring system employs the Sharpless AD reaction. This radical-inhibited procedure allows an asymmetric construction of a quaternary carbon centre by Table 1 Isolation of Amaryllidaceae alkaloids Species Alkaloid (structure) Ref.Amaryllis belladona (whole plant) Anhydrolycorin-7-one 7 6·-Hydroxybuphanisine 8 6-Hydroxycrinine 9 Crinine 10 Galanthine 11 Hippadine 12 Ismine 13 Pratorimine 14 Pratosine 15 4 Brunsvigia orientalis (bulbs) Lycorine 3 Crinine 10 Buphanisine 16 Buphanidrine 17 Epibuphanisine 18 Undulatine 19 Crinamidine 20 Crinamine 21 6-Hydroxycrinamine 22 1-Epibowdensine* 23 1-Epidemithoxybowdensine* 24 1-Epideacetylbowdensine* 25 5 Crimum firmifolium var hygrophiluim (whole plant) Lycorine 3 Criwelline 26 Crinamine 21 6-Hydroxycrinamine 22 Hamayne 27 Ismine 13 Trisphaeridine 28 3-Hydroxy-8,9-methlenedioxyphenthridine* 29 6 Hippeastrum solandrilorum Lycorine 3 Hamayne 27 Vittatine 30 Ismine 13 Ungeremine 31 7 Hymenocallis littoralis (bulbs) Littoraline* 32 Trazettine 6 O-Methyllycorenine 42 Pretazettine 43 Macronine 44 Lycorine 3 Homolycorine 36 Lycorenine 37 Hippeastrine 38 Lycoramine 45 Demethylmaritidine 46 Haemanthamine 5 Vittatine 30 5,6-Dihydrobicolorine (ismine) 13 8 Narcissus cantabricus Vittatine 30 Crinamine 21 6·-Hydroxybuphanisine 8 6‚-Hydroxybuphanisine 34 Tazettine 6 Cantabricine* 33 9 Narcissus confusus Galanthamine 1 N-Formylgalanthamine 2 1 Narcissus CV salome 2·-Hydroxy-6-O-methyloduline* 41 Norgalanthamine 35 Crinamine 21 Pseudolycorine 48 Hippeastrine 38 Tortuosine 39 Vasconine 40 10 Sternbergia clusiana Lycorine 3 Haemanthidine 4 Haemanthamine 5 Tazettine 6 2 *New alkaloids.N O O O 49 Br O O Br O O O O O O BuO O O O O O O O O O O CO2H OR OH N3 NH O O N N H N Me Me ii i iii iv v vi vii viii ix x + 50 51 52 53 R = H 54 R = Ac Heat 55 56 57 58 60 + 59 I– Scheme 1 Reagents: i, BunLi, Et2O–THF, 78 )C, then room temp.; ii, NaBH4, CeCl3 · 7H2O, MeOH, 0 )C; iii, AcCl, py; iv, LDA, THF; v, TBSCl, "78 )C]reflux; vi, LiAlH4, THF; vii, Ph3P, DEAD, (PhO)2P(O)N3, CH2Cl2; viii, toluene, reflux, 24 h; ix, NaBH3CN, AcOH; x, Eschenmoser’s salt, THF, 50 )C, 48 h Lewis: Amaryllidaceae and Sceletium alkaloids 109diastereoselective formation of the „-lactone in moderate yields when double dihydroxylation is applied to the diene ester 61 (Scheme 2).13 Thus treatment of 61 with the chirally modified osmium tetroxide reagent developed by Sharpless, namely AD-mix-‚, gave a mixture of diols and tetrols 62 and 63 which spontaneously cyclised to a mixture of lactone diols and triols.If excess of reagent AD-mix-‚ was used, triol lactones predominated. The triol lactones 64 were a mixture of isomers with the R conformer predominating, which upon oxidation with Pb(OAc)4 gave aldehyde 65 and thence with N-methylbenzylamine and NaBH3CN gave tertiary amine 66. Hydrogenation in the presence of di-tert-butoxycarbonic anhydride yielded urethaine 67 which upon reduction gave diol 68. Removal of the oxygen functionality was achieved by first converting 68 into the thiohemiketal 69 whereby oxidation to the ketone 70 allowed a radical initiated reductive cleavage of the thiohemiketal using But 3SnH in the presence of AIBN to give 71 which was then cyclised to cyclohexanone 72.A final treatment with dilute HCl produced (")-mesembrine 73. 4 References 1 S. Bergonon, C. Codina, J. Bastida, F. Viladomat and E. Mele, Plant Cell, Tissue Organ Cult., 1996, 45, 191 (Chem.Abstr., 1996, 126, 46 341). 2 M. Tanker, G. Citoglu, B. Gumusel and B. Sener, Int. J. Pharmacogn., 1996, 34, 194 (Chem. Abstr., 1996, 125, 316 966). 3 J. Wagner, H. L. Pham and W. Döpke, Tetrahedron, 1996, 52, 6591. 4 O. R. Queckenberg, A. W. Frahm, D. Mueller-Dobbies and U. Mueller-Dobbies, Phytochem. Anal., 1996, 7, 156 (Chem. Abstr., 1996, 125, 5487). 5 F. Viladomat, A. Francese, R. Giovanna, C. Codina, J. Bastida, W. E. Campbell and S. M. Mathee, Phytochemistry, 1996, 43, 1379. 6 J. Razafimbelo, M. Andrianlsiferana, G. Baoudouin and F. Tillequin, Phytochemistry, 1996, 41, 323. 7 J. Bastida, C. Codina, C. L. Porras and L. Paiz, Planta Med., 1996, 62, 74 (Chem. Abstr., 1996, 124, 226 600). 8 L-Z. Lin, S-F. Hu, H-B. Chai, T. Pengsuparp, J. M. Pezzuto, G. A. Cordell and N. Ruangrungsi, Phytochemistry, 1995, 40, 1295. 9 J. Bastida, J. L. Contreras, C. Codina, C. W. Wright and J. D. Phillipson, Phytochemistry, 1995, 40, 1549. 10 G. R. Almanza, J. M. Fernandez, E. W. T. Wakori, F. Viladomat, C. Cordina and J. Bastida, Phytochemistry, 1996, 43, 1375. 11 H. W. Shao and J. C. Cai, Chin. Chem. Lett., 1996, 7, 13 (Chem. Abstr., 1996, 124, 289 955). 12 J. M. Schkeryantz and I. V. H. Pearson, Tetrahedron, 1996, 52, 3107. 13 T. Yoshimitsu and K. Ogasawara, Heterocycles, 1996, 42, 135. Ar CO2Et Ar CO2Et OH H H OH H OH Ar CO2Et H HO H OH CO2Et Ar H HO OH H OH H O O O CHO HO O OMe MeO OMe MeO O N O HO OMe MeO N Boc Me OMe MeO N Boc Me O O OMe MeO O N SPh X OMe MeO O N Me HO H Ar OH H O H H OH HO H O H Ar OH H H OH H ii i iii iv v vi vii viii ix x + OMe MeO 61 O N OH HO 62 63 68 65 71 66 R = Bn 67 R = Boc 72 R Me Boc Me Boc Me 73 (–)-Mesembrine S-64 R-64 + i 69 R = H,OH 70 R = O Scheme 2 Reagents: i, AD-mix-‚ (2 equiv.), MeSO2NH2 (1 equiv.); ii, Pb(OAc)4, PhH, room temp.; iii, BnNHMe, NaBH3CN, 4 Å mol. sieve, MeOH, room temp.; iv, H2, Pd(OH)2, (Boc)2O, AcOEt–EtOH; v, DIBALH, CH2Cl2, "78 )C; vi, PhSH, BF3OEt, CH2Cl2, "78 )C]room temp.; vii, Swern oxidation; viii, But 3SnH (6 equiv.), AIBN, PhH, reflux; ix, 10% KOH–MeOH (1:2), room temp.; x, HCl–EtOH, reflux 110 Natural Product Reports, 1998

ISSN:0265-0568    

DOI:10.1039/a815107y

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Book review Natural product chemistry. A mechanistic, biosynthetic and ecological approach Kurt B. G. Torssell, Apotekarsocieteten, Stockholm, 1997, 2nd edn, 480 pp. ISBN 91 8627 463 5 This is a new edition of a book first published in 1983. The book aims to provide a mechanistic, biosynthetic and ecological approach to natural product chemistry. In selecting additional material for this new edition the author has concentrated on chemical ecology and on oxidative mechanisms in biological chemistry.Following an introduction on the principal reactions and pathways of biosynthesis, there is a chapter on chemical ecology. There are then chapters on carbohydrates, the shikimic acid pathway, the polyketides, the terpenoids, amino acids, peptides and proteins, the alkaloids and finally N-heteroaromatics such as the pyrimidines and purines, pyrroles and porphyrins. At the end of each chapter there are some problems and some literature references. The introductory chapter contains a general list of natural product books and some journals.In writing a book of this kind the problem is to be selective and yet maintain an even balance between the diVerent facets of the subject. This is particularly diYcult for a single author covering such a wide area. This book concentrates on presenting the biosynthetic and mechanistic rationale for natural product structures. Although some biosynthetic experimental evidence is described, the distinction between this and plausible biogenetic speculation is not always made.Although the title of the book is ‘Natural product chemistry’, the concentration on biosynthesis leads to the exclusion of a great deal of chemistry. The partial synthesis of biologically active natural products from more readily available natural substances is a major area of current natural product chemistry. This has revealed many aspects of conformational analysis and of unusual reactivity arising from a unique juxtaposition of functional groups found in a natural product.The chemistry of the terpenoids and steroids has a great deal to teach in this context. The synthetic work on the steroid hormones and their biological function receives barely a mention. Although the author has made some eVort to include material other than biosynthesis, it is not presented in a particularly systematic way. For example, there is a jump from three pages on the carotenoids immediately to five pages on optical rotatory dispersion.In this context it would have been much better to bring all the methods of structural elucidation together into one chapter. The elucidation of natural product structures is another area of current natural product chemistry which receives relatively little attention in this book. There are a number of mistakes. Some are relatively trivial such as the formula of cadinene (p. 49) but others are more serious such as the biosynthesis of the gibberellins (Fig. 22). In presenting biosynthetic schemes the letter P is used loosely for both phosphate and pyrophosphate whilst many arrows denoting electron movement do not clearly indicate which centres and which bonds are involved (e.g. structure 15, p. 92). There is a tendency to coalesce the mechanistic rationalization for several steps into one structure and this can be confusing to the student. Despite these criticisms, this book contains a wealth of information across a broad range of natural product biosyntheses. It is an interesting book providing background reading for a chemist starting natural product research or giving background material for a lecture course on natural product biosynthesis. James R. Hanson University of Sussex, Brighton, UK 111

ISSN:0265-0568    

DOI:10.1039/a815111y

出版商:RSC    

年代:1998

数据来源: RSC