摘要:

The Biosynthesis of Shikimate Metabolites P. M. Dewick Department of Pharmacy University of Nottingham Nottingham NG7 2RD Reviewing the literature published during 1984 (Continuing the coverage of literature in Natural Product Reports 1984 Vol. 1 p. 45 1) 1 The Shikimate Pathway 1.1 DAHP Synthase 1.2 3-Dehydroquinate Synthase 1.3 Shikimate Dehydrogenase 1.4 5-Enolpyruvylshikimate-3-phosphateSynthase 1.5 Chorismate Mutase 1.6 Prephenate Dehydrogenase 1.7 Anthranilic Acid 1.8 Phenylalanine Tyrosine and Tryptophan 2 Phenols and Phenolic Acids 3 Indole-3-acetic Acid and Related Metabolites of Tryptophan 4 Phenylpropanoids 4.1 Phenylalanine Ammonia-lyase 4.2 Cinnamic Acid Esters 4.3 Phenylpropane Phenylethane and Phenylmethane Derivatives 4.4 Lignins 4.5 Lignans 4.6 Coumarins 5 Flavonoids 5.1 Flavanones Dihydroflavonols Flavonols Flavones and Anthocyanidins 5.2 Procyanidins 5.3 Methylation of Flavonoids 5.4 Glycosylation of Flavonoids 5.5 Quinol Ethers 5.6 Isoflavonoids 6 Stilbenes and Phenanthrenes 7 Quinones and Quinols 8 Miscellaneous Shikimate Metabolites 8.1 Ketomycin 8.2 Bakuc hiol 8.3 9-Phenylp henalenones 8.4 Tuberin and Xanthocillin 9 References This Report reviews the literature published during 1984 on the biosynthesis of non-nitrogenous aromatic compounds that are derived wholly or partly from shikimate and continues the coverage in Volume 1 of Natural Product Reports.' 1 The Shikimate Pathway 1.1 DAHP Synthase Crude extracts of the yeast Rhodotorula glutinzs contain three differentially regulated species of 3-deoxy-~-arabino-heptulo-sonate-7-phosphate synthase (DAHP synthase) [phospho-2- dehydro-3-deoxyheptonatealdolase (E.C.4.1.2.15)].* Two of these isozymes are relatively stable and are inhibited by tyrosine (DAHP synthase-Tyr) or tryptophan (DAHP synth- ase-Trp) respectively but the third which is inhibited by phenylalanine (DAHP synthase-Phe) is extremely labile. DAHP synthase appears to be the only site for control of the biosynthesis of phenylalanine in this organism. This is co; CS; I I 6H 0 I HO COi x OH 3 -Dehydroquinate (2) Scheme I apparently sufficient since a spontaneous mutant that is resistant to the growth-inhibitory phenylalanine analogue p-fluorophenylalanine has been found to contain a feedback- resistant DAHP synthase-Phe and it can cross-feed a phenylalanine auxotroph of Bacillus subtilis.The herbicide glyphosate is a reversible inhibitor of DAHP synthase-Tyr from the yeast Candida rnalt~sa.~ In the presence of glyphosate growth slows down and the yeast cells excrete shikimic acid. The DAHP synthase from cells of carrot (Daucus carota) was found to be activated by tyrosine and tryptophan the extent varying during gr~wth.~ By exploiting an auxotroph of Escherichia coli that lacks 3-dehydroquinate synthase substantial amounts of DAHP (1) may readily be obtained offering an easier approach than chemical synthesis for hundred-milligram quantities of this metab~lite.~ 1.2 3-Dehydroquinate Synthase This enzyme [E.C.4.6.1.31 catalyses the conversion of DAHP (1) into 3-dehydroquinate (2) which is a sequence requiring an oxidation a 0-elimination a reduction and an intramolecular aldol condensation (Scheme 1). The protein normally occurs in Nature at very low concentrations and has thus been unavailable in reasonable quantity for any detailed examin- ation of this multi-step sequence. To overcome this problem recombinant DNA techniques have been applied to produce two plasmid-bearing strains of E. coli that over-produce the enzyme by factors of 20 and 100O.‘j Fifty grams of cells containing the subcloned gene are reported to have yielded 1 50,mg of homogeneous enzyme.1.3 Shikimate Dehydrogenase The isolation of shikimate dehydrogenase [E.C. 1.1.1.251 which catalyses the reversible reduction of 3-dehydroshikimate to shikimate from the fruit of the tomato (Lycopersicon esculentum) has been described.’ With shikimate as its substrate NADP+ is a necessary cofactor and could not be replaced by NAD+. Protocatechuic acid competitively inhibits the enzyme. 1.4 5-Enolpyruvylshikimate-3-phosphateSynthase The steric course of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase) [3-phosphoshikimate l-carboxy- vinyltransferase (E.C. 2.5.1.19)] which is the enzyme that catalyses the condensation of shikimate 3-phosphate (3) with phosphoenolpyruvate (4) has been investigated by two separate group^.^.^ Although experimental approaches varied the two groups used the same type of reasoning to study the problem.The reaction is thought to proceed via the addition- elimination mechanism that is shown in Scheme 2. During the addition step C-3 of phosphoenolypyruvate becomes a transient methyl group which can rotate (5). If the enzyme is supplied with stereospecifically doubly labelled [3-*H1 3-3H ]phosphoenolpyruvate the addition reaction will give a Shik imate 3 -phosphate (3) NATURAL PRODUCT REPORTS 1985 chiral methyl group from which loss of hydrogen via the elimination reaction will be subject to isotope effects. The labelled methylene group of EPSP (6) may now be analysed by suitable degradation procedures involving stereospecific intro- duction of lH thus generating a chiral methyl group in some molecules (Scheme 3); the chirality of the methyl group can be assessed via its conversion into acetic acid.The same conclusions were reached in the two studies i.e. that the configuration in the side-chain of the labelled EPSP (8) was predominantly the same as that in the labelled phosphoenol- pyruvate (7) (Scheme 4) and the addition and elimination reactions must therefore proceed with opposite stereoche- mistry. If the addition is anti then the elimination is syn or vice versa. Knowles’ groups used an enzyme preparation from Klebsiella pneumoniae (Aerobacter aerogenes 62-1) and specifi- cally labelled phosphoenolpyruvates for the studies whereas Floss and co-workers9 used the whole organism and analysed the chorismate that was obtained from shikimate and specifically labelled glycerol i.e.the phosphoenolpyruvate was produced in situ. The EPSP synthase from K. pneumoniae has been purifiedlO and the inhibitory effects of the broad-spectrum non-selective herbicide glyphosate [N-(phosphonomethy1)glycinel on this enzyme have been studied.’ The inhibition is competitive with respect to phosphoenolpyruvate and non-competitive with respect to shikimate 3-phosphate and EPSP. It has been proposed that glyphosate (9) binds to the phosphoenolpyru- vate-binding site of EPSP synthase as an analogue of the transition state (10) of phosphoenolpyruvate.Similar conclu- sions were reached in studies with the enzyme from cell suspension cultures of Nicotiunu siluestris. * However a strain of K. pneumoniae that is resistant to inhibition of its growth by glyphosate has been described.I3 This strain contains a glyphosate-insensitive form of EPSP synthase and the organ- EPSP (6) Scheme 2 R-0-1~ ’H ’”&” [ ~ [-HO@A 2HxH‘“d“ + 3HxH @o COZH @o CO,H RO COZH RO CO2 H OR (only tritiated molecules are shown 1 ’H ’H chiral ac hi ral methyl methyl Scheme 3 NATURAL PRODUCT REPORTS 1985 -P. M. DEWICK (7) OH (8) Enzymes i 3-phosphoshikimate 1-carboxyvinyltransferase (EPSP syn thase) Scheme 4 0 0- O\\r/O-+P/ L 'L ism does not excrete shikimate 3-phosphate if it is exposed to the herbicide.Significant differences in physical properties between the sensitive and insensitive forms of the enzyme were noted. The purification of EPSP synthase from seedlings of the pea (Pisumsutiuurn) has been described. Access to relatively large amounts of the enzyme has been achieved by using genetic engineering techniques using recombinant plasmids in cells of Escherichia coli. These cells over-produced the enzyme some 100-fold and allowed the homogeneous enzyme to be isolated in milligram amounts. The complete amino-acid sequence for this enzyme has been determined. 1.5 Chorismate Mutase The rearrangement of chorismate (1 1) to prephenate (12) (Scheme 5) is perhaps the only example of a pericyclic reaction in primary metabolism,' and the detailed stereochemistry of this process which is catalysed by chorismate mutase (CM) [E.C.5.4.99.51 has been investigated. Transition states with boat- or chair-like geometry are possible (Scheme 6). Samples of (E)-and (2~)-[9-~H Ichorismate [prepared chemically or enzymically (uiu EPSP ~ynthase)~] were incubated with the bifunctional enzyme chorismate mutase-prephenate dehydro- genase from E. coli in the absence of NAD+ thus yielding prephenate. By carrying out the reaction at pH 6 in the presence of phenylpyruvate tautomerase [E.C. 5.3.2.13 the prephenate that was initially formed was spontaneously converted into phenylpyruvate (13) and on to the enol form (14) with the loss of the pro-R-hydrogen from the methylene group (Scheme 7).By monitoring the appearance of tritium label in the solvent from the labelled chorismates it was possible to deduce that the reaction that is catalysed by chorismate mutase must therefore proceed through a chair-like transition state (Scheme 6; 6) since the (pro-Z)-hydrogen of chorismate became the (pro-S)-hydrogen of prephenate. Studies of the sequence of amino-acid residues in the chorismate mutase-prephenate dehydrogenase from E. coli support earlier proposals that there is a close spatial relation- ship between the active sites. A particularly reactive cysteine residue has been identified and shown to be essential for both activities of the enzyme.*O Two isozymes of chorismate mutase exist in Nicotiunu siluestris; CM-1 constitutes the major fraction in suspension- cultured cells and is very sensitive to allosteric control by aromatic amino acids whereas CM-2 which is the major bH Chorismate (11) Prephenate (12) Enzymes i chorismate mutase Scheme 5 H$ co; CO -(a)via boat F OH OH I" U I OH OH Scheme 6 A c0,-HS YR A !R I 6 >O i f' 1 I OH PhenyL pyruvate (13) (14) (12) Reagents i H+; ii phenylpyruvate tautomerase Scheme 7 co; I NAD' NADH I OH AH (12) p -Hydroxyphenylpyruvate (15) Enzymes i prephenate dehydrogenase Scheme 8 fraction in green leaf tissue is not inhibited by aromatic amino acids.The regulatory properties are consistent with different roles for the two activities CM-1 controlling the biosynthesis of phenylalanine and tyrosine and CM-2 the formation of secondary phenylpropane metabolites uiu overflow produc- 498 ti~n.~ Differential regulation of two chorismate mutase 1-23 activities in the yeast Rhodotorula glutinis has also been noted.2 In Cundida multosa the chorismate mutase activity like the prephenate dehydrogenase and prephenate dehydratase activi- ties is dependent on the presence of trypt~phan.~~ 1.6 Prephenate Dehydrogenase The two activities of chorismate mutase-prephenate dehydro- genase [E.C.5.4.99.5 and 1.3.1.12 respectively] can be studied separately because of the requirement for NAD+ in the prephenate dehydrogenase (PDH) step that leads to p-hydroxyphenylpyruvate (1 5) (Scheme 8).The mechanism of PDH has been investigated by observing I3C kinetic isotope effects on the decarboxylation of both deuteriated and non- deuteriated substrate^.^^ The results that were obtained lead to the conclusion that it is a concerted mechanism rather than a stepwise one in which dehydrogenation precedes decarboxyla- tion. Removal of the keto function of the side-chain from the substrate had little effect on the reaction but if one or both double-bonds in the ring were reduced oxidation occurred but decarboxylation was prevented. In contrast the acid-catalysed decarboxylation of prephenate that leads to phenylpyruvate (13) is a stepwise process proceeding through a carbo-cation intermediate (16) (Scheme 9).1.7 Anthranilic Acid The biosynthesis of anthranilate (19) from chorismate is believed to proceed via the sequence that is shown in Scheme 10. Anthranilate synthase (AS) [E.C. 4.1.3.271 from enteric bacteria usually consists of two dissimilar subunits AS-1 and AS-2 and the native enzyme will utilize either glutamine or NH,+ to transform chorismate into anthranilate. The subunit AS-1 by itself requires NH,+ and cannot use glutamine. The amino-alcohol (18) had been isolated earlier but the postulated intermediate (17) was unknown. The synthesis of this unstable compound from (18) has now been achieved,26 and its transformation into anthranilate by AS-1 from Serratia murcescens in the absence of NH,+ has been demonstrated. A breakdown product of (1 7) in aqueous solution is (20) which appears to be an inhibitor of the enzymic reaction.Anthanilate synthase was employed in conjunction with the investigations on the steric course of EPSP synthase (see Section 1.4).9 The results from that study suggest that the enzyme-catalysed protonation of the enolpyruvyl side-chain of the labelled chorismate (21) which liberates the pyruvate (22) occurs from the re face (Scheme 11). This contrasts with the steric course of other reactions that have been studied in which attack at C-3 of phosphoenolpyruvate is involved. An N-malonyltransferase that is capable of malonylating anthranilic acid has been isolated from seedlings of the peanut (Aruchis hypogae~).~’ 1.8 Phenylalanine Tyrosine and Tryptophan Cell-free extracts of the yeast Cundida multosa yielded three aromatic-amino-acid aminotransferases.28 These had overlap- ping specificities each being capable of transamination with phenylpyruvate p-hydroxyphenylpyruvate prephenate or in- dole-3-pyruvate as the amino-acceptor but they were regulated differently by the amino acids. The biosyntheses of tyro~ine~~ and tryptophan30 have been reviewed. Oxidative coupling of two tyrosine molecules giving small yields of bityrosine (23) has been observed to occur in a tyrosine-horseradish peroxidase-H 202system. 2 Phenols and Phenolic Acids In plants 3,4,5-trihydroxybenzoic acid (gallic acid) may arise by two major pathways i.e. by dehydrogenation of 3-NATURAL PRODUCT REPORTS 1985 Jc COZ 6 \ (13) Scheme 9 coz-COT I I I OH (11) (17) co Anthranilate (19) (18) Scheme 10 COzH I p””‘ (732-’H OH (21) (19) (22) Enzymes i anthranilate synthase Scheme 11 NATURAL PRODUCT REPORTS 1985 -P.M. DEWICK OH OH Tryptophan Indole -3 -pyruvic acid Tryptamine H CO-, CO- H (23) (24) dehydroshikimic acid or by &oxidation of a phenylpropane/ cinnamic acid precursor. Results of studies using leaves of Acer buergerianum and Rhus succedanea3* have again confirmed these findings and in addition it has been suggested that the age of the leaf tissue determines the preferred pathway. In young leaves shikimic acid is a better precursor than phenylalanine the reverse situation being observed in mature and autumnal leaves.Phenyl-lactic acid is metabolized almost as well as phenylalanine. Similar results were noted with these substrates for the formation of ellagic acid (24) which is a dimer of gallic acid but gallic acid proved to be the most effective precursor. An enzyme that was isolated from Aspergillusniger catalysed the oxidative deamination and dihydroxylation of anthranilic acid to 2,3-dihydroxybenzoic acid.33The enzyme incorporated one atom of oxygen each from '*02and H2180 into the product and represents a new type of NADPH-linked non-haem iron-containing mono-oxygenase. Cell cultures of Gardenia jasminoides are capable of glucos-ylating salicyl alcohol on either the phenolic or the alcoholic group producing salicin and isosalicin respectively in a ratio of about 2 :1.When a partially purified enzyme preparation was incubated with salicyl alcohol and UDPglucose only salicin was produced however and no isosalicin was de-te~ted.~~ Despite this specificity the enzyme must have some other function since salicin is not endogenously synthesized by cells of G. jasminoides. 3 Indole-3-acetic Acid and Related Metab-olites of Tryptophan Studies of metabolism in needles of Pinus syl~estris~~ show that tryptophan and tryptamine may be converted into indole-3-ethanol which in turn may serve as a precursor of indole-3-acetic acid (IAA) (Scheme 12). However label from IAA was not detected in indole-3-carboxylic acid (ICA) suggesting that at best only a minor portion of the endogenous JCA pool originates from IAA.This contrasts with results for wheat (Triticum compacturn) where IAA may be metabolized to indole-3-methano1,indole-3-aldehyde and indole-3-carboxylic acid.36A glucoside which released indole-3-methanol if it was treated with P-glucosidase was also observed as a metabolite of IAA. Oxidation of indole-3-acetic acid (25) to oxindole-3-acetic acid (26) further hydroxylation to 7-hydroxyoxindole-3-acetic acid (27),and glucosylation to (28) have been observed to occur in seedlings of Zea mays (Scheme 1 3).37The glycoside (28) was also isolated from the seedlings and appears to be a natural metabolite. 4 Phenylpropanoids 4.1 Phenylalanine Ammonia-lyase The regulation of induction of phenylalanine ammonia-lyase (PAL) [E.C.4.3.1.51and its role in the development of plants have been reviewed.38 4.2 Cinnamic Acid Esters Glucose esters of phenolic acids represent an activated form of the acids and can be involved in metabolic processes instead of I/ Indole -3 -acetaldehyde f-) Indole -3-ethanol 1 Indole -3-acetic acid Scheme 12 Indole -3-acetic acid (25) (26) I /CH,CO- H (28) (27) Scheme 13 HO OMe (29) the more commonly encountered esters of coenzyme A. Thus 1-0-trans-cinnamoyl-P-D-glucose has been implicated in the biosynthesis of chlorogenic acid in the root of sweet potato (Ipomoea batatas). The enzyme from this source has been partially purified39 and shown to have broad substrate specificity glucosylating p-coumaric o-coumaric benzoic ferulic and vanillic acids as well as the preferred substrate i.e.cinnamic acid. Uridinediphosphoglucose was the glucose donor. The glucose esters may then serve as precursors in the biosynthesis of other esters via transesterification. An enzyme that catalyses the formation of a p-coumaroylquinic acid from 1-0-p-coumaroyl-D-glucose and quinic acid has been detected in the same plant,40 but the site of attachment of the p-coumaroyl group on quinic acid has not been established. In suspension cultures of cells of Chenopodium rubrum glucose esters of p-coumaric acid and ferulic acid accumulate in large amounts and moreover are subject to high t~rnover.~' A considerable portion of the ferulic acid content was eventually found to be in an insoluble form probably being bound to cell-wall material.1,2-Di-~-sinapoyl-~-~-glucose (29) accumulates in cotyle-dons of seedlings of radish (Raphanus satiuus) that have grown in the dark.42 This arises as demonstrated by enzymic from two molecules of 1 -0-SinapOyl-p-D-glUCOSe by a disproportionation reaction in which the substrate is used as both acyl donor and acyl acceptor. This observation parallels recent work on the origins of di-O-galloyl-p-D-glucose (see ref. 1). 4.3 Phenylpropane Phenylethane and Phenylmethane Derivatives A feruloyltyramine-synthesizingenzyme that was isolated from tobacco (Nicotiana tabacum) that was infected with tobacco mosaic virus had relatively low specificity for cinnamoyl-CaA thioesters and aromatic amines and was thus capable of synthesizing a wide range of amides.Feruloyl-CoA cinna- moyl-CoA p-coumaroyl-CoA and sinapoyl-CoA were effec- tive sources of the acid function and amines that were well utilized included tyramine octopamine dopamine and n~radrenaline.~~ Tropic acid (3 1) is a rearranged phenylpropane derivative that is found in the form of its esters in a number of tropane alkaloids. The rearrangement has been shown to involve an intramolecular migration of the carboxyl group of phenylalan- ine (30) (Scheme 14). Recent experiments with stereospecific- ally labelled L-phenylalanine substrates in Datura stramonium plants45 have indicated that the conversion occurs with loss of the P(pro-PR)-hydrogen and inversion of configuration at the benzylic centre as the carboxyl group migrates.Whilst loss of the P(pro-PR)-hydrogen was almost total some 30% of P(pro-PS)-label was also lost although complete retention might have been expected. Possible exchange processes were suggested e.g. via transamination or reversible exchange via phenylalan-ine ammonia-lyase. Analogous experiments with Taxus baccata showed that the biosynthesis of (3R)-3-dimethylamino-3-phenylpropionic acid (Winterstein's acid) (32) which is a component of the taxine group of alkaloids gave almost identical results. The P(pro-PI?)-hydrogen of phenylalanine was almost completely lost and some 67% of the P(pro-PS)-label was retained. Cinnamic acid was a poor precursor. In this case a 2,3-shift of the amino-group with stereospecific loss of hydrogen and inversion of configuration at C-p are proposed.45 Related studies on the biosynthesis of tropic acid in both Datura stramonium and D.in no xi^^^ employed as the precursor a mixture of all four diastereoisomers of DL-[Carbox~~-'~c, p-3H Iphenylalanine. Incorporation into the tropane alkaloids scopolamine and hyoscyamine was accompanied by a high retention of tritium significantly more than the expected 50%. Further degradation of these esters of tropic acid showed tritium to be located also on the methylene of tropic acid demonstrating that when the carboxyl group migrates there is also migration (33) of one of the hydrogen atoms from C-P of phenylalanine to form the methylene of tropic acid.The two (30) Tropic acid (31) Scheme 14 NATURAL PRODUCT REPORTS 1985 -P. M. DEWICK sets of data45,46 appear contradictory but the interpretation of results is complicated by the facile racemization of tropic acid for which due allowance must be made. In purified intact chloroplasts of spinach (Spinacia oleracea) phenylalanine and tyrosine were transformed into phenylacetic acids rather than cinnamic However cinnamic acids could be detected along with phenylacetic acids in leaf homogenates indicating the presence of phenylalanine am-monia-lyase and tyrosine ammonia-lyase activities and thus localizing the biosynthesis of secondary metabolites outside the chloroplasts. The formation of toluene from phenylalanine via phenylacetic acid in cultures of Clostridium aerofoetidum has been dem~nstrated.~~ Evidence for the role of phenylalanine was obtained by the detection of [2H,]toluene when cultures were supplied with [2H,]phenylalanine.4.4 Lignins Lignins are natural polymers that are believed to be derived by phenolic oxidative coupling of monomer hydroxycinnamyl alcohol units. The most important of these are p-hydroxycinna- my1 alcohol coniferyl alcohol and sinapyl/syringyl alcohol. These arise from cinnamic acid precursors via the correspond- ing CoA esters and aldehydes. The formation of sinapic acid (36) from ferulic acid (34) proceeds via 5-hydroxyferulic acid (35) (Scheme 19 and although 0-methyltransferases that catalyse the second reaction have been characterized the ferulic acid 5-hydroxylase (trans-ferulate 5-mono-oxygenase) which catalyses the conversion of (34) into (35) has eluded detection.It has now been characterized for the first time in a microsomal fraction of the stem of a hybrid poplar (fopulus x euramericana) and shown to be a cytochrome-P-450-dependent mixed-function mono-~xygenase.~~ Comparison of the regulat- ing properties and the tissue distribution of ferulic acid 5- hydroxylase and cinnamic acid 4-hydroxylase [trans-cinnamate 4-mono-oxygenase (E.C. 1.14.13.1 I)] suggested that the two hydroxylase activities depend on two distinct cytochrome P-450 systems and that ferulic acid 5-hydroxylase is probably involved in the quantitative control of the monomer compo- sition of the lignin that is synthesized in different parts of the plant stem.In contrast cinnamoyl-CoA reductase [E.C. I .2.1.44] and cinnamyl-alcohol dehydrogenase (CAD) [E.C. 1.1.1.1951 from xylem and sclerenchyma tissues of this plant showed little variation in substrate specificities whereas earlier results had indicated that the hydroxycinnamate-CoA ligase isozymes do have varying substrate specificities. This suggests that the reductive enzymes probably play little part in controlling the differing compositions of the lignins of these tissues.so The cinnamoyl-CoA reductase exhibited decreasing affinity towards substrates in the order feruloyl-CoA > sinapoyl-CoA > p-coumaroyl-CoA and CAD catalysed the reduction of all three cinnamaldehydes with highest efficiency towards coniferaldehyde.Only one form of each enzyme was identified. p-Coumaric acid is the best substrate for the hydroxycinna- mate-CoA ligase that has been isolated from cultures of the stems of Salix babyloni~a.~' This was also linked to its possible role in controlling the composition of lignin monomers. No multiple forms of the enzyme were observed in contrast to the C02H CO H CO2H I I I Ferulic acid (34) (35) Sinapic acid (36) Winterstein's acid (32) (33) Scheme 15 NATURAL PRODUCT REPORTS 1985 -P. M. DEWICK results that have been reported for other plants e.g. soybean pea and species of the genus Petunia. The corresponding ligase from wounded fruits of the tomato (Lycopersicon esculentum) acted preferentially on p-coumaric and ferulic acidss2 An 0-methyltransferase that was also studied was particularly active on 5-hydroxyferulic acid.These enzymes contribute towards the rapid lignification of the cells bordering the wounded zones leading to the healing of lesions. A lignin-specific 0-methyltransferase that was isolated from callus homogenates of needles of Douglas fir (Pseudotsuga menziesii)was found to be in a membrane-bound state that was associated with the cell-wall fraction.53 This enzyme was very specific for caffeic acid but had a high K for this substrate. Coupled with the low levels of endogenous substrate that were found in the callus cinnamic acids appear to be channelled into the production of tannins rather than into the synthesis of lignins.4.5 Lignans In a series of feeding experiments using whole plants of Podophyllum he~andrum,~~ it has been shown that phenylalan- ine cinnamic acid and ferulic acid were good precursors of the two major tumour-inhibitory aryltetralin lignans podophyllo- toxin (39) and 4-0-demethylpodophyllotoxin(40). Sinapic and 3,4,5-trimethoxycinnamic acids were poorly incorporated suggesting that the substitution patterns in the pendent aryl rings were built up after coupling of the two phenylpropane units. Degradation of a sample of podophyllotoxin (39) that had been derived from [methyl-’4C]ferulic acid showed equal labelling in both halves of the molecule which implies that the two phenylpropane units that form the lignan skeleton have the same substitution pattern at the coupling stage.This is likely to be the ferulic (4-hydroxy-3-methoxy) substitution pattern. Incorporation of 3,4-methylenedioxycinnamicacid was shown to occur via initial degradation from which the carbon of its methylenedioxy-group entered the C pool and then labelled the methylenedioxy and methoxy substituents of the lignan. 501 The rest of its skeleton was probably incorporated via caffeic and ferulic acids. Interrelationships amongst some of the ten aryltetralin lignans that have been isolated from Podophyllum hexandrum were investigated by feeding labelled lignan~.~~ Deoxypodo-phyllotoxin (37) was hydroxylated to podophyllotoxin (39) which in turn was oxidized to podophyllotoxone (41) though the latter step was reversible (Scheme 16).A similar sequence exists for the 4-0-demethyl series [(38) (40) and (42)] but lignans of the 4’4-dernethyl series are not methylated to form the 4-0-methyl series. The Podophyllum lignans may thus be subdivided biogenetically into two groups i.e.those with 3,4,5- trimethoxy-substitution in the pendent aryl ring and those with a 4-hydroxy-3,5-dimethoxy-substituted pendent ring although these would seem to arise from a common precursor that has not yet been identified. The synthesis of a range of natural coumarinolignans via chemical and enzymic oxidations has been described.56 The enzymic methods employing horseradish peroxidase (with or without H202) and chloride peroxidase are presumably analogous to the natural processes and usually result in regioselectivity in contrast to the corresponding chemical conversions.The production of propacin (45) from the coumarin fraxetin (43) and isoeugenol(44) is shown in Scheme 17. 4.6 Coumarins The biosynthetic pathway to the 6,7,8-trioxygenated coumarin puberulin (50) in shoots of Agathosma puberula has been investigated by feeding experiment^.^^ Ferulic sinapic and caffeic acids were all more poorly utilized thanp-coumaric acid (46) and the coumarins umbelliferone (47) and scopoletin (49) were well incorporated. A pathway (Scheme 18) has been proposed in which a prenyl ether is formed at a late stage. The implication that ferulic acid (34) is not involved contrasts with the known role of this compound [but not esculetin (48)] as a precursor of scopoletin in tobacco.However it is consistent OR OR (37) R = Me Podophyllotox in (39)R = Me (41) R = Me (38)R = H 4’-0 -Demethylpodophyllotoxin (40)R = H (42)R =H Scheme 16 MePo? HO HO (431 + OMe (44) Me’ XOMe OH Propacin (451 Reagents i horseradish peroxidase H202 Scheme 17 with the observation that caffeic acid is not incorporated into esculetin in chicory (Cichorium intybus). The glucosylation of esculetin to the 7-0-glucoside esculin by cell suspension cultures of Lithospermum erythrorhizon Garde- nia jusminoides and Nicotiana tabacum has been reported. 58 The Lithospermum culture was particularly effective convert- ing nearly half of the substrate that was administered within 24 hours.Aspects of the biosynthesis of coumarins in the Rutaceae have been discussed in a recent review.59 (46) Umbelliferone (47) NATURAL PRODUCT REPORTS 1985 5 Flavonoids 5.1 Flavanones Dihydroflavonols Flavonols Flavones and Anthocy anidins Cell suspension cultures of carrot (Daucus caruta) that have grown in the presence of gibberellic acid fail to synthesize flavonoids because of the low activity of chalcone synthase.60 The biosynthesis of anthocyanins could be restored by supplying appropriate precursors e.g. (2s)-naringenin (5l) eriodictyol (52) and 2,3-dihydroquercetin (54). White-flower- ing mutants of species of the genera Dahlia Streptocarpus Verbena and Zinnia are blocked in the 3-hydroxylation of flavanones to di hydroflavonols by flavanone 3-dioxygenase [E.C.1.14.11.91Y The appropriate enzyme was readily detected in cyanic strains but absent from acyanic mutants and is similar to enzymes from other sources belonging to the 2- oxoglutarate-dependent dioxygenases. The formation of the flavonols kaempferol (55) and quercetin (56) from the dihydroflavonols 2,3-dihydrokaempferol (53) and 2,3-dihydro- quercetin (54) (Scheme 19) could be detected in young flower buds of Matthiola incana but enzyme activity rapidly declined during the development of the flower.62 The enzyme had the same cofactor requirements i.e. 2-oxoglutarate ascorbate and Fe2+ as had been reported earlier for an enzyme from parsley. Homo HO \ Red-flowering strains of Verbena hybrida contain a range of Esculetin (48) Scopoletin (49) Puberulin (50) Scheme 18 0 (2s)-Noringenin (51) R = H Eriodictyol (52) R = OH I 4’-hydroxylated and 3’,4’-dihydroxylated flavonoids.Enzymic studies revealed that a small proportion of the 3’,4’-dihydroxy- flavonoids can be formed directly from caffeoyl-CoA in that the chalcone synthase would accept this substrate.63 The more common route (via p-coumaroyl-CoA and with 3’-hydroxyla- tion occurring at the flavonoid level) accounts for the greater proportion however. Flavanone oxidase enzymes which could transform (2q-naringenin (51) into the flavone apigenin (57) or eriodictyol (52) into luteolin (58) were also demonstrated (Scheme 19).These reactions required NADPH as the cofactor. 5.2 Procyanidins An extract from cell suspension cultures of the needles of Douglas fir (Pseudolsuga menziesii) contained reductase en- zymes that were capable of converting (+)-2,3-dihydroquerce-tin (54) into the 2,3-trans-flavan-3,4-cis-diol leucocyanidin (59) and (59) into (+)-catechin (60) (Scheme 20).64The reactions were NADPH-dependent. The same intermediate (59) was 0 2,3 -Oihydrokaempferol (53) R = H 2,3-Oihydroquercetin (54) R = OK 1 Apigenin (57) R = K KaempferoC (55) R = H Luteolin (58) R = OK Quercetin (561 R = OH Scheme I9 NATURAL PRODUCT REPORTS 1985 -P. M. DEWICK detected in experiments utilizing mutants of barley (Hordeurn ~ulgare)~~ which showed that the biosynthesis of procyanidins could be blocked via four different genes.Two of these controlled the conversions that are illustrated in Scheme 20. The incorporation of phenylalanine substrates that were labelled stereospecifically with 3H at C-f3 into (+)-catechin (60) and (-)-epicatechin (61) was shown to be accompanied by an apparently random loss of the 3H label at least half being lost in some cases.45 This pattern was consistent in all of the plants that were studied i.e. Salix caprea and Taxus baccata for (+)-catechin and Aesculus x carnea and Prunus laurocerasus for (-)-epicatechin. For comparative purposes the biosyntheses of Winterstein’s acid (see Section 4.3) in T. baccata and the cyanogenic glucoside prunasin in P. laurocerasus were also studied and shown to proceed with stereospecific loss of hydrogen from phenylalanine contrasting markedly with the random loss that was observed for (60) and (61).This suggested that rapid equilibration of the P(pro-PR)-and P(pro-pS)-hydrogens in phenylalanine probably catalysed by a trans- aminase occurred during the biosynthesis of procyanidins and raised the possibility of alternative pathways to dihydrofla- vonol intermediates. A pathway involving an or-hydroxychal- cone that is derived from phenylpyruvic acid is one proposal. In this case phenylalanine ammonia-lyase would no longer be an obligatory enzyme in the biosynthesis of flavonoids. 5.3 Methylation of Flavonoids A flavonoid 0-methyltransferase (OMT) system that was isolated from suspension cultures of cells of apple fruit66 had properties rather different from those of most other flavonoid OH (541 J (59) (+I-Catechin (60) &heme 20 (-) -Epicatechin (61) (62) OH HO GlcO OGlc -0-Rha -p-Coum (63) p -Coum = p -coumaroyl OMT’s that have been reported.It was specific for flavonols with hydroxyl groups at both C-3’ and C-4 attacked mainly the 3-hydroxyl of quercetin (56) and not that at the 3’-position and accepted partially 0-methylated substrates such as 3-0-methyl- and 7-0-methyl-quercetin giving 3,7-di-O-methyl- and 3,4’,7- tri-0-methyl-derivatives ; this indicates that several OMT activities are present. An 0-methyltransferase that catalyses the methylation of the 7-hydroxyl of the flavone C-glycoside vitexin 2”-0-rhamnoside (62) has been isolated from primary leaves of oat (Auena Biosynthetic intermediates e.g.naringenin and vitexin were not methylated suggesting that this reaction is probably the last step in the pathway to this flavonoid. Four 0-methyltransferases that are involved in the methylation of anthocyanins in flowers of Petunia hybrida have been separated and studied.68 Methylation in vitro of the delphinidin 3-0-[(p- coumaroyl)rutinoside] 5-0-glucoside (63) reflected the accumu- lation patterns of methylated anthocyanins in vivo and established the regulatory roles of the 0-methyltransferases in Petunia hybrida. 5.4 Glycosylation of Flavonoids An anthocyanin Os-glucosyltransferase from flowers of Petunia hybrida glucosylated 3-0-[ (p-coumaroyl)rutinoside]-derivatives of delphinidin and petunidin [cf.(63)] but delphinidin 3-0-rutinoside and delphinidin 3-0-glucoside were not substrate^.^^ Anthocyanidin 03-glucosyltransferase and flavonol 03-gluco- syltransferase activities [E.C. 2.4.1.11 5 and 2.4.1.91 respec- tively] from this plant were proved to arise from the same enzyme though it exhibited higher activity towards flavonols than towards anthoc~anidins.~O The anthocyanidins delphini- din and cyanidin and the flavonols kaempferol quercetin and myricetin were glucosylated. Although only one enzyme is involved in P. hybrida the literature suggests that flavonol- specific and anthocyanidin-specik 03-glucosyltransferases exist in other plants. 5.5 Quinol Ethers In the previous review,’ the formation of chalaurenol (67) which is the quinol ether of 2-formyl-6-hydroxycoumaranone via the peroxidase-catalysed oxidation of 2’,4,4‘-trihydroxy- chalcone (64)7 was discussed.Enzymically oxidized deriva- tives from (64)had been isolated previously72 and structures had been proposed. Re-analysis in the light of the more recent study has resulted in two of these being assigned to chalaurenol and the spiro-epoxide (66) with the third being reaffirmed as the dioxetane (65). The sequence in Scheme 21 has been suggested.73 The structure of chalaurenol had been likened to those of capillarisin (68) and conyzorigun (69). Attempts to study the biosynthesis of conyzorigun in whole plants of Ageratum c~nyzoides~~ showed that structure (69) must be incorrect and the constitution of conyzorigun has now been revised to that of the known flavone eupalestin (70).Acetate and phenylalanine precursors were incorporated as expected. 5.6 Isoflavonoids Isoflavonoids are formed in Nature from flavonoid C&-c6 precursors but during the biosynthesis the aromatic ring that is derived from phenylalanine/cinnamic acid migrates to the adjacent carbon of the C3 unit. Over the years many hypotheses have been proposed for the mechanism of this aryl migration though none has accommodated all of the available biosynthetic evidence. A significant breakthrough in this field is the long-awaited isolation of the enzyme that catalyses this crucial step. An isoflavone synthase activity was detected in a microsomal preparation from cell suspension cultures of soybean (Glycine max) that had been challenged with a glucan elicitor of phytoalexins from Phytophthora megasperma f.sp.NATURAL PRODUCT REPORTS 1985 0 0 (65 1 (66) Chalaurenol (67) Scheme 21 HOP 0 Me0Me0 It &Me Me0 \ gly~inea.~~ The enzyme activity had a half-life of only about 10 minutes at 30 "C required NADPH and molecular oxygen as cofactors and transformed the flavanone substrates (2s)- naringenin (51) or (2S)-liquiritigenin (7 1) into the isoflavones genistein (72) or daidzein (73) respectively. Although the microsomal preparation also contained chalcone isomerase [E.C. 5.5.1.61 it is believed that flavanones rather than chalcones are the true substrates.The cofactor requirements indicate that the enzyme is a mono-oxygenase and a hypothetical pathway via epoxidation of the enol form of the flavanone has been proposed (Scheme 22). Isoflavone synthase was also detected in untreated cell cultures and seedlings but the levels of the activity increased significantly in both tissues after they had been challenged with the glucan elicitor. Feeding experiments in CuC1,-treated seedlings of red clover (Trifoliurn pratense) using ,C- and 2H-labelled 2',4,4'-trihy- droxychalcone confirmed that the rearrangement process in the biosynthesis of isoflavonoids was an intramolecular migration of an aryl Thus the [I 3C,]trihydroxychal-cone (74) was transformed into the labelled isoflavone formononetin (75) and the corresponding labelled pterocarpan phytoalexins medicarpin (76) and maackiain (77) with reten- tion of the intact 3C-1 3C linkages (Scheme 23) demonstrating the aryl migration.The intramolecular nature of the migration was proved by the isolation of 2H3-labelled formononetin and medicarpin after feeding the [2H3]chalcone (78) to the plants as shown by mass-spectral analysis. The sample of maackiain that was produced in the same experiment consisted of 'mOMe MeoY? Me0 0 Yo OJ approximately equal amounts of 2H2- and *H,-labelled species the latter molecule (79) undoubtedly arising via a [2H3]formon- onetin through an NIH shift during the build-up of the more complex substitution pattern in this pterocarpan.This was confirmed in a further experiment via the use of ,H n.m.r. spectroscopy which showed the presence of *H at both position 7 and position 10. Experiments with the [2H,]isofla- vanone (80) and subsequent m.s. analysis showed that this compound was converted into a [2Hl]formononetin and a [2H2]medicarpin and gave direct proof of the existence of a metabolic grid of isoflavones and isoflavanones in the sequence to this compound (Scheme 24). Based on incorporation data the sequence from formononetin (82) to medicarpin (85) via the isoflavone (84) and the isoflavanone (83) would seem more important than that via the isoflavanones (81) and (83). The [*H2]chalcone(86)was transformed into formononetin medi- carpin and maackiain with retention of the P-hydrogen but the a-hydrogen was lost making route b of Scheme 24 more likely than route a.The results are best explained in terms of an oxidative process for the aryl migration in which an isoflavone is the first isoflavonoid intermediate to be formed and although a chalcone substrate was postulated here the data are fully consistent with the enzymic studies in Glycine max (Scheme 22).75 The biosynthetic pathway to the glyceollins which are the phytoalexins of soybean has been shown to proceed via the pterocarpans (87) and (88) followed by the introduction of isoprenyl substituents (see ref. 1). An enzyme preparation from NATURAL PRODUCT REPORTS 1985 -P. M. DEWICK -0 OH (2s)-Naringenin (51) R = OH (2s)-Liquiritigenin (71) R = H Genistein (72) R = OH Ocridttin (731 R = ti Scheme 22 Maackiain (77) Scheme 23 H elicitor-challenged suspension cultures of soybean cells has been shown to catalyse the 6a-hydroxylation of (87) to form (88).77 Essential cofactors were NADPH and dioxygen and the enzyme was specific for the (6aR,llaR)-isomer (87); the (6aS,1la$)-isomer from racemic substrate did not react.Analysis of the optical rotatory dispersion of the product confirmed that the hydroxylation proceeded with retention of configuration. The hydroxylase activity was very unstable with a half-life of only 9 minutes at 30 "C and was inhibited by cytochrome c indicating that the enzyme is probably a cytochrome-P-450-dependent mono-oxygenase.Enzyme acti- vities in cells and seedlings were increased several-fold by treatment with the elicitor. A malonyltransferase that catalyses the malonylation of isoflavone 7-0-glucosides at position 6 of the glucose using O D malonyl-CoA as the acyl donor has been purified from roots of chick pea (Cicer arietin~rn).~~ Best substrates for the enzyme were formononetin 7-0-glucoside and biochanin A 7-0-glucoside giving the products (89) and (90) respectively. Isoflavone 4'-0-glucosides were not malonylated and other flavonoid 7-0-glucosides proved to be poorer substrates. A detailed review of the biosynthesis of rotenoids has been published elsewhere in Natural Product Reports,79 and the biosynthesis of pterocarpans and isoflavans in fungally infected legumes has been discussed.80 6 Stilbenes and Phenanthrenes Stilbenes are formed from a cinnamoyl-CoA starter and three molecules of malonyl-CoA in a similar manner to chalcones NATURAL PRODUCT REPORTS 1985 (81) Formononetin (82) I (83) (84) un HOvOMe HOvOMe > Medicarpin (85) Scheme 24 0 (86) (87) R = H (88) R = OH and it might be assumed that stilbene-forming activity could be a side-reaction that is catalysed by a modulated form of chalcone synthase.That this is not the case has been adequately demonstrated in two plants. Cultured cells of Picea excelsa are capable of forming both stilbenes and flavonoids. Although needles of intact plants contain piceatannol (3,3’,4’,5’-tetrahy- droxystilbene) and stilbene glycosides the cultured cells converted phenylalanine and p-coumaric acid primarily into the resveratrol monomethyl ether (91) and the flavanone naringenin.* Partially purified enzyme preparations were assayed for both chalcone synthase and stilbene synthase activities and although the enzymes use the same substrates and exhibit similar molecular properties (molecular weights of entire enzyme and of subunits) they are undoubtedly two different proteins.The purified stilbene synthase was most effective with p-coumaroyl-CoA as its substrate giving resveratrol (92) but other phenylpropanes were accepted especially feruloyl-CoA and dihydro-p-coumaroyl-CoA giving 3,4’,5-trihydroxy-3’-methoxystilbene(93) and 3,4’,5-tri hydroxybi benzyl (94) respectively.The synthesis of stilbenes in cell suspension cultures of peanut (Arachis hypogaea) can be selectively induced by dilution with fresh medium. After this induction the stilbene synthase was extracted and purified to apparent homogene- ity.82 The enzyme showed high selectivity towards p-coumar- oyl-CoA as substrate (producing resveratrol) other structurally related CoA esters being converted less efficiently (by factors of 10 or more). Acetyl-CoA could not be substituted for malonyl- CoA. The purified enzyme was shown to be free from chalcone synthase activity. Furthermore antibodies that had been raised against stilbene synthase were monospecific and did not cross- react with chalcone synthase. The formation of the bibenzyls batatasin I11 (98) and 3,3’,5- trihydroxybibenzyl (97) from m-coumaric acid (95) and dihydro-m-coumaric acid (96) was demonstrated to occur in aerial bulbils of Dioscorea macruora and in tubers of Dioscorea rot~ndafa,*~ but p-coumaric acid (46) was incorporated into resveratrol only.Cell-free preparations showed bibenzyl synthase activity when they were supplied with dihydro-m- coumaroyl-CoA and malonyl-CoA yielding (97) and stilbene synthase activity when they were incubated with p-coumaroyl- CoA and malonyl-CoA yielding resveratrol. The crude preparation also methylated (97) to (98) using S-adenosylmeth- ionine (SAM) as the methyl donor. In the presence of fungal inducers of the production of phytoalexins aerial bulbils synthesize greater amounts of the dihydrophenanthrene hir- cinol (99) along with the phenanthrene batatasin I (100) [or isobatatasin I (lol)].Feeding experiments showed good incorporations of the precursors phenylalanine p-coumaric acid and resveratrol into the phenanthrene and of m-coumaric CH2OCOCHz C02H H HO o HO *ow ‘ OMe (89) R = H (90)R = OH OH (93) (91) R = Me OH Resvemtrol (92) R = H (94) NATURAL PRODUCT REPORTS 1985 -P. M. DEWICK acid into the dihydrophenanthrene. The data indicate that two pathways exist in species of the genus Dioscorea for the biosynthesis of the basic phenanthrene skeleton (Scheme 25). There is a pathway from m-coumaric acid to dihydro-m- coumaric acid to bibenzyls and then to dihydrophenanthrenes analogous to that which has been discovered in species of the genus Orchis (see ref.l) and a pathway fromp-coumaric acid to stilbenes and ultimately to phenanthrenes. The latter pathway (951 probably operates where an oxygen function is present that is para to the ethylene bridge. Full details concerning the nature of prelunularic acid (1 02) which had been isolated from cell cultures of the liverwort H0$ Marchantia polyrnorpha and has now been detected in several other liverworts have been published.84 This compound malonate appears to be a ‘pre-aromatic’ link between ‘phenylpropanoid- polymalonate’ intermediates and the dihydrostilbene (biben- zyl) lunularic acid (1 03). The very facile conversion of (102) intc; HO (103) under acidic or basic conditions has probably masked the ,b OH HO detection of prelunularic acid.Careful re-examination of various liverworts has shown that the true content of (961 (971 prelunularic acid is in fact much greater than that of lunularic acid.8s 7 Quinones and Quinols Cell suspension cultures of Galium mollugo have been shown to produce anthraquinones e.g. lucidin 3-0-primeveroside (1 07) and the naphthoquinol derivatives (1 10) and mollugin (109).86 The production of the biglucoside (1 10) was much increased by HO administering the precursor o-succinylbenzoic acid (OSB) (104) Samples of labelled (107) (109) and (110) that were OMe HO isolated after the feeding of 13C-labelled OSB (104) were shown Batatasin III (981 Hircinol (99) to be remarkably enriched (specific incorporations of 80-90%> and *3C was localized as indicated in Scheme 26.It was thus proven that the biosynthetic sequence involves prenylation at the position corresponding to C-3 of OSB and the sequence in Scheme 26 has been proposed. The high enrichments are probably caused by over-production of 1,4-dihydroxy-3-prenyl- naphthoic acid (106) due t0 administration of OSB and then the levels of (106) are regulated by greater synthesis of the metabolites (107) (109) and (110) especially the last. The OH quinone ester (108) is also known to be a minor metabolite of G. (46) cis -Resveratrol mollugo. The mode of prenylation that is encountered during the biosynthesis of quinone derivatives in Galium mollugo is quite different from that observed in some other organisms where prenylation has been demonstrated to occur at C-2 of OSB.Full details of studies of the prenylation in Streplocarpus dunniz J where this alternative type of pathway occurs (see ref. l) have now been p~blished.~’ The alkylation steps (Scheme 27) that are involved in the biosynthesis of phylloquinol(l1 l) which is the quinol form of vitamin K 1 in spinach (Spinacia oleracea) have been shown to take place at different sites in the chloroplast.88 The prenylation is localized in the envelope membranes whereas Batatasin I (100) R’ = OW R2=OH the methylation occurs in the thylakoid membranes. In plastids of Capsicum annuum the biosynthesis of phylloquinone from Isobatatasin I (101) R1= OH,R2= OMe naphthoquinone naphthoquinol and the dihydroxynaphthoic Scheme 25 acid (105) could be demonstrated to occur in the presence of SAM and phytyl diph~sphate.~~ Other quinone metabolites may be formed in Nature from 4- hydroxybenzoic acid (1 12) and isoprenoid precursors.Cultured cells of Lithospermum erythrorhizon produce the naphthoquin- 0 one shikonin (114) and related materials by such a sequence (Scheme 28). However the cells stop synthesizing quinones when grown in a liquid medium instead of an agar-containing one until a small amount of activated carbon is added.90 A new benzoquinone derivative echinofuran B (1 151 is then pro- duced This material is considered to be an abnormal metabolite from geranylquinol(113) which is a3 intermediate on the pathway to shikonin.The activated carbon is suggested (102 1 (103; to absorb some inhibitor of secondary metabolism. NATURAL PRODUCT REPORTS 1985 0 OH OH OSB (104) (105) 0 OH Olt (107) R = primeverosyl OGlc OH ( 0 =’3c) OGlc (110) Mollugin (109) Scheme 26 geranyl diphosphate CO;! ti OH OH 6” -L q (105) OH OH J (1 12) f$&Ytyl 1 OH 0 OH (111) Scheme 27 OH \ *-q Isolated mitochondria from tubers of potato (Solanurn OH tuberosurn),leaves of spinach (Spinaciaoleracea) and petals of 0 daffodil (Narcissus pseudonarcissus) form a range of interme-diates of the biosynthetic pathway to ubiquinones (1 16) when Shikonin (114) (113) supplied with isopentenyl diphosphate and endogenous or exogenous 4-hydroxy benzoate.In contrast 5-dip hospho- mevalonate which is the immediate precursor of isopentenyl diphosphate was not accepted as a substrate. The compounds that were produced included prenylated 4-hydroxybenzoate prenylated phenols and quinone derivatives with prenyl chains in the range C10-C50. The results suggest that plant mitochondria have their own prenyltransferase and prenyla- tion system similar to the plastid compartment which also utilizes isopentenyl diphosphate. 8 Miscellaneous Shikimate Metabolites 8.1 Ketomycin Ketomycin [(1’R)-cyclohex-3’-enylglyoxylic acid] (1 19) is an Echinofuran B (115) antibiotic metabolite from strains of Streptomyces antibioticus. Scheme 28 NATURAL PRODUCT REPORTS 1985 -P.M. DEWICK HO4 Me H Bakuchiol (121) NHCHO CO2H I CO H I Tuberin (122) AH OH Ii OH (117) ovco2HXanthocillin (123) R’ = R2= H (124) R’ = R2= Me H02ClJ pro-S-R -S edge t I OH (1 18) /.I. CO?H 5 4 (120) Ketomycin (119) Scheme 29 In a series of feeding experiment^,^^ it has been shown to be derived from shikimate via chorismate and prephenate. Phenylalanine was poorly utilized by the organism. Degrada- tion of a sample of ketomycin that had been derived from [U-14C]shikimic acid indicated that only the ring portion was labelled and the side-chain presumably arises by shortening of the side-chain of prephenate. Analysis of the labelling pattern that was produced after administration of [1,6-14C]shikimic acid (1 17) to the bacterium showed that the labelled prephenic acid (1 18) is converted into ketomycin with stereospecific discrimination between the two enantiotopic edges of the ring of prephenic acid the pro-R-S edge giving rise to the C-2’-C-3’ edge of the cyclohexene ring in ketomycin (Scheme 29).Similar specific utilization of prephenic acid is encountered during the biosynthesis of 2,5-dihydrophenylalanine(1 20). This however was shown not to be an intermediate in the pathway to ke tomycin. 8.2 Bakuchiol The novel phenolic meroterpene bakuchiol (121) is known to arise from mevalonic acid and phenylalanine the latter losing its carboxyl carbon during the sequence. Further studies of (125) R1= Me,R2= H biosynthesis in Psoralea corylifolia plants93 have demonstrated the incorporation of phenylalanine cinnamic acid and p-coumaric acid with tyrosine being a much poorer precursor.8.3 9-Phenylphenalenones The biosynthesis of this group of plant pigments and their possible derivation from 1,7-diarylheptanes has been reviewed.94 8.4 Tuberin and Xanthocillin Tyrosine is the precursor of the mould metabolites tuberin (122) and xanthocillin (123) in each case accompanied by loss of the carboxyl group of the side-chain. These compounds are structurally similar in that an extra carbon atom is then attached to the nitrogen of tyrosine giving an N-formyl derivative in the case of (122) and an isocyanide in (123). In recent studies,95 the origins of the unsaturation that is introduced into the side-chain of tyrosine have been investiga- ted.In Streptomyces amakusaensis neither threo-nor erythro-P-hydroxytyrosine acted as an intact precursor of tuberin. Tyramine and octopamine were similarly not implicated in the biosynthesis of (1 22) nor of xanthocillin dimethyl ether (1 24) in Aspergilfus clavatus. Experiments with stereospecifically deu- teriated tyrosines demonstrated that (as)-tyrosine (126) but not (ctR)-tyrosine was incorporated and that specific removal of the P(pro-bR)-proton occurred (Scheme 30). Retention of some of the label at C-cr indicated that the introduction of a double-bond must be associated with loss of the carboxyl group and an antiperiplanar elimination of hydrogen and carbon dioxide has been suggested.In contrast the biosynthesis of xanthocillin monomethyl ether (125) in cultures of Dichotomo-myces cejpii was accompanied by loss of the p(pro-pS)-hydrogen of tyrosine (Scheme 30). This result is consistent with the formation of the Zdouble-bonds of this metabolite by a similar antiperiplanar elimination. The two C units of tuberin can be supplied by C-2 of glycine but not from formate. The use of glycine as a precursor has been further investigated by feeding Streptomyces amakusaensis with [2-I 3C,2-2 H Igly~ine.~~ Carbon- 1 3 n.m. r. analysis indicated that both the methyl group and the formyl group contained species with one or no deuterons attached and the methyl group also contained 3C1H2H2species illustrating that there 510 NATURAL PRODUCT REPORTS 1985 A 24 R.Bode C. Melo and D. Birnbaum Hoppe-Seyler’s 2. Physiol. H Chern. 1984 365 799. 25 J. D. Hermes P. A. Tipton M. A. Fisher M. H. O’Leary J. F. Morison and W. W. Cleland Biochemistry 1984 23 6263. 26 C.-Y.P. Teng and B. Ganem J. Am. Chem. Soc. 1984 106 2463. 27 U. Matern C. Feser and W. Heller Arch. Biochem. Biophys. 1984 235 218. 28 R. Bode and D. Birnbaum Z. Allg. Mikrobiol. 1984 24 67. 29 H. Camakaris and J. Pittard In ‘Amino Acid Biosynthesis and Genetic Regulation’ ed. K. M. Hermann and R. L. Sommerville -+ Addison-Wesley New York 1983 p. 339. 30 R. L. Sommerville in ‘Amino Acid Biosynthesis and Genetic (126) Regulation’ ed. K. M. Hermann and R. L. Sommerville Addison- Wesley New York 1983 p.351. 31 Y. Ushijima M. Nakano and T. Goto Biochem. Biophys. Res. Commun. 1984 125 916. 32 N. Ishikura S. Hayashida and K. Tazaki Bot. Mag. 1984,97,355. 33 V. Subramanian and C. S. Vaidyanathan J. Bacteriol. 1983 160, (125) 651. Scheme 30 34 T. Terao H. Ohashi and M. Mizukami Plant Sci. Lett. 1984 33 47. 35 G. Sandberg Planta 1984 161 398. is some loss of 2H label by exchange during biosynthesis. In 36 B. Langenbeck-Schwich and H. J. Grambow Physiof. Plant. 1984 addition whereas (2S)-[2-*11,Iglycine gave tuberin that was 61 125. labelled in the methyl group only (2R)-[2-2H,Iglycine labelled 37 H. M. Nonhebel and R. S. Bandurski Plant Physiol. 1984,76,979. 38 D. H. Jones Phytochemistry 1984 23 1349. both the formyl and the methyl group. Thus the N-formyl 39 T.Shimizu and M. Kojima J. Biochem. (Tokyo) 1984 95 205. group cannot originate via an isocyanide function and the 40 M. Kojima and R. J. A. Villegas Agric. Biol. Chem. 1984,48 2397. biosynthesis involves stereospecific loss of the 2(pr0-2S)-41 D. Strack M. Bokern J. Berlin and S. Sieg 2.Naturjorsch. Sect. hydrogen from glycine although partial non-specific exchange C 1984 39 902. of hydrogen from C-2also occurs (see above). This fits well with 42 D. Strack B. Dahlbender L. Grotjahn and V. Wray Phytoche-known data on tetrahydrofolate intermediates in C metabo-mistry 1984 23 657. lism. In marked contrast the carbon atoms of the isocyanide 43 B. Dahlbender and D. Strack J. Plant Physiol. 1984 116 375. groups of xanthocillin monomethyl ether are not supplied by @-44 J.Negrel and C. Martin Phytochemistry 1984 23 2797. 2 of glycine C-3of serine or formate despite the incorporation 45 R. V. Platl C. T. Opie and E. Haslam Phytochemistry 1984 23 2211. of these compounds into (1 25). Methionine labelled the methyl 46 E. Leete J. Am. Chem. Soc. 1984 106 7271. group only. The origin of the carbon of the isocyanide groups in 47 A. Bitsch R. Trihbes and G. Schultz Physiol. Plant. 1984 61, (125) remains unknown. 617. 48 J. L. Pons A. Rimbault J. C. Darbord and G. Leluan Ann. Microbioi. (Peris) B 1984 135 219. 9 References 49 C. Grand FEBS Lett. 1984 169 7. 1 P. M.Dewick Nut. Prod. Rep. 1984 1 451. 50 F. Sarni C. Grand and A. M. Boudet Eur. J. Biochem. 1984,139 2 M. J. Fiske and J. 1;.Kane J. Bacteriol. 1984 160 676.259. 3 R. Bode C. M. Ramos and D. Birnbaum FEMS Microbiol. Lett. 51 A. Feutry and R. Letouze Phjltochemistry 1984 23 1557. 1984 23 7. 52 A. Fleuriet and J. J. Macheix Physiol. Plant. 1984 61 64. 4 J. A. Suzich R. Ranjeva P. M. Hasegawa and K. M. Herrmann 53 S. H. Monroe and M A. Johnson Phytochemistry 1984 23 1541. Plant Physiol. 1984 75 369. 54 D. E. Jackson and P. M Dewick Phytochemistry 1984 23 1029. 5 J. W. Frost and J. R. Knowles Biochemistry 1984 23 4465. 55 D. E,. Jackson and P. M.Dewick Phytochemistry 1984 23,>1037. 6 J. W. Frost J. L. Bender J. T. Kadonaga and J. R. Knowles 56 L.-J. Lin and G. A. Cordell J. Chem. Soc. Chern. Commun. 1984 Biochemistry 1984 23 4470. 160. 7 E. J. Lourenco and V. A. Neves Phytochemistry 1984 23 497. 57 S.A.Brown D. E. A. Rivett and H. J. Thompson 2.Naturforsch. 8 C. E. Grimshaw S. G. Sogo S. D. Copley and J. R. Knowles J. Sect. C 1984 39 3 1. Am. Chem. Soc. 1984 106 2699. 58 M. Tabata Y. Umetani K. Shima and S. Tanaka Plant Cell 9 J. J. Lee Y. Asano T.-L. Shieh F. Spreafico K. Lee and H. G. Tissue Organ Cult. 1984 3 3. Floss J. Am. Chem. Soc. 1984 106 3367. 59 M.F. Grundon in ‘Chemistry and Chemical Taxonomy of the 10 H. C. Steinrucken and N. Amrhein Eur. J. Biochem. 1984 143 Rutales’ ed. P. G.Waterman and M. F. Grundon Academic Press 341. London 1983 p. 9. 11 H. C. Steinrucken and N. Amrhein Eur. J. Biochem. 1984 143 60 W. Hinderer M. Petersen and H. U. Seitz Planta 1984 160 544. 351. 61 G. Forkmann and G. Stotz Planta 1984 161 261. 12 J. L. Rubin C. G. Gaines and R.A. Jensen Plant Physiol. 1984 62 R.Spribille and G. Forkmann 2.Naturforsch. Sect. C 1984 39, 75 839. 714. 13 A. Schulz D. Sost and N. Amrhein Arch. Microbiol. 1984 137 63 G. Stotz R. Spribille and G. Forkmann J. Plant Physiol. 1984 121. 116 173. 14 D. Sost A. Schulz and N. Amrhein FEBS Lett. 1984 173 238. 64 H. A. Stafford and €3. H. Lester Plant Physiol. 1984 76 184. 15 D. M. Mousdale and J. R. Coggins Planta 1984 160 78. 65 K.N. Kristiansen Carlsberg Res. Commun. 1984 49 503. 16 K. Duncan A. Lewendon and J. R. Coggins FEBS Lett. 1984 66 J. J. Macheix and R. K. Ibrahim Biochem. Physiol. Pflanz. 1984 165 121. 179 659. 17 K. Duncan A. Lewendon and J. R. Coggins FEBS Lett. 1984 67 W. Knogge and G. Weissenbijck Eur. J. Biochem. 1984,140 113. 170 59.68 L. M. V. Jonsson M. E. G. Aarsman J. E. Poulton and A. W. 18 J. R. Knowles Proc. Robert A. Welch Found. Con$ Chem. Res. Schram Plunta 1984 160 174. 1984 27 149. 69 L. M. V. Jonsson M. E. G. Aarsmaq J. van Diepen P. de 19 S. G. Sogo T. S.Widlanski J T. Hoare C. E. Grimshaw G. A. Vlaming N. Smit and A. W. Schram Planta 1984 160 341. Berchtold and J. R. Knowles J. Am. Chem. Soc. 1984 106 270. 70 I,. M.V. Jonsson M E. G. Aarsman J. Bastiaannet W. E. 20 G. S. Hudson V. Wong and B. E. Davidson Biochemistry 1984 Donker-Koopman A. G. M. Gerats and A. W. Schram Z. 23 6240. Naturforsch. Seci. C 1984 39 559. 21 S. K. Goers and R. A. Jensen Planta 1984 162 109. 71 M. J. Begley L. Crombie M.London J. Savin and D. A. Whiting, 22 S. K. Goers and R. A. Jensen Planra 1984 161 117.J. Chem. Soz. Cheni. Commun. 1982 1319. 23 T. A. d’Amato R. J. Ganson C. G. Gaines and R. A. Jensen 72 E. Wong and J. M. Wilson Phytochcmistry 1976 15 1325. Pluntu 1984 162 104. 73 E. Wong Tetrahedrorr Leti. 1984 25 2631. NATURAL PRODUCT REPORTS 1985 -P. M. DEWICK 74 A. V. Vyas and N. B. Mulchandani J. Chem. SOC. Perkin Trans. 1 1984 2945. 75 M. Hagmann and H. Grisebach FEBS Lett. 1984 175 199. 76 H. A. M. Al-Ani and P. M. Dewick J. Chem. SOC. Perkin Trans. 1 1984 2831. 77 M.-L. Hagmann W. Heller and H. Grisebach Eur. J. Biochem. 1984 142 127. 78 J. Koester R. Bussmann and W. Ban Arch. Biochem. Biophys. 1984 234 513. 79 L. Crombie Nat. Prod. Rep. 1984 1 3. 80 S. A. Popravko G. P. Kononenko and S. A. Sokolova Prikl.Biokhim. Mikrobiol. 1984,20 723 (Chem. Abstr. 1985 102,42806). 81 C. H.Rolfs and H. Kindl Plant Physiol. 1984 75 489. 32 A Schoppner and H. Kindl J. Biol. Chem. 1984 259 6806. 83 K. H. Fritzemeier H. Kindl and E. Schlosser Z. Naturforsch. Sect. C 1984 39 217. 34 Y. Ohta S. Abe H. Komura and M. Kobayashi Phytochemistry 1984 23 1607. 85 S. Abe and Y. Qhta Phytochemistry 1984 23 1379. 511 86 K. Inoue Y. Shiobara H. Nayeshiro H. Inouye G. Wilson and M. H. Zenk Phytochemisrry 1984 23 307. 87 K. Inoue S. Ueda H. Nayeshiro N. Moritome and H. Inouye Phytochemistry 1984 23 3 13. 88 S. Kaiping J. Soll and G. Schultz Phytochemistry 1984 23 89. 89 J. P. Gaudilliere A. d’Harlingue B. Camara and R. Monegar Plant Cell Rep. 1984 3 240. 90 H.Fukui N. Yoshikawa and M. Tabata Phytochemistry 1984,23 301. 91 F. Lutke-Brinkhaus B. Liedvogel and H. Kleinig Eur. J.Biochem. 1984 141 537. 92 Y. Takeda V. Mak C.-C. Chang C.-J. Chang and H.G. Floss J. Antibiot. 1984 37 868. 93 A. Banerji and G. J. Chintalwar Phytochemistry 1984 23 1605. 94 U. Weiss Proc. Indian Acad. Sci. Chem. Sci. 1984 93 1159. 95 R.B. Herbert and J. Mann Tetrahedron Lett. 1984 25 4263. 96 R. B. Herbert and J. Mann J. Chem. SOC. Chem. Commun. 1984 I474.

ISSN:0265-0568    

DOI:10.1039/NP9850200495

出版商:RSC    

年代:1985

数据来源: RSC

摘要:

The Biosynthesis of C.-C.o Terpenoid Compounds D. V. Banthorpe and S. A. Branch Department of Chemistry University College 1ondon 20 Gordon Street 1ondon WC7 H OAJ Reviewing the literature published during 1984 (Continuing the coverage of literature in Natural Product Reports 1984 Vol. 1 p. 444) 1 Introduction 2 Hemiterpenoids 3 Monoterpenoids 4 Sesquiterpenoids 5 Diterpenoids 6 Tissue Culture Biotransformations Genetics and Chemotaxonomy 6.1 Tissue Culture 6.2 Biotransformations 6.3 Genetics 6.4 Chemotaxonomy 7 References 1 Introduction Studies of the incorporation of presumed biosynthetic inter- mediates in vivo or the labelling of specific positions in products by appropriately labelled precursors are becoming less common as knowledge of the outlines of most metabolic pathways (and of the existence of metabolic grids of relatively unspecific enzymes that can functionalize intermediates in different sequences) has accumulated.In contrast the use of cell-free extracts continues to increase and to give important informa- tion on the details and the enzymology of several routes to lower terpenoids in higher plants (see Sections 3 and 4 especially). Much work is now appearing concerning the production of terpenoids and of secondary metabolites in general in tissue cultures of higher plants and a few studies deal with more fundamental points such as the control of synthesis and degradation and the influence of environmental factors on these processes.This work is reviewed in Section 6 together with other more biological aspects of the subject. Another feature is the use of stable isotopes which are detected in intermediates or in products by n.m.r. to provide biosynthetic information. Biogenetic schemes are often presented to account for the formation of new and not so new terpenoids. These are only reported where in our opinion some novel point of view is involved. Monographs have become available on secondary metab- olism in micro-organisms plants and animals’ and on hydroxymethylglutaryl-CoA reductases [E.C. I. 1.1.88 and E.C. 1.1.1.341.’ Chapters in symposium-type volumes deal with the chemistry and biogenetic relationships of terpenoids in gene~al,~ the biosynthesis and catabolism of m~noterpenes,~ the biosynthesis of sesquiterpene lac tone^,^ and the accumu- lation of terpenoids especially sesquiterpenes in fungally infected potato.6 A review assesses the achievements and prospects for the study of plant metabolism in vivo by n.m.r.techniques. Much work has been carried out on hydroxymethylglutaryl- CoA reductase (NADPH) [E.C. 1.1.1.341 as befits its importance as the catalyst of what is generally considered to be the rate-limiting step in the biosynthesis of terpenoids and which is subject to feedback control. Most studies have used rat liver preparations but the conclusions are believed to be generally applicable for all mammals birds and higher plants OH in which this enzyme occurs. The modulation of the enzymic activity by phosphorylation and dephosphorylation has been investigated in great detail,*-’ and has been shown to control the overall rate of synthesis of terpenoids’ ’-I4 and the allosteric properties of the proteins.* Immunological and biochemical studies of the active and inactive forms of the reductase have revealed that the latter is not an artefact of isolation but occurs in oioo in rat liver in four-fold excess over the active species. ’. The. reductase and its kinases are regulated by mevalonic acid (MVA) in rat liver;I9 structurally related drugs such as compactin and mevinolin are competitive inhibitors for the enzyme in explants of Hefiunthus tuberosus resulting in inhibition of growth.’O One diastereoisomer of cyclomevalonic acid (1) is a potent inhibitor of the reductase (Ki = mol dm-3) whereas the other has no effect.21 Two distinct species of the reductase -one chloroplastic and the other extrachloroplastic -were found in Nepeta cutaria; a similar distribution existed for mevalonate kinase and the situation may be general for higher plants.*? The use of doubly labelled precursors in wheat seedlings has provided evidence that the sequence of recycling of mevalonate to HMG-CoA and thence viu C moieties to acetate and long-chain (predominantly c?2-c26) fatty acids is analogous to the similar shunt in animals.It was concluded that in both plants and animals the route has real metabolic significance in the control of the synthesis of terpenoids (especially steroids) and is not a minor vestigial pathway.*3 Full details have appeared of a synthesis of (RS)-mevalono- lactone that is especially useful for the preparation of 3- and/or 3’-labelled compounds.24 2 Hemiterpenoids Isopentenyl-diphosphate A-isomerase and prenyltransferase activities (mol. wt 34 and 64 kdalton respectively) have been purified up to 250-fold from the plastids of tomato fruits.’ Prenyltransferase in particular seems to vary widely (mol. wt of 45 to 80 kdalton) in preparations that have been described from different animal and plant sources. Certain enzymes that are involved in the formation of isopentenyl diphosphate could only be detected as membrane-bound forms in the endoplasmic reticulum (e.g. mevalonate kinase and phosphomevalonate kinase) of purified spinach chloroplasts.Presumably the isopentenyl diphosphate that is formed in the cytoplasm is distributed to other cellular components (endoplasmic reticu- lum plastids and mitochondria) for further biosynthetic elaboration.26 A cytosolic fraction of rat liver has been demonstrated to convert mevalonate into isoprene.27 An astonishing claim was made in 1983 that exceptions had been discovered to a ‘rule’ that the formation of double-bonds in the extension of terpenoid chains by C5 units requires the loss of 4( pro-4s)- or 4( pro-4R)-hydrogen atoms of mevalonate accord- ing to whether the configuration of the double-bond is E or 2 respectively. In fact abrogations of this ‘rule’ have been known since 1972 in many instances and the more recent claim has been retracted.28 A detailed review covers the sequence of formation and functionalization and the biological activities of prenylated coumarins flavonoids and phenols occurring in the members of the flora of the USSR.29 3 Monoterpenoids The biosynthesis of monoterpenes has been and a compendium of properties biosynthetic routes and pharma- cological activities of the class has become generally avail- able.33 The role of the glandular trichomes in the biosynthesis of terpenoids has been assessed34 and the secretory glands of Saluia oficinalis have been shown to consist of two types of trichome each type being capable of producing the four major skeletal types of monoterpene (p-menthane pinane cam-phane and thujane) that are characteristic of the species.35 Kinetic analysis of the non-enzymic solvolysis of neryl and geranyl diphosphates showed that the dissociation constants and rates of subsequent reactions of the bis-metallic complexes of these esters with Mg2+ and Mn2+ were larger for the former ester by ca 10-fold.Similar behaviour occurred for Co2+ and in general elimination and cyclization were catalysed by metal ions. The carbocyclase-catalysed formation of cyclic monoter- penoids from the acyclic precursors invariably involves metallic ions as cofactors and it has been suggested that the ‘true’ substrate is the magnesium complex of neryl diphos- phate whereas complexes of geranyl diphosphate lead to other reactions in the isoprenoid pathways.36 Many halogeno- monoterpenes (especially mixed bromo-chloro-derivatives) occur in marine organisms (especially seaweeds and other algae).Experiments with model compounds have shown that bromide peroxidases exist that do not directly oxidize C1- but which in the presence of Br- C1- and HzOz react with alkenes to give bromo-chloro-compounds. The same reaction is catalysed when seawater is the source of the halide ions and Br Br (5) NATURAL PRODUCT REPORTS 1985 this suggests that a bromonium-ion-induced biosynthesis of halogenated monoterpenes (and higher terpenoids) occurs. Thus the organisms that are involved in such biosyntheses possess bromide peroxidases that ignore the vast excess of C1-; these enzymes control the synthesis uia the minute ambient concentration of Br- to yield the very small amounts of chlorinated products that are necessary (?)for the well-being of the organism.The advantage of utilizing a bromo-chloro-rather than a dichloro-intermediate aside from control factors is that bromine is more easily removed in subsequent steps than is chlorine. Thus it has been suggested that chlorinated monoterpenes are formed by the addition of BrC1 followed by the loss of bromine rather than by direct chlorination. Bromonium-ion intermediates were suggested to account for the known occurrence of plocamene D (2) and violacene (3) and for the acyclic compounds (4) and (5). Evidence was also adduced for an iodonium-ion-mediated pathway to chlorinated monoterpenes but previous claims for the presence of a chloride peroxidase in marine organisms have been discounted.a-Terpinene (6),which is a major component of the fruit oil was converted into ascaridole (7) in cell-free extracts from the fruit of Chenopodium arnhrosioides (wormseed). The peroxidase complex that is responsible for this conversion was fractionated and the activity was shown to be susceptible to proteolytic destruction only after it had been treated with a periodate; this suggests that the enzyme is associated with carbohydrate material. The biosynthesis of ascaridole was inhibited by CN- catalase and reducing agents but not by compounds that trap 0 or which quench singlet oxygen; consequently it was concluded that a peroxide-transfer reaction that is initiated by a peroxidase-generated I+ (or its equivalent) was re~ponsible.~~ Paraquat and diquat stimulate the formation of ‘resin’ and other secondary metabolites in species of the genus pi nu^.^^ A long-standing intriguing problem in Pinus species is that a-pinene occurs as the (+)-or (-)-isomer but there is a wide range of optical purities of this terpene in different species (ca 90%enantiomeric excess at the extremes) whereas 0-pinene is found almost invariably as the (-)-isomer (>90% enantio- meric excess) and typically co-occurs with (+)-a-pinene of the opposite absolute configuration.Feeding experiments with several Pinus species showed that C-3 of both (+)-and (-)-a-pinene and of (-)-P-pinene was derived from C-2 of mevalonate.This pattern is consistent with two enzymic routes for bicyclization of the acyclic precursor -one leading to (-)-P-pinene (8) (where 0 indicates 14C from [2-1SC]mevalonate) and the other to (+)-r-pinene (9). (-)-a-Pinene (10) was con- sidered to result from subsequent isomerization of the (-)+ pinene; very small amounts of (+)-P-pinene (1 1) arise from a similar (but thermodynamically unfavourable) isomerization of the (+)-a-isomer. Thus the (-)-a-pinene that feeds into the product from the ‘P-pinene’ route could largely dilute the optically pure (+)-a-pinene that is formed directly (as the p-t a NATURAL PRODUCT REPORTS 1985 -D. V. BANTHORPE AND S. A. BRANCH rearrangement is very favourable) whereas any ( -)-P-pinene that is directly formed is optically diluted to a very limited extent by the (+)-P-pinene that feeds in from the ‘a-pinene’ pathway.40 This self-consistent model may however have to be adjusted or abandoned if conclusions from studies on the biosynthesis of pinenes in Salvia oficinalis are generally applicable.A soluble enzyme preparation from immature leaves of this sage was fractionated into two pinane cyclases (I and 11). Component I (mol. wt ca 96 kdalton) catalysed the conversion of geranyl diphosphate into ( +)-a-pinene together with smaller quantities of the rearranged alkene (+)-camphene (1 2) and the monocyclic (+)-limonene (1 3). In contrast component I1 (mol. wt ca 50 kdalton) transformed the acyclic precursor into (-)-P-pinene and (-)-a-pinene and also into smaller amounts of (-)-camphene (14) (-)-limonene (19 and rnyrcene (1 6).The minor multiple biosynthetic activities could be co-purified with those of the pinane cyclases as far as was studied; the abilities to accept geranyl neryl and linaloyl diphosphates as substrates were likewise coincident throughout purification. Detailed speculations involving the intermediacy of formal carbo-cations have been proposed to accommodate these findings.41 It will be instructive to determine if similar multiple activities occur in species of the genus Pinus and other genera. Geranyl and linaloyl diphosphates are preferred over neryl diphosphate as substrates for monoterpene cyclases in various species. Cell-free preparations from Foeniculum uulgare (fennel) yielded (-)-endo-fenchol (1 7) from geranyl diphosphate; despite vigorous attempts to obtain cyclase preparations that were free of phosphatases fenchyl diphosphate could not be detected as an intermediate.This situation differs from that for cyclization to form the camphane skeleton where bornyl diphosphate is formed and has been shown to be a precursor of borneol. In F. uulgare presumably for steric reasons direct attack by water on the presumed bicycloterpinyl cation :pyro-phosphate ion-pair leads to the alcohol. Consistent with this hypothesis no label was found in the fenchol if [l-180]geranyl diphosphate was used as the substrate; thus the oxygen in the product did not arise from that of the diphosphate group in the precursor.As all of the plant phosphatases that have been (18) $HO (20)R = Me (21) R = CHO CHO studied catalyse the cleavage of 0-P bonds this indicates that fenchyl diphosphate is not an intermediate.42 Details of the pathways to the iridoid glucosides and of the incorporation of these units into terpene alkaloids (the latter are not covered in this Report) still remain to be elucidated. The administration of 3H-labelled precursors to Catharanthus roseus and Lonicera morrowi showed that loganin (22) secologanin (23) and other seco-iridoids arose uia cyclization of 10-oxogeranial (18) and 10-oxoneral (19) with iridodial (20) and iridotrial (21) as intermediate^.^^,^^ This contradicts previous work (in 1983) which claimed to demonstrate the intermediacy of 9,lO-dihydroxygeraniol and its 0x0-derivative in the biosynthesis of secologanin in cell cultures of C.roseus.Also the incorporation of 3H-labelled cyclopentanoid mono- terpenes (24) (25) and (26) into dolicholactone (27) in Teucrium marum conformed the previously proposed route.45 More detailed studies of the incorporation of *H-and 13C- labelled monoterpenes into iridoids in cell cultures of Gardenia jasminoides demonstrated the route (18)/(19) -P (20) + 8-epi-iridodial (28) -+ 8-epi-iridotrial (29) -+ boschnaloside (30) + dehydroiridotrial 0-glucoside (3 I) + more highly oxygenated glucosides such as gardenoside (32).46 Photocitral-A (33) may be a significant precursor of (27) and of nepetalactone (34) and its dihydro-derivative in Teucrium marum and Nepeta ~ataria.~’ 10-Hydroxycitronellol (35) was a more effective precursor of (31) in N.cataria than was either 10-hydroxygeraniol or 10-hydroxynerol and 3H-labelling showed that iridodial(20) was a key intermediate to (34) and that a shift of hydride from C-1 to C-10 preceded the lactonization step.48 Previously it was shown that (-)-menthone is reduced to (-)-menthol and (+)-neomenthol in leaves of Mentha spicata and that part of the former product is converted into menthyl acetate whilst the neomenthol is converted into its P-D-glucoside which is then transported to the rhizome. The glucoside is subsequently metabolized in the rhizome,49 being hydrolysed re-oxidized to menthone and lactonized to the lactone (36) en route to more polar products; all of the individual steps have been demonstrated to occur in cell-free extracts of rhizomes.The lactonization step is of particular interest because the p-menthane ring is cleaved to give an HO 3) A (16) (17) OGlc + OHC ;r COzMe (221 (23) / (24) T t (25) (26) CIS + I; CHO / (28) R = Me (30) (29) R = CHO I %OH (33) (34) acyclic skeleton that can be further degraded by modification of the P-oxidation sequence. Lactonization of (+)-camphor and conversion of the corresponding hydroxy-acid into a glucoside glucose ester also occurs in Salvia ~ficinalis.~~ This resembles a similar route of catabolism of camphor that is utilized by micro-organisms but is the first report of this type of reaction in higher plants.The biosynthesis of the propylcanna- binoid acid (37) in Cannabis sativa and its relationship with methyl- and pentyl-cannabinoid acids have been demon-~trated.~' 4 Sesquiterpenoids Differing profiles of time-incorporation of 4C from C02 acetate and mevalonate into a variety of sesquiterpenes (including cubebenes cadinenes longifolenes and copaenes) NATURAL PRODUCT REPORTS 1985 OH (27) HO b; HOGo / I CHO C02Me (31) (32) I I (35) (36) in needles of Pinus pinaster were interpreted as indicating that the detailed mechanisms for the various classes of products differed for the individual precursors; this presumably results from compartmentation and overloading effects.The use of [14C]C02 (which is the only precursor that can be fed at physiological concentrations) was claimed to reveal the importance of trans-P-farnesene (38) as a transitory intermedi- ate prior to cy~lization.~~ These conclusions extend previous proposals that ocimene and myrcene [i.e.the Cl0analogue of (38) and an isomer] were similarly significant en route to a-and P-pinenes. However there appears to be no evidence from other tracer or enzymic studies to suggest that these views are generally applicable. In contrast the cyclization of (2E 6E)-[ 12,13-l4C2;9-3H2]farnesyl diphosphate (39) to pentalenene (40) which is the parent hydrocarbon of the pentalenolactone family of sesquiterpene antibiotics in cell-free extracts from NATURAL PRODUCT REPORTS 1985 -D.V. BANTHORPE AND S. A. BRANCH t @O * (39) (not free 1 (421 1 (43) (44) i (46) = C2H3 species of the genus Streptomyces resulted in the retention of both equivalents of 3Hat the positions shown (as established by a combination of chemical and microbiological methods) and this was considered to show that cyclization from the precursor occurred at a single active site of a unique enzyme and that there is no intervention of kinetically free intermediates.53 The cyclization of farnesyl diphosphate to humulene (42) and caryophyllene (43) has been demonstrated to occur in cell-free extracts from Saluia oficinalis and the specificity of labelling of the terpenes was proved by enzymic degradation of the products that were formed from specifically labelled precursor.The enzyme (system ?) was dependent on divalent metal cations and represents the first example of the preparation of a soluble C ,-cyclase from a higher plant.s4 y-Bisabolene (44) which is a notional intermediate in the formation of several classes of sesquiterpene may be formed either by cyclization of (2E 6E)-farnesyl diphosphate (41) or of its (22 62)-isomer (45). Very detailed studies with intact callus cultures and derived enzymic extracts from Andrographis paniculata have revealed that (a) the y-bisabolene that was biosynthesized indeed had the 2 configuration (as had been previously shown); (b)the immediate precursor is the (6E)- rather than the (62)-farnesyl diphosphate; and (c) consistent with (b),in the paniculide B that was derived (and probably in the y-bisabolene) the carbon atom in the ring that was derived from C-2 of mevalonate was anti to the side-chain (H as shown).In addition both mevalonate and (41) were incorporated into (44) without loss of hydrogen respectively from C-5 or from C-1 cyclization to the hypothetical bisabolenyl cation therefore did not involve a prior E -+ 2isomerization of the terminal double- bond by a redox mechanism. Thus there must exist a (2E,6E)- farnesyl-diphosphate isomerase-cyclase linked system that is independent of a previously identified (2E 6E) -+ (22 6E) isomerase system that is responsible for the formation of (2E 6E)-farnesol in these culture^.^^ These studies illustrate some of the particular advantages of plant tissue cultures in biosynthe- tic studies -the ready preparation of an active cell-free system and high incorporations of tracers.In another example of the application of a plant tissue culture the injection of [6,6,6-2HH,]mevalonate into callus of Perilla jiutescens and g.c.-m.s. examination of the distribution of tracer in the products revealed (not unexpectedly) that the cuparene (46) that was biosynthesized was labelled as shown.s6 Feeding [ 1-13C]- and [2-’ 3C]-acetate to Stereum purpureum showed that sterpuric acid (48) and sterpurene-3,12,14-triol (49) which are members of the unique class of sterpurenes that is produced by this basidiomycete fungus were probably formed via humulene (42) and the protoilludane cation (47); variants of the pathway were also discu~sed.~’A new hydrocarbon isopatchoul-3-ene (50),that has been obtained from Cyperus scariosus may be a biogenetic precursor of isopatchoulenol (51).ss Phytoalexins of Ipomoea batatas (mainly furanosesquiter- penes) and the general metabolism of the class of compounds have been re~iewed.~~.~~ The incorporation of ‘H-labelled mevalonate into ipomeamarone (59 which is the major phytoalexin of fungally infected I.batatas revealed an unusual migration of a double-bond involving a stereospecific 1,3-hydride shift the whole being envisaged to occur whilst the intermediates were enzyme-linked; cj (52) + (59 where H’ and HA refer to the prochiral hydrogen atoms at C-2 of mevalonate.61 Derivatives of (55) have been investigated,6’ and tracer studies indicated that a new phytoalexin (56) [= (52) the latter presumably being released from the postulated enzyme complex?] from I.batatas was a close precursor of dehydroip~meamarone.~~ Ipomoea batatas also yielded a NADP-dependent farnesol dehydrogenase that efficiently converted (2E 6E)-farnesol into the corresponding aldehyde but which also showed broad substrate specificity. The enzyme (mol. wt 90 kdalton) was present with low activity in intact tissue of sweet potato but the level rapidly increased if the tissue was damaged or infected with Cvratocvstis .fimbriata.6J NATURAL PRODUCT REPORTS 1985 Work on abscisic acid (ABA) (57) continues unabated; some resuspended mycelium of the fungus but (2E 6E)- and (2E of the most interesting has exploited the fungus Cercospora 62)-farnesol (2E,62)-farnesyl diphosphate and (2E 6E)- and rosicola (a plant pathogen) which synthesizes large quantities (2E 6Z)-farnesoic acids were not; these findings delineated the of the hormone.Carbon-1 4-labelled (2E 6E)-farnesyl phos- sequence of reactions and in particular suggested that the phate and diphosphate were both converted into ABA by transformations involving the terminal carbon atoms of the side-chain occurred subsequent to functionalization elsewhere in the molecule.65 A sample of ABA that had been produced from [ 1,2-l 3C,]acetate by the fungus was analysed by detailed INEPT and n.0.e. techniques to determine the labelling pattern and to establish the absolute configurations at C-1' and C-6'.The configurations that were determined were compat- ible with a chair-folded intermediate during the cyclization H+ procedure similar to that which had previously been proposed for the construction of the p and E end-groups of carotenoids.66 A micro-organism that was discovered in soil (Corynebacterium sp.) metabolized ABA to dehydrovomifoliol (59) as the main product. As a cell-free extract of this organism also demon- strated vomifoliol dehydrogenase activity it is likely that vomifoliol (58) is the immediate precursor of (59).67Abscisic acid accumulates in wilted detached leaves of Xunthiurn strumarium but only one of the four oxygen atoms is derived from O2;this is in the C02H group.This suggests that either the remaining oxygen atoms are obtained from water at the time of stress or there exists a stored precursor in which oxygen (47) is already present in the corresponding positions for ABA that is converted into the latter under conditions of water stress. On rehydration of the leaves the excess of hormone is deactivated by the formation of phaseic acid (60) inter alia now the oxygen H atom of the bridge is derived from 02,i.e. an oxygenase is directly involved.68 Cell suspension cultures of Lycopersicon peruuzunum deactivated ABA to (60) and the novel metabolite (61).69 The metabolism of (-)-(R)-and (+)-(S)-ABA has been compared70 and the pathways of deactivation of exogenously supplied mate~ial~l-~~ and of that which has accumulated R' while plants are under stress have been re-expl~red.~~~~~ A (48)R'= Me,R2=C02H convenient radioimmunoassay-h.p.1.c.technique for the detec- (49)R'= R2= CH20H tion of ABA has been described.76 Certain fungi produce a group of biosynthetically related metabolites of mixed polyketide-terpenoid origin :77,78 thus andibenin B (64) from Aspergillus variecolor comprises a moiety I that has been derived from farnesyl diphosphate [cf. (62) + (64)]. Detailed information concerning the later functionaliza- tion of the molecule the intermediate oxidation states and mechanisms has been deduced from impressive analyses of the isotope shifts that are induced in the 13C n.m.r. spectra by l80 that has been introduced into andibenin B from acetate and (51) from gaseous oxygen.77 The incorporation of [2-I4C]acetate (53) A H 519 NATURAL PRODUCT REPORTS 1985 -D.V. BANTHORPE AND S. A. BRANCH and the effect of exogenous farnesol and farnesoic acid on the formation of juvenile hormone JH-III(65) has been reported.79 5 Diterpenoids The full paper on the biosynthesis of the interesting and possibly important antiviral and tumour-inhibitory agent aphidicolin (69) by the micro-organism Cephalosporium aphidi- cola has now been published.80 The incorporation of label from [I-’ 3C]- [2-l3CC]- and [ 1,2- 3C2]-acetate led to the characteriza- tion of the component C5 units and the numbers of 2- and 5-mevalonoid hydrogen atoms that were incorporated were determined by 3H-labelling.These data enabled the outline of the pathway to be deduced and the zH-13C spin coupling that was observed in the *H n.m.r. spectrum of product that had been biosynthesized from [3-’3C 4-2H2]mevalonate further proved the migration of a 9b-hydrogen to C-8. Thus the full pathway summarized here from geranylgeranyl diphosphate [(66) -,(69)] was established. The occurrence of the hydride shift led to an interesting proposal that cyclic diterpenoids in which the initial cyclization forms a bicyclic intermediate can be divided into two classes according to whether geranylger- any1 diphosphate adopts a chair-chair or a chair-boat conformation in this initial step.81 Parvinolide (70) which is a new sesquiterpene lactone from Pogostemon parugorus could arise from an enzymatic Baeyer-Villiger oxidation of the ketone that is derived from 10-hydroxycaryophyllene.82Whilst (56) a number of examples of sesquiterpenoid &lactones are known this is the first example of the natural formation of such a compound by the (presumed) oxidation of a cyclobutanone ring.New diterpenes from marine sponges of the genus Dendri//a have been characterized and have led to biogenetic speculations whereby norrisane (72) and dendrillane (73) are derived from a common precursor (7 1p3Geranylgeranyl phosphate and diphosphate and phytyl diphosphate were effectively (3 12 and 12% respectively) incorporated into the chlorophyll of tobacco cell cultures within 12 hours. Tracer was present in the phytyl moiety throughout which indicated that effective hydrogenation of the alcohol moiety of the first two substrates had occurred.84 The remaining advances in the biosynthesis of diterpenoids concern the gibberellin plant growth hormones and much additional detail although no surprises have accrued.Informa- tion on most steps of the abbreviated scheme of biosynthesis from geranylgeranyl diphosphate (74) to gibberellin A aldehyde (78) and thence to the gibberellins A to A, (79) has been obtained. Reviews cover the biochemistry and physiology of these compounds;85 their biosynthesis in cell-free extracts in generaP and in those from immature seedlings of Cucurbita maxima and Pisum sati~urn,~’ and of the effect of growth retardants in the latter;87 and their general metabolism88 and the metabolic pathways beyond compound (78).89 The last review covers the properties reaction mechanisms and substrate specificities of plant fungal bacterial and mamma- lian dioxygenases that utilize 2-oxoglutarate and Fe2+ as cofactors and which are involved in the functionalization steps.In the following account structures of only the especially significant gibberellins are given a list of structures from gibberellin Al to A53is available.g0 Lysed chloroplasts from several higher plants synthesized ent-kaurene (76) from copalyl diphosphate (79 but not from geranylgeranyl diphosphate (74) the kaurene synthetase for CO 2H 0MoH (57) (58) 1 1 0wco*H OH (59) 0 520 the former transformation was most stable from Pisum sativum and although it was largely located in the stroma of the plastids the presence of membrane material was necessary for optimal conver~ion.~~ Crude extracts from seedlings of Helianthus annuus that had been stored in liquid nitrogen for several days had greatly enhanced kaurene synthetase activity for the conversion of (74) + (76); however the activity for the step (75) -+ (76) was little affected by such freezing and storage.The enzymes that are responsible for the two steps could not be resolved although there is a different pH optimum for each reaction .9 The metabolism of several ring-c- and -D-functionalized ent- kaur-16-en-19-oic acids [derivatives of (77)] by mutants of Gibberella fujikuroi led to the corresponding derivatives of the normal fungal gibberellins and ent-ka~renoids.~~ 12a-Hydroxy-derivatives of gibberellins A12 (80) A14 (81) and A37 (82) and the 12P-hydroxy-derivatives of ent-7a-hydroxy- and ent-6a,7a- di hydroxy-kaurenoic acid were found in seeds of Cucurbita maxima.The main metabolite of gibberellin A9 (84)that was formed in seeds of Pisum sativum and in cultures of mutants of G. fujikuroi was identified as the 12~-hydroxy-derivative. ent-11P-Hydroxy- and ent-22-0x0-kaurenoic acids were also con- verted by the fungus into the corresponding 11-oxygenated derivatives of the normal fungal ent-kaurenoids and gibberel- lin~.~~ A previously unknown pathway for the biosynthesis of 12a-hydroxylated gibberellins has been discovered using cell- free extracts of the endosperm of Cucurbita maxima.94 The microsomal fraction converted (78) into the corresponding acid (gibberellin Al 2) and 12a-hydroxygibberellin A aldehyde.The latter was further functionalized by soluble enzymes to 12a-hydroxy-derivatives of gibberellins A14 (81) A, (83) and A37 (82). Incubation of gibberellin A9 (84)with a preparation comprising both microsomal and soluble enzymes yielded gibberellins A (85) and A58 (86). A soluble fraction from immature seeds of Phaseolus vulgaris converted gibberellin A20 (87) into gibberellins A (88) and A (89) but it did not metabolize gibberellin Al or gibberellin A29 (90) to gibberellin A,. The 3P-hydroxylation and the formation of a double-bond required 02,Fe2+ and 2-oxoglutarate and both processes were I (65) +‘U (661 NATUZAL PRODUCT REPORTS 1985 stimulated by ascorbate.The enzymes that catalyse these reactions had properties very similar to those of the oxidases from species of the genera Cucurbita and Pi~um.~ Resuspended cultures of a strain of G. fujikuroi metabolized [3-l 3C]mevalon-ate to 3C-enriched gibberellins plus 3C02 (resulting from the loss of C-20). The formation of gibberellin A3 (91) could be observed in uiuo as could that of C02 if the filtered medium was ~oncentrated.~~ Other studies have mapped the metabo- lism of endogenous gibberellins in immature barley grain and led to the proposal of a metabolic pattern in sit^,^^ have shown changes in the levels of endogenous gibberellins during the growth of cultured tobacco cells,98 and have demonstrated that gibberellins A, A8,A17 A19,A20,A23 A29 andA530ccurred in Leucaena leucocephala and that several were present as their glucosyl esters or ethers.99 The glucosides may occur as storage forms of the hormone in immature seeds.The glucosylation of gibberellins by extracts from Phaseolus coccineus’ O0 and genetic studies on the 3P-hydroxylation of gibberellin A20 to form gibberellin Al in mutants of species of the genera Pisum and Zea101*102 have also been described. The inhibition of biosynthesis of gibberellins has potential importance in the discovery and design of regulators of plant growth. Paclobutrazol which is a broad-spectrum retardant inhibits the formation of gibberellins by blocking the oxidation of ent-kaurene to kaurenoic acid [(76) -+ (77)] and it also inhibits cell division in suspension cultures of plant cells.Io3 Cytokinins that have some structural resemblance to ancymi- dol which is a known growth retardant also inhibit the oxidation of kaurene in microsomal extracts from seed endosperm,Io4 and other more exotic growth inhibitors have been synthesized.lo5V lo6 6 Tissue Culture Biotransformations Genetics and Chemotaxonomy 6.1 Tissue Culture Considerable progress has been achieved in establishing tissue cultures of higher plants that can sustain often impressive levels of biosynthesis of terpenoids. It seems likely that callus and cell suspension cultures will become the materials of choice within the next decade for studies of biosynthetic pathways and of control mechanisms and for the accumulation of biomass for enzymic studies.Unfortunately many doubtful claims (based on quite inadequate assay methods) are still made and it is often not clear whether synthesis de nouo rather than ‘carry- over’ occurs. Nevertheless a corpus of information is slowly accumulating. The use of tissue cultures has been recorded in previous Sections for the biosynthesis of monoterpenes in Gardenia j~sminoides,~~ for the formation of sesquiterpenes in (67) I NATURAL PRODUCT REPORTS 1985 -D. V. BANTHORPE AND S. A. BRANCH Andrographis paniculata5 and Perilla jrute~cens,~~ and for the formation of diterpenes in Nicotiana t~bacum.*~ Biotransforma-tions of exogenously supplied metabolites by tissue cultures are covered in Section 6.2.The biosynthesis of secondary metabolites (in general) in plant cultures,107 the formation of anti-tumour agents,Io8 the relationship between the application of stress and the forma- \ 0 (70) H (71) (72) 111 H H I (73) tion of secondary metabolites,lo9 and the potential industrial applications of the method' O have been reviewed. An important compendium of techniques has appeared. * Callus cultures of Pinus radiata that synthesized monoterpenes de nouo and which were stable (morphologically and genetically) for at least 1 year have been established. l3 The products differed from those of parent plants in that a-pinene (up to 100%)rather than 0-pinene was the main component of the extractable oil but the best lines accumulated monoterpenes in yields comparable with those from parent stem and needles.The composition of the essential oil depended on the light regime. After culture in total darkness toluene and acetone were formed but exposure to light induced the re-accumulation of a-pinene. Toluene and acetone also occur (at low levels) in the seeds of P. radiata and in dark-grown seedlings and are presumably found by ring-opening aromatization and cleav- age of the a-pinene skeleton. Presumably these culture lines contain an effective photoreceptor. Cell-free extracts of the most active culture lines converted 4C-labelled isopentenyl diphosphate into geraniol and nerol together with a-and p-pinenes in up to 46% total yield.These are the most active crude extracts that have been recorded from either tissue cultures or higher plants. Most tissue cultures do not accumulate terpenoids even if the parent material was a potent source. One reason may be that lack of organization or compartmentation within the culture (especially for cell suspensions) leads to the degradation of any such nascent secondary metabolite. Consistent with this theory is the demonstration that cell-free extracts from Jasminum oficinale contained epoxidase activities for which isopentenyl diphos- phate geraniol and nerol were substrates and also contained hydratase activities towards the resulting terpene oxides.The extractable activities were up to 100-fold greater than those that had been obtained from the foliage of the plant.' l4 This is another example of the observation that the enzymes that are associated with the metabolism of terpenoids (and with secondary metabolism in general?) may be extracted from cell cultures with activity levels that are several hundred-fold greater than the levels that can be extracted (and assayed) under the same conditions from the parent plants. This may not be due to any intrinsic enhanced activity :it may result from the greater ease of extraction owing to the fragility of the cell walls and the relatively low levels of phenolics (which rapidly deactivate enzymes) in the cultures. Callus tissues of Lauandula (74) (75) H 1 2? 11 functionalized gi bberell ins HO C H02C 17 (79) 522 R .'*R2 'e' i H02C C02H C02H (80) R = H (82) RL OH,R~= H (81) R = g-OH (83) R'= R2= H OH (84)R'= R2= H (85)R' = -OH R2= H (87) R'= H ,R2= OH (88) R'= p-OH R2= OH (89) A2 ;R' = H R2= OH (90)28-OH ; R' = HI R2= OH (91) A'; R'= p-OH R2= OH angustijolia and of Rosmarinus ojicinalis that were maintained under a variety of regimes of diurnal changes of light and temperature and on many different media did not accumulate or secrete the terpenes that are characteristic of the parent tissue over a period of up to two years in culture.However such terpenoids did accumulate (up to 12% of the level in the parent) in shoots that had regenerated (after treatment with appropri- ate hormones) from the two-year-old callus.11s This is a conclusive demonstration of both the necessity for organogene- sis for the induction of accumulation of monoterpenes and the chemical totipotency of these lines.The production of oils in buds that had been induced to grow from callus tissues of other species was subsequently recorded.' l6 Cultures have often been assayed for the accumulation but only very rarely for the secretion of secondary metabolites. Suspension cultures of Thuja occidentalis excreted a-and p-pinenes myrcene limonene and certain oxygenated monoter- penes (but no thujyl compounds that are characteristic of the parent) into the medium but most of these were lost (by volatilization or by secondary reactions) before they could be assayed unless they were trapped in a layer of Miglyol (a water- insoluble non-toxic trap for lipophilic compounds).' The use of this trap showed that the cells excreted up to 3 mg of terpenoids per gram of dry weight per day; continuous extraction with the solvent also allowed the quantification of a tris-thujaplicinato-iron(1Ir)complex.This important work may lead to a re-assessment of many of the negative results that have previously been reported for cells of various species in suspension culture. Limonene was also secre?ed into the medium when suspension cultures of Apium graueolens (celery) were cultured at 4 "C at the start of the cell cycle.l18 Radioimmunoassays for loganin and secologanin (the first such assays to be achieved for monoterpenoids) have allowed the time course of biotransformations of these compounds in cell suspensions of Lonicera tatarica to be defined.' l9 The produc- tion of monoterpenes in photomixotrophic suspension cultures of Ruta graueolens' 2o and in Genipi clones of Artemisia species have also been reported.Little is known about the biosynthesis of sesqui- and di- terpenes in culture (although much work has been done on the production of triterpenoids and steroids). Callus tissue of the NATURAL PRODUCT REPORTS 1985 @ oQ#-I 0 A (92) R = H (94) (93)R = OH stem and the flowers of Matricaria chamomilla produced an oil that contained chamomillol caryophyllene oxide and other sesquiterpenes that were characteristic of the roots of the parent and these compounds were associated with cells and with morphological structures that are characteristic of the root.Suspension cultures of Cryptorneriu japonica (Japanese cedar) yielded an extractable oil containing low levels of abietatriene (92) and ferruginol (93)' 23 6.2 Bio t ransform a t ions Work in this field involving micro-organisms or cell suspen- sion cultures is largely confined to the higher terpenoids (especially steroids). Suspension cultures of Nicotiana tabacum hydrolysed the 'foreign' substrates carvoxime and dihydrocar- voxime to the corresponding ketones or reduced these to the alcohols. 24 The anti-tumour agent (-)-a-santonin (94) was reduced to its 1,2-dihydro-derivative by Streptomyces cinereo- crocatus 25 and the microbial metabolism of a-terpineol by Pseudomonas incognira' 26 and of the sesquiterpene quadrone' 27 and the production of volatile flavour compounds by cultures of Kluyueromyces lactis' 28 have been reported.The biohydroxyla- tion of terpenoids in mammals has been reviewed.129 6.3 Genetics The formation of monoterpenes by species of the genus Mentha has been studied in detail especially the analysis of hybridiza- tion between different species.' 309 31 The demonstration that Mendelian ratios of offspring produced the characteristic terpenes of each parent in crossing experiments confirms that there is strict genetic control of the formation of 2-and 3-0x0- compounds.32 A theoretical genetic analysis of cultured cells that produce useful secondary metabolites has been described.' 33 6.4 Chemotaxonomy An excellent monograph is available. 34 Many reports deal with quantitative analyses of the terpenoid content of individual species or groups of species but few investigations go beyond this to seek answers to questions concerning the introgression of species gene-flow clonal variations etc. or deal with the incidence of marker compounds that are specific to species or to genera. 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Zeevaart Plant. Physiol. 1984 75 166. 69 H. Lehrmann and P. V. Vlasov Wiss.Z. Ernst-Moritz-Arndt-Univ. Griejsw. Mat. Naturwiss. Reihe 1983,32 102 (Chem. Abstr. 1984 101 126 849).70 G. T. Vaughan and B. V. Milborrow J. E.xp. Bot. 1984,35 110. 71 J. Daie R. Wyse M. Hein and M. L. Brenner Plant Physiol. 1984 74 810. 72 M. Orlandini P. Barthe and C. Bulard Physiol. Plant. 1984,62 553. 73 J. Daie and R. Wyse A.C.S. Symp. Ser. 1984 257 127 (Chem. Abstr. 1984 101 188 093). 74 H. Lehmann and H. R. Schuette J. Plant. Physiol. 1984 117,201. 75 G. J. P. Murphy Planta 1984 160 250. 76 T. Kannangard G. M. Simpson K. Rajkumar and B. D. Murphy J. Chromatogr. 1984 283 425. 77 C. R. McIntyre T. J. Simpson R. N. Moore L. A. Trimble and J. C. Vederas J. Chem. Soc. Chem. Commun. 1984 1498. 78 T. J. Simpson D. J. Stenzel R. N. Moore L. A. Trimble and J. C. Vederas J. Chem. Soc. Chem. Commun. 1984 1242. 79 R. Feyereisen R.P. Ruegg and S. S. Tobe Insect Biochem. 1984 14 657. 80 M. J. Ackland J. R. Hanson and A. H. Ratcliffe J. Chem. Soc. Perkin Trans. 1 1984 275 1. 81 J. R. Hanson Nut. Prod. Rep. 1984 1 443. 82 B. Nanda S. A. Patwardhan A. S. Gupta K. R. Acharya N. N. Dhaneshwar S. S. ‘Tavale and T. N. G. Row J. Chem. Res. (S) 1984 394. 83 B. Sullivan and D. J. Faulkner J. Org. Chem. 1984 49 3204. 84 J. Benz U. Lempert and W. Rudiger Planta 1984 162 215. 85 ‘Biochemistry and Physiology of Gibberellins’ ed. A. Crozier Praeger New York 1983 Vol. 11. 86 Y. Yamiya Shokubutsu no Kagaku Chosetsu 1984 19 66. 87 J. E. Graebe Ber. Dtsch. Bot. Ges. 1984 97 67. 88 J. MacMillan in ‘Analysis of Plant Hormones and Metabolism of Gibberellins’ ed. A. Crozier and J.R. Hillman Society for Experimental Biology Cambridge England 1984 Vol. 23 p. 9. 89 J. MacMillan Monogr. Br. Plant Growth Regul. Group 1984,11 13 (Chem. Abstr. 1985 102 41 885). 90 T. Goodwin and E. Mercer ‘Introduction to Plant Biochemistry’ Pergamon Oxford 1983 2nd edn. p. 582. 91 I. D. Railton B. Fellows and C. A. West Phytochcmistry 1984 23 1261. 92 J. Shen-Miller and C. A. West Plant Physiol. 1984 74 439. 524 93 P. Gaskin M. Hutchinson N. Lewis J. MacMillan and B. 0. Phinney Phytochemistry 1984 23 559. 94 P. Hedden J. E. Graebe M. H. Beale P. Gaskin and J. MacMillan Phytochemistry 1984 23 569. 95 Y. Kamiya M. Takahashi N. Takahashi and J. E. Graebe Planta 1984 162 154. 96 P. Lewer and J. MacMillan Phytochemistry 1984 23 2803.97 S. J. Gilmour P. Gaskin V. M. Sponsel and J. MacMillan Planta 1984 161 186. 98 K. H. Park S. Fujisawa A. Sakurai I. Yamaguchi and N. Takahashi Plant Cell Physiol. 1984 25 1303. 99 S. Arigayo S. Fujisawa A. Sakurai Y. Kamiya S. Adisewojo and N. Takahashi Plant Cell Physiol. 1984 25 1395. 100 H. D. Knoefel E. Schwarzkopf P. Mueller and G. Sembdner J. Plant Growth Regul. 1984 3 127. 101 T. J. Ingram J. B. Reid I. C. Murfet P. Gaskin C. L. Willis and J. MacMillan Planta 1984 160 455. 102 C. Spray B. 0. Phinney P. Askin S. J. Gilmour and J. MacMillan Planta 1984 160 464. 103 J. Dalziel and D. K. Lawrence Monogr. Br. Plant Growth Regul. Group 1984 11 43 (Chem. Abstr. 1985 102 19513). 104 R. C. Coolbaugh J.Plant Growth Regul. 1984 3 97. 105 T. Mita and H. Shibaoka Plant Cell Physiol. 1984 25 1531. 106 K. Izumi I. Yamaguchi A. Wada H. Oshio and N. Takahashi Plant Cell Physiol. 1984 25 61 1. 107 B. E. Ellis Can. J. Bo?. 1984 62 2912 I08 J. P. Kutney Pure Appl. Chern. 1984 56 1011. 109 F. DiComo and G. H. N. Towers Recent Adv. Phytochem. 1984 18 97. 110 M. W. Fowler Biotechnol. Genet. Eng. Rev. 1984 2 41. 11 I K. W. Fuller Chem. Ind. (London) 1984 825. 112 ‘Cell Culture and Somatic Cell Genetics of Plants’ ed. I. K. Vasil Academic Press Orlando Florida 1984 Vol. I. I13 D. V. Banthorpe and V. C. 0.Njar Phytochemistry 1984,23,295. 114 D. V. Banthorpe and M. G. Osborne Phytochemistry 1984 23 905. 115 J. K. Webb D. V. Banthorpe and D. G.Watson Phytochemistry 1984 23 903. 116 Kanebo Ltd. Jpn. Kokai Tokkyo Koho 59 213 393 (1984) (Chem. Abstr. 1985 102 129 117). 117 J. Berlin L. Witte W. Schubert and V. Wray Phytochemistry 1984 23 1277. NATURAL PRODUCT REPORTS 1985 118 M. J. Watts I. J. Calpin and H. A. Collin New Phytol. 1984,98 583. 119 T. Tanahashi N. Nagakura H. Inouye and M. H. Zenk Phytochemistry 1984 23 19I 7. 120 F. Drawert R. G. Berger R. Godelmann S. Collin and W. Barz 2. Naturjorsch. Sect. C 1984 39 525. 121 C. Leddet C. Paupordin and R. Gautheret C.R. Seances Acud. Sci. Ser. 3 1984 299 621 122 J. Reichling W. Bisson and H. Becker Planta Med. 1984 50 334. 123 N. Ishikura K. Nabeta and H. Sugisawa Phytochemistry 1984 23 2062. 124 T. Suga T. Hirata and M.Futatsugi Phytochemistry 1984 23 1327. 125 Y. Sato T. Oda J. Inoue M. Kunugi and K. T. Suzuki Chem. Pharm. Bull. 1984 32 504. 126 K. M. Madyastha and V. Renganathan Can. J. Microbiol. 1984 30 1429. 127 J. M. Beale Jr. J. M. Hewitt and J. P. Rosazza Enzyme Microb. Technol. 1984 6 543. 128 H. P. Hanssen E. Sprecher and A. Klingenberg 2.Natucfbrsch. Sect. C 1984 39 1030. 129 T. S. Santhanakrishnan Tetrahedron 1984 40,3597. 130 S. A. Reznikova L. A. Bugaenko and V. V Makarov Rastit. Resur. 1984 20 544 (Chem. Abstr. 1985 102 59 441). 131 L. A. Bugaenko and S. A. Reznikova Genetika (Moscow) 1984 20 2018 (Chem. Abstr. 1985 102 59447). 132 L. A. Bugaenko and S. A. Reznikova Genetika (Moscow) 1984 20 1857 (Chem. Abstr. 1985 102 43 048).133 M. Mino and Y. Yamada Shokubutsu No Kagaku Chosetsu 1983 18 91 (Chem. Abstr. 1984 101 18 223). 134 J. B. Harborne and B. L. Turner ‘Plant Chemosystematics’ Academic Press London 1984. 135 A. D. Dembitskii Izv. Akad. Nauk Kaz. SSR Ser. Khim. 1984 No. 4 p. 4 (Chem. Abstr. 1984 101 167 163). 136 R. P. Adams T. A. Zanoni and L. R. Hogge Biochem. Syst. Ecol. 1984 12 23. 137 R. W. Scora and J. Kumamoto Annu. Proc. Phytochem. SOC. Eur. 1983 22 343. 138 J. Kumamoto and R. W. Scora J. Agric. Food Chem. 1984 32 418.

ISSN:0265-0568    

DOI:10.1039/NP9850200513

出版商:RSC    

年代:1985

数据来源: RSC

摘要:

The Biosynthesis of Triterpenoids and §teroids D. M. Harrison Chemistry Department The University of Ulster at Coleraine Coleraine Co. Londonderry Northern Ireland BT52 7SA Reviewing the literature published between January 1979 and December 1983 (Continuing the coverage of literature in Biosynthesis,Vol. 6 p. 95) 1 Introduction 2 Mevalonic Acid 3 The Biosynthesis of Squalene from Mevalonic Acid 4 The Biosynthesis of Triterpenoids from Squalene 5 The Formation of Sterols in Vertebrates 5.1 The Biosynthesis of Cholesterol 5.2 The Biosynthesis of Steroidal Hormones 5.3 The Biosynthesis of Bile Acids 6 Triterpenoids and Steroids in Higher Plants Algae and Fungi 6.1 The Biosynthesis of Triterpenoids 6.2 The Biosynthesis of Phytosterols 6.3 The Biosynthesis of Sterols in Fungi 6.4 Alkylation of the Sterol Side-chain 6.5 Further Metabolism of Steroids 7 Triterpenoids and Steroids in Invertebrates 7.1 Insects 7.2 Other Invertebrates 8 References 1 Introduction The material of this Report is organized in the manner that was used previously,' while the scope has been widened somewhat by the inclusion of brief discussion on the biosynthesis of the bile acids and steroidal hormones.The papers that are discussed were selected mainly through the perusal of Chemical Titles for the appropriate period. The choice of whether or not to include discussion on a particular publication has often been difficult particularly when the work that was reported is indirectly relevant to biosynthesis.Such areas include studies on inhibitors of biosynthesis and enzymological studies related to biosynthesis. In such cases I have considered the likely interest of the work to the biosynthetic chemist in deciding whether to include the reference. In keeping with previous reports,' the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (both E.C. 1.1.1.34 and E.C. 1.1.1.88) is discussed owing to its unique importance in the regulation of the biosynthesis of steroids. Many useful reviews have appeared during the period that is surveyed and will be cited in the appropriate parts of this Report. One excellent two-volume work on the biosynthesis of isoprenoids is relevant to most of this Report and deserves mention at this stage.' Shorter accounts have been published on the biosynthesis of steroid^.^ 2 Mevalonic Acid Mevalonic acid (la) is formed from acetyl-coenzyme A uia The mammalian reductase is subject to complex regulatory controls (see below) and the reduction of HMG-CoA to mevalonate is generally regarded as the rate-limiting step in the biosynthesis of steroids.Consequently the reductase is a key target in efforts directed towards finding clinically useful hypocholesterolemic agents. H ydroxymethylglutaryl-CoA re-ductase has been reviewed exten~ively.~~~~ The activity of mammalian HMG-CoA reductase in uiuo is regulated (in part) by low-density lipoprotein. Recent evidence suggests that the earlier enzymes of the biosynthesis of mevalonate may also be regulated by low-density lipoprotein and by 25-hydroxycholesterol (2a) in cultures of mammalian cells.In rat glial cells hydroxymethylglutaryl-CoA synthase (E.C. 4.1.3.5) (and not the reductase) directs the rate of biosynthesis of cholesterol under serum-free growth conditions. The solubilization isolation and purification of HMG-CoA reductase (NADPH) from avian' and from human8 liver have been reported. The reductase of human liver was isolated as a dimer of molecular weight 104 OO0.8 There is considerable controversy surrounding the nature of the native microsomal enzyme. Thus earlier reports on the purified reductase from rat liver suggested that it was a tetrameric protein with subunits of molecular weight ca 51 OO0.9 However the popular freeze- thaw procedure that was used for solubilizing the microsomal enzyme is believed to result in partial degradation of the native enzyme through the release of proteases;l0 solubilization of the reductase in the presence of inhibitors of proteases (using a non-ionic detergent and avoiding the freeze-thaw technique) gave a preparation with significantly different properties.Recent studies suggest that the native rat liver reductase has a molecular weight of ca 94 000,' * which is in close agreement with the most recent isolation of HMG-CoA reductase (mol. wt. 92 000) from hamster kidney cells that had been grown in culture.13 The reductase of chicken is believed to be synthe- sized as a pro-enzyme of molecular weight 102 000 which is cleaved subsequently to give the active enzyme which has a molecular weight of 94000.'4 It was shown that 25-hydroxy- cholesterol (2a) inhibits the biosynthesis of cholesterol (2b) by increasing the rate of degradation of HMG-CoA reductase and by decreasing the rate of synthesis of the same enzyme.14 The fungal metabolite compactin (3a) (also known as ML- 236B) acts as a competitive inhibitor of HMG-CoA reductase.' The related mould metabolites mevinolin' (3b) (also called monacolin K16) dihydrocompactin,' 'and dihydromevinolin' inhibit the activity of HMG-CoA reductase in a similar H acetoacetyl-coenzyme A and (3S)-3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA).In mammalian cells and in those of birds and higher plants HMG-CoA (lb) is reduced to mevalonic acid (la) by NADPH; the reaction is catalysed by the enzyme hydroxymethylglutaryl-CoA reductase (NADPH) [E.C.1.1.1.341. In micro-organisms hydroxymethylglutaryl- CoA reductase [E.C. 1.1.1.881 utilizes NADH as the cofactor. b; R = COSCOA (2)a;R=OH b;R=H NATURAL PRODUCT REPORTS 1985 HO H tl' (3)a;R = H b; R = Me manner. Mevinolin has been shown to act as a hypocholestero- lemic agent in human subjects. Compactin suppressed the biosynthesis of sterols from [U-14C]acetate or from [U-14C]leucine in tissue cultures of sycamore (Acer pseudopla- tanus). The post-mevalonate phase of biosynthesis of sterols was unaffected.*O Mevinolin inhibits normal growth of seedlings of radish (Raphanus sativus) and of wheat (Triticum aestivum) by inhibiting the HMG-CoA reductase.2 Studies in uitro with radish microsomal red~ctase~','~ and with the partially purified enzyme of a yeast (Saccharomyces cereui-si~e)~~ confirmed the site of inhibition.Cultures of mammalian cells that were incubated with compactin or mevinolin responded by increasing the activity of HMG-CoA reductase as assayed subsequently in cell-free systems in the absence of the inhibitor. This effect occurs pre- sumably because there is a reduced level of feedback inhibi- as serine residues."' Astonishingly the HMG-CoA reductase kinase is itself subject to control by a phosphorylation-dephosphorylation cycle; the active form of the kinase is ph~sphorylated.~~ The validity of the phosphorylation-dephos- phorylation sequence in the control of the activity of HMG-CoA reductase has been ~hallenged."".~~ A common assay for HMG-CoA reductase involves catalysis of the reduction of I4C- or 3H-labelled HMG-CoA to furnish mevalonic It has been suggested that the inadvertent presence of mevalonate kinase (see Section 3) as a contaminant of the inactivator protein [HMG-CoA reductase (NADPH)] kinase could render unreliable the assay of phosphorylated HMG-CoA red~ctase.~~ Support for this view has been given by other workers who have also demonstrated that FeZ+ ions inhibit HMG-CoA reductase."' Other problems in the assay of HMG-CoA reductase have been reported.58 However it has been shown that mevalonate kinase can be separated from the reductase kina~e,~~ and assays which would exclude artefactual results that are due to the possible presence of mevalonate kinase have been de~cribed.~~,~~ Evidence has been presented to show that the microsomal HMG-CoA reductase of rat brain cells5 and of livers of other vertebrates5' is also subject to modulation of activity via a phosphorylation-dephosphorylation sequence.It has been shown that dephosphorylation (activation) of the reductase of rat liver microsomes is inhibited by its substrates i.e. HMG-CoA and NADPH.53 The question of the relevance of these studies in vitro to the control of HMG-CoA reductase activity in vivo is a subject of current interest. Administration of mevalonolactone to the rat stomach results in the rapid inactivation of the liver microso- tion of the HMG-CoA reductase activity by ch~lesterol.?~-~~ The increases in activity of HMG-CoA reductase may be due to an increase in the rate of synthesis of the reducta~e'~ or to a decrease in the rate of degradation of the reductase,26 or both.27 Studies were made also on the reductase of mouse liver in vivo.28 Strains of compactin-resistant hamster cells have been developed that show a dramatic and sustained increase in the activity of HMG-CoA reductase (NADPH),29 which has facilitated the isolation and study of the enzyme (e.g.ref.13). It was shown that low-density lipoprotein or 25-hydroxycholes-terol inhibits the activity of HMG-CoA reductase in these cell cultures almost exclusively by suppression of the synthesis of the enzyme.30 Many new inhibitors of HMG-CoA reductase activity have been identified including 3,3,5-trimethyl~yclohexanol,~~ di-chloroacetic acid,32 5-phenylpentanoic acid,33 monoesters of substituted glutaric acids13" and other analogues of mevalonic Amongst the latter are the sulphide (4a)36 and the disulphide (4b).37 Benzylsulphonyl fluoride inhibits the production of steroids from acetate in rat liver homogenates by acting on the enzymes acetate thiokinase [acetate-CoA ligase (E.C.6.2.1. l)] HMG-CoA synthase and HMG-CoA reductase (NADPH). 38 Rat liver microsomal HMG-CoA reductase is inhibited in vitro by ATP-Mg'+ in the presence of a cytosolic protein. Re- activation of the reductase can be achieved with the aid of an acid phosphatase or with a cytosolic activator protein called [hydroxymethylglutaryl-CoA reductase (NADPH)]-phospha- tase (E.C.3.1.3.47).1.39 It is believed that the inactivation process involves covalent phosphorylation of the reductase and the cytosolic inactivator protein has been called [hydroxy- methylglutaryl-CoA reductase (N ADPH)] kinase (E .C. 2.7.1.109). When [y-32P]ATP was used in the inactivation process the phosphorylated reductase which was 70% inacti- vated could be purified to homogeneity without loss of 31P. Upon incubation of the inactivated reductase with [HMG-CoA reductase (NADPH)]-phosphatase the reductase was re-activated and [3'P]phosphate was released.jO Studies have been reported on the isolation purification and properties of [HMG-CoA reductase (NADPH)]-phosphatases of rat liver.41 During the inactivation of HMG-CoA reductase with ATP- Mg2+ catalysed by the reductase kinase phosphorylation occurs at several sites in the reductase;"' these were identified ma1 HMG-CoA reductase.This inactivation (first phase) could be reversed in vitro by the addition of phosphatases and so must arise via phosphorylation of the reductase. However after longer time intervals the inactivation was permanent (second phase) and was not reversed by the action of phosphatase~.~~ Qualitatively similar results were obtained when rats were fed a It diet that was rich in chole~terol.~~ was shown (using immunotitration techniques) that the second phase of inhibi- tion was due to further inactivation of existing enzyme.s6 Similar results were reported for mevalonolactone by another group but perfusion of 25-hydroxycholestero1 through rat livers resulted in rapid and irreversible inhibition of the HMG-CoA reduc tase.Two key enzymes of cholesterol metabolism are regulated by phosphorylation-dephosphorylation cycles namely cholesterol 7a-mono-oxygenase (E.C. 1.14.13.17) and cholesterol acyl- transferase (E.C. 2.3.1.26). Both of these enzymes are deactivated by dep hosp horyla t ion and activated by p hosp hory -lation. It has been suggested that when the cholesterol level rises in the liver in vim the latter enzymes become phosphorylated leading to increased conversion of cholesterol into bile acids and cholesteryl esters respectively; at the same time phosphorylation of HMG-CoA reductase occurs leading to decreased synthesis of mevalonate and hence of cholesterol to restore the status quo.Conversely when the cholesterol level falls each enzyme becomes dephosphorylated leading to decreased usage and increased production of ch~lesterol.~~ It has been claimed by other workers that the diurnal variation in reductase activity and the fall in reductase activity that is consequent upon the feeding of cholesterol in uivo are due solely to changes in the number of enzyme molecules and that inactivation by ATP-Mg2+ has no physiological signifi- ~ance.~~ Furthermore in rat liver cells the HMG-CoA reductase activity did not vary during the circadian cycle and the ratio between phosphorylated and unphosphorylated forms remained constant.Treatment with 25-hydroxycholestero1 resulted in inhibition of reductase activity but did not alter the ratio between phosphorylated and unphosphorylated en-zymes.60 Extensive use has been made of immunotitration techniques using antibodies that had been raised to pure HMG-CoA reductase (NADPH) to distinguish between changes in the number of reductase molecules and changes in NATURAL PRODUCT REPORTS. 1985 -D. M. HARRISON the level of activation of existing molecules of the en~yme.~~.~~ Inhibition of HMG-CoA reductase activity by 25-hydroxychol- esterol or by low-density lipoprotein in cultured hamster cells may be due to an acceleration in the rate of degradation of the enzyme6* or to repression of synthesis of the enzyme.63 The regulation of HMG-CoA reductase (NADPH) by the phos- phorylation-dephosphorylation sequence has been reviewed extensively.64 Aspects of the control of the HMG-CoA reductase of a yeast (Saccharomyes cereviszae) have been investigated.6s The fungicides clotrimazole and triadimefon are known to block the demethylation of precursors of ergosterol.A secondary effect in this yeast is the feedback inhibition of HMG-CoA reductase by the precursors of ergosterol that accumulate.66 The reductase of the yeast is rapidly inactivated by coenzyme A disulphide in uitro. Re-activation follows the addition of thi01s.~~ Rat liver microsomal HMG-CoA reductase (NADPH) is similarly inactivated by disulphides and re-activated by thiols.68 Kinetic analyses have been reported on HMG-CoA reductase that was isolated from yeast69 and HMG-CoA reductase (NADPH) from rat liver.70 Studies have been reported on the regulation of the HMG-CoA reductase of pea seedlings7’ and on the effects on the reductase of radish seedlings of those herbicides that are effective by blocking photosystem II.72 The reductase from the latex of Hevea brasiliensis has also been studied.73 3 The Biosynthesis of Squalene from Mevalonic Acid Rat serum contains a heat-labile 6-lactonase which catalyses the interconversion of mevalonolactone and mevalonic The ATP-dependent conversion of mevalonate into 5-phospho- mevalonic acid (5a) is catalysed by the enzyme mevalonate kinase (E.C.2.7.1.36). The mevalonate kinase of hog liver is inactivated by pyridoxal phosphate which implies the pres- ence of an essential amino-group in the enzyme. The enzyme is inactivated also by reagents that are directed at free thiol groups.7s Phosphomevalonate kinase (E .C. 2.7.4.2) which catalyses the conversion of (R)-5-phosphomevalonic acid (5a) into (R)-5-diphosphomevalonic acid (5b) has been isolated from pig liver and purified. The enzyme consists of a single polypeptide chain of molecular weight 22000 and contains free thiol groups.76 Evidence has been presented for the role of both thiol and amino functions in the catalytic Phosphomeva-lonate kinase is specifically inhibited by 3-hydroxy-3-methyl-6- phosphonohexanoic acid (5c) which is a structural analogue of the natural substrate in a cell-free rat liver h~mogenate.~~ Diphosphomevalonate decarboxylase (E.C.4.1.1.33) which catalyses the conversion of (R)-5-diphosphomevalonic acid into isopentenyl diphosphate (6) has been purified to homogeneity from rat liver79 and from chicken liver.80 The avian enzyme exists as a dimer (mol. wt. 85400); the optimum pH for catalytic activity is between 4.0 and 6.5 and the enzyme does not require thiols for activation or for its stability.80 An essential arginyl residue was detected by inactivation of the enzyme with phenylglyoxal and it was found that its substrates protect the enzyme from inactivation.8 Some aromatic carboxylic acids and phenols were shown to inhibit the diphosphomevalonate decarboxylase of rat liver ;under certain conditions phosphomevalonate kinase was also inhibited.82 The activity of the rat liver decarboxylase is regulated in vivo by dietary cholesterol.The equilibration of isopentenyl diphosphate (6) with dimethylallyl diphosphate (7) is catalysed by the enzyme isopentenyl-diphosphate A-isomerase (E.C. 5.3.3.2) while the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to furnish geranyl diphosphate (8a) and the condensation of the latter with isopentenyl diphosphate to yield farnesyl diphosphate (10a) are both catalysed by dimethylallyl- transtransferase (E.C. 2.5.1. I) which is often described as prenyltransferase. The prenyltransferase of pig liver exists in / CO2H R (510; R = 08 b; R = Om c ; R = CH,PO(OH) (racemic) (6) (7 1 two interconvertible forms (A and B) each of which is a dimer (mol.wt. 77 000) and which differ by the number of free thiol groups that they contain. The subunits of form B are held together by a disulphide bond. Both forms are deactivated by phenylglyoxal which implies the presence of an essential arginyl residue.84 A prenyltransferase has been solubilized with Triton X-100 from pig liver micro some^.^^ Human liver prenyltransferase has been purified to homogeneity and described in detail. The enzyme is similar to the B form of the pig liver enzyme and antibodies to the latter cross-react with human prenyltransferase. The human enzyme has an absolute requirement for Mn2+ (best) or Mg2+ ions; it is inactivated if it is treated with phenylglyoxal or iodoacetic acid and the presence of thiols is required to maintain the enzymic activity.86 The prenyltransferase of chicken liver also exists as a dimer with two identical subunits.The active site has been modified with a photoaffinity reagent and the modified protein was then partially degraded.87 The non-productive binding of farnesyl diphosphate to the allylic or homoallylic active sites of the avian enzyme has been studied.88 A detailed kinetic analysis on the prenyltransferase of chicken liver has been reported; the work involved in part the use of fluorine-containing inhibi- tor~.~~ Further kinetic studies have been published in support of an ordered sequential mechanism in which the allylic substrate-Mg2+ binds first followed by the binding of isopentenyl diphosphate and a Mg2+ Poulter and his colleagues have gathered compelling evi- dence to show that the enzyme-catalysed condensation of isopentenyl diphosphate with geranyl diphosphate proceeds by the mechanism that is summarized in Scheme 1.91-94 Thus the rate of condensation of isopentenyl diphosphate with 2-fluorogeranyl diphosphate (8b) is 8.4 x times that of the condensation of isopentenyl diphosphate with geranyl diphos- phate despite the fact that the Michaelis constant (K,) for the fluorinated compound is similar to that of the natural substrate.This result is consistent with a unimolecular ionization of geranyl diphosphate to form an allylic cation (9a) which subsequently reacts with isopentenyl diphosphate (6) to furnish farnesyl diphosphate (10a).91 Complementary evidence was obtained with the avian prenyltransferase using the fluori- nated analogues (8c)-(8e) of geranyl diphosphate @a).The relative rate constants (k@,)= 1) of the catalytic step for the reactions between isopentenyl diphosphate and each of the allylic diphosphates @a) (8c) (8d) and (8e) were compared with the relative rate constants (lq1 = 1) for solvolysisof each of the corresponding mesylates (1 1a) (1 1c) (1 1d) and (1 1 e) in aqueous acetone. The results demonstrated the complete lack of participation of the rc-bond of isopentenyl diphosphate in the rate-determining step of the conversion of (8) into The nature of the ion-pair (9a) was investigated with the aid of the diphosphoric ester of [I 80]geraniol.Excess of the [ 80,]geranyl diphosphate (1 1 b) was incubated with the avian prenyltrans- ferase in the presence of isopentenyl diphosphate and it was shown that the geranyl diphosphate that was recovered retained virtually all (94%) of the I8Olabel at C-1. It was argued that a substantial proportion of the [I 80,]geranyl diphosphate NATURAL PRODUCT REPORTS 1985 -R2 -O@ I+ R’ (81 Throughout a; R’= H,R2= Me b ;R’ = F R2= Me c;R’=H,R2=CH2F d; R’= H R2= CHF2 e; R’= H,R2= CF3 Scheme 1 had undergone reversible ionization at the active site of the enzyme and thus the lack of significant scrambling of the lS0 label was evidence of a rigid highly structured ion-pair intermediate.93 Attention has been directed also to events in the isopentenyl diphosphate molecule that undergoes condensation.The reaction is known to involve suprafacial alkylation-deprotona- tion of isopentenyl diphosphate. In principle the reaction could involve (i) a concerted addition-elimination sequence (as shown in Scheme 1) or (ii) a trans-addition process [(7) + (6) -+ (12)] followed by a trans-elimination [(12) .+(8a)I (as Scheme 2) where Enz-X- represents an unidentified nucleophilic group on the surface of the enzyme. It has been suggested following studies with model compounds that the base that is involved in the elimination step of mechanism (ii) could be an a-phosphoryl oxygen atom (Scheme 2; arrows).95 However this ‘x-group’ mechanism appears to be ruled out by the non- appearance of an irreversible component of inhibition of prenyl- transferase when either racemic 2-fluoroisopentenyl diphos- phate (13a) or the 2,2-difluoro-analogue (1 3b) is incubated with the enzyme in the presence of geranyl dipho~phate.~~ Further studies have been reported on the substrate specificity of a prenyltransferase from pig liver.Thus analogues of isopentenyl diphosphate can replace the natural substrate in condensations with dimethylallyl diphosphate or geranyl diphosphate. The best artificial substrates were (14a) (14b) and (l4~).~~ A prenyltransferase has been isolated and purified 106-fold from seedlings of pea (Pisum sativum).The enzyme is dimeric with a molecular weight of 96 OO0.97 The prenyltransferase of Gossypium hirsutum is part of a multi-enzyme complex which includes the isopentenyl-diphosphate A-isomera~e.~~ The flavedo of Citrus sinensis yielded a prenyltransferase activity which furnished farnesyl diphosphate and the (22,6E)-’ isomer when it was incubated with the appropriate substrates. The E and 2 synthetase activities both required Mg2+ ions and could be partially separated which suggests that they belong to different enzymes.99 Prenyltransferase was inactivated if it was treated with reagents that attack thiol group^.^^.^^^ The geranyltranstransferase (E.C. 2.5.1.10) of Bacillus subtilis has been purified 120-fold. The enzyme has a molecular weight of 67 000 requires Mg2+ ions for optimal activity and is activated by thiols.lol Details have been published on the ability of squalene Hii (10) AJAR* (11) a; R’= Me R2 = OS02Me 0 0 II ii b ; R’= Me R2 !80-p-~-p-~~ I I OH OH c; R’ = CH,F R2= OS0,Me d ; R1= CHF2 R2= OS02Me e ; R’ = CF3 R2 = OS02Me JiEnz I H’ R’ synthetase of pig liver to accept modified analogues of farnesyl diphosphate as substrates.The best artificial substrates were (1 5a) (1 5b) (1 5c) and (1 5d) which were each converted enzymically into the appropriate squalene-like analogues in good yield.Io2 Presqualene diphosphate (1 6) has long been recognized as an intermediate in the conversion of farnesyl diphosphate (10a) into squalene (19) catalysed by squalene synthetase [farnesyl- diphosphate farnesyltransferase (E.C.2.5.1.21)]. The ion-pair (13) a ; R’ = F ,R2= H (14)a; R’= R2=Me ,R3= H b; R’= R2= F b; R’=R3=Me,R2=H c ; R’ R2= [CH2]3 ,R3= H (17) is a possible intermediate in this process (Scheme 3). The ammonium diphosphate salt (20) acts as an efficient inhibitor of the squalene synthetase of yeast microsomes presumably owing to its structural similarity to the ion-pair (17).Io3 It is known that (122)-12,13-didehydrosqualene (21) is formed NATURAL PRODUCT REPORTS 1985 -D. M. HARRISON R2 10 O@ (15) a;R'= R2= Me b ;R' = HI R2= Et c ;R' = R2 = H ;10,ll -dihydro d ;R' = HI R2= Me ;10,ll- dihydro 2 x (10a) I (cl1Hl9 = ) Scheme 3 when farnesyl diphosphate or presqualene diphosphate is incubated with yeast microsomes in the presence of Mn2+ ions but in the absence of NADPH.IoS It has now been shown that the dehydrosqualene arises from a squalene-synthetase-medi-ated reaction.Presumably the intermediate allylic cation (18) is reduced to squalene in the presence of NADPH but eliminates a proton to form the dehydrosqualene (21) in the absence of NADPH. The dehydrosqualene (21) was also formed from farnesyl diphosphate by pig liver microsomes in the absence of NADPH.IoS It has been shown that low-density lipoprotein 25-hydroxy- cholesterol (2a) and cholesterol (2b) each suppress squalene synthetase activity in cultured human fibroblasts. This effect is in addition to the rapid suppression of the HMG-CoA reductase activity by these agents.O6 Cholesterol also inhibits the biosynthesis of the triterpene tetrahymanol in the proto- zoon Tetruhyrnenu pyrifbrrni~,~~' owing to inhibition of the synthesis of squalene synthetase.I0* Excellent reviews have been published on the biosynthesis of isopentenyl diphosphate from acetyl-coenzyme A,2bon prenyl- transferase,*C.lo9 and on squalene synthetase.2d,1 lo 4 The Biosynthesis of Triterpenoids from Squalene The first step in the biosynthesis of sterols from squalene involves oxidation of the latter to furnish (3s)-squalene 2,3-epoxide (22a) in a reaction that requires molecular oxygen and NADPH. Squalene epoxidase [squalene mono-oxygenase (E.C. 1.14.99.7)] has now been isolated and purified from the microsomal fraction of rat liver homogenates.I I The mono- oxygenase was isolated as a protein of molecular weight cu 47 000. This protein required NADPH-cytochrome P-450 reductase FAD and Triton X-100for reconstitution of the squalene mono-oxygenase activity. The conversion of acetate into cholesterol by human kidney cancer cells was found to be inhibited by the presence of exogenous choles- terol.Il2 One site of inhibition was the HMG-CoA reductase; evidence was presented to show that squalene mono-oxygenase also was inhibited in this cell culture."' Squalene mono-oxygenase was inhibited in the fungi Cundidu purupsifosis and Trichophyton rnentugrophytes by the antimycotic agent nafti- fine.' l3 (3s)-Squalene 2,3-epoxide has been isolated from the green alga Cuuferpa prolijeru' and has been prepared in low optical yield by the asymmetric epoxidation of squalene.' (22) (23) Throughout ; a ; R' = Me,R2= [CHz],CH=CMe2 b;R' = Et R2= Me c;R' =Me,R2= Et P\ d; R' = Me R2=[CH2I2CH-CMe2 Further studies have been reported on the mechanism of the enzyme-catalysed cyclization of squalene 2,3-epoxide to fur- nish lanosterol (23a).l l6 Thus the (18Z)-tetranorsqualene epoxide (22b) was cyclized by a crude preparation of lanosterol synthase (E.C.5.4.99.7) from hog liver to give the unnatural (20s)-tetranorlanosterol (23b) as the sole tetracyclic product. In contrast the (18E)-epoxide (22c) furnished the (20R)-tetranor- lanosterol (23c) when submitted to enzyme-catalysed cycliza- tion.Thus the stereochemistry that is induced at C-20 in the products (23) of the cyclization reaction is determined not by the relative sizes of the substituents at C-19 in the analogues of squalene 2,3-epoxide but by the stereochemical disposition of these substituents about the A' *double-bond. This conclusion is not consistent with the formation of the classical carbo- cation (24) as an intermediate in the biosynthesis of lanosterol (23a); it is consistent with a pathway that involves the stereospecific participation of an enzyme-bound nucleophile in the cyclization step i.e. (22a) -+ (25); elimination of Enz-X- occurs after bond rotation i.e. (25) (26) to initiate the rearrangement step (Scheme 4).' l6 A mechanism that involves non-classical carbo-cations' could also account for the stereochemical preference that is exhibited by lanosterol syn t hase.It is known that 18,19-dihydrosqualene 2,3-epoxide (27a) is cyclized by mammalian lanosterol synthase to form the tricycle (28) which contains a five-membered ring c.'I8 It has now been shown that the homologous substrate (27b) is converted into a single tricycle (29) in 7% yield by a soluble enzyme preparation from rat liver. I ' These observations were inter- preted in terms of the relative ease of elimination of a proton or transfer of hydride in the non-classical carbo-cation interme- diates (30a) and (30b) respectively. For the natural substrate squalene 2,3-epoxide it is the proximity of the A18 double-bond (squalene numbering) to the cation site in (~OC) which is imposed by the synthase that ensures that a six-membered ring c is formed contrary to Markovnikov behaviour.' 2o The bicyclic analogue (3 1) of squalene 2,3-epoxide was converted into the p-onocerine derivative (32) by the same synthase.The decalin derivative (33) is known to be a specific inhibitor of mammalian lanosterol synthase. 22 Squalene 2,3-epoxide and squalene 2,3 :22,23-diepoxide (22d) both accumulated in rat liver homogenates that were incubated with labelled mevalonate in the presence of this inhibitor.123 The squalene diepoxide was cyclized to form 24,25-epoxylanostero1 (23d) when incubated with rat liver homogenates in the absence of oxygen;24,25-epoxydesmosterol (34) was formed in aerobic incubations.'23 The latter epoxide has been shown to be a normal product of metabolism of acetate in rat liver homogenates.I (27)a;R =Me (28) b;R=H NATURAL PRODUCT REPORTS 1985 It has long been recognized that the microsomal enzymes of the biosynthesis of cholesterol in vertebrates require the participation of non-enzymic soluble proteins for full activity.Such a cytosolic protein has been implicated in the epoxidation of squalene by rat liver microsomes. 25 Furthermore a 'soluble protein factor' which stimulates squalene mono-oxygenase has been isolated from hog liver' 26 and a cytosolic 'sterol carrier protein' has been implicated in the transportation of labelled squalene from artificial liposomes to microsomes during studies on the biosynthesis of sterols in vitro.' 27 The isolation and purification of a 'supernatant protein factor' (SPF) that stimulates microsomal squalene mono-oxygenase was reported previously; it has now been shown that the protein SPF stimulates also the lanosterol synthase of rat liver microsomes A X-Enz H H Me (25) li H (23a) 'Enz (26) Scheme 4 b; R = H ; 18,19 -dihydro c; R =Me NATURAL PRODUCT REPORTS 1985 -D.M. HARRISON 531 I + 7 HO +A (35) ? / (37) (38) a; R = H (39) a; R' = OH R2= H,R3=CO H b; R =OH b; R1= H,R2=OH,R3=Me Scheme5 in the presence of anionic phospholipids.128 It was shown subsequently that SPF does not act as a carrier protein for squalene or squalene 2,3-epoxide7 nor does it bind to microsomes though it does interact with anionic phospho- lipids.129 The mode of action of SPF remains uncertain.Attention has also been given to the formation of pentacyclic triterpenes from squalene 2,3-epoxide in higher plants. Ursolic acid (38a) 2a-hydroxyursolic acid (38b) and 3-epi-maslinic acid (39a) as their methyl esters were examined by I3C n.m.r. spectroscopy following the incorporation of [4-' 3C]mevalonate or [1,2-I3C2]acetate into these acids in tissue cultures of Zsodon japonicus.I3O The labelling patterns that were observed and their mechanistic interpretations are summarized in Scheme 5. It is apparent that rings D and E are formed in these triterpenes by the routes that were predicted by Eschenmoser et ~1."~ rather than by plausible alternative routes.Furthermore the labelling pattern for carbons 4,23 and 24 in 3-epi-maslinic acid (39a) is consistent with the formation of the latter from (3s)- squalene 2,3-epoxide7 with epimerization (at some stage) of the 3P-hydroxy function which must be formed initially; the evidence precludes the alternative namely the direct formation of a 3a-hydroxy function uia cyclization of the (3R)-squalene epoxide.' 30 The early theoretical treatment predicted that the 12a-protons of intermediates (36) and (37) should be eliminated in the formation of the oleanane (39) and urs-12-ene (38) skeleta respectively.' These predictions also have been probed by feeding [5-'3C,5-2H2]mevalonate to tissue cultures of I.japonicus.' The triterpenoid metabolites were examined by I3C n.m.r. with simultaneous H-and 2H-decoupling which permits the determination of the positions of the 'H labels. The urs- 12-ene derivative 2a-hydroxyursolic acid (38b) was isolated with the labelling pattern shown [(40a) + (40b)l; this reveals unexpectedly that the 12P-proton was lost from the intermedi- ate (37). The oleanane derivative 3-epi-maslinic acid (39a) had the expected labelling pattern [(41a) + (41b)] consistent with the loss of the 12a-proton of (36) during its biosynthesis. The steroid 0-sitosterol (42) also was isolated and was labelled as shown [(42a) + (42b)l in Scheme 6.j3' NATURAL PRODUCT REPORTS 1985 D D 9 R2 D \ I D R1- \ I \ D I D HO b 0D 2 H 0 D* D D4=H,and~ 2 =D R'= D,R~= HI HOX 4 HO' (40)a; R = H (41)a; R1= H.R2= D b;R=D D b; R1= D,R2= H HO D (42)a; R' = H ,R2= D b; R'= D,R2= H Scheme 6 0Lq (43) Y (451 (44) 1 (46) (47) Scheme 7 NATURAL PRODUCT REPORTS 1985 -D.M. HARRISON The unnatural analogue (43) of squalene 2,3-epoxide has been used to elucidate the degree of control that is exercised by the cyclase during the formation of ring E of 0-amyrin (39b).132 A microsomal extract from seedlings of pea (Pisurn satiuurn) converted (43) into the analogue (46) of P-amyrin. Thus the 'normal' pathway which would require cyclization of the intermediate cation (44) to the primary carbo-cation (47) is diverted owing to the greater stability of the tertiary carbo- cation (49 as shown in Scheme 7.It was concluded that the formation of the tertiary carbo-cation (35) which is an intermediate in the biosynthesis of p-amyrin is a consequence of the thermodynamic stability of that cation rather than the result of control by the cyclase.'32 The squalene 2,3-epoxide p-amyrin-cyclase from microsomes of pea cotyledons is effi-ciently inhibited by the amines (48a) and (48b) and by the ammonium cation (48c). 33 Mechanistic aspects and evolutionary implications of the cyclization of squalene to tetrahymanol(49a) by the protozoon Tetruhyrnenupyriformzs have been reviewed. 34 It is known that an equatorial deuterium atom is incorporated at C-3 of tetrahymanol [as (49b)l when squalene is cyclized by a cell-free enzyme preparation from the same organism.' This result is consistent with an all-chair conformation (50a) for the enzyme- bound squalene molecule.Details of this work have now been published.' 35 The epimeric 3-hydroxytetrahymanol derivatives (49c) and (49d) were formed by a similar enzyme extract when (3RS)- squalene 2,3-epoxide was supplied. 36 Since T. pyriforrnis is incapable of the biosynthesis of squalene 2,3-epoxide it is assumed that the enzyme squalene cyclase brings about this transformation. The hexadeuteriotetrahymanol(49e) with two equatorial CD3 groups was formed when the stereospecifically labelled squalene (50b) was cyclized with the same cell-free enzyme system.The 3P-hydroxytetrahymanol that was bio- synthesized was similarly labelled [as (4901 when the (3RS)- hexadeuteriosqualene 2,3-epoxide (50c) was supplied as a substrate; these results are consistent with all-chair conforma- tions for the cyclization of squalene and (3S)squalene 2,3- epoxide. However the 3or-hydroxytetrahymanol(49g)that was formed from (3RS)-squalene 2,3-epoxide contained a 4p- (axial) CD? group which is consistent with its formation by cyclization of (3R)-squalene 2,3-epoxide via a boat-chair-chair-chair-chair mode of folding of the substrate [as (5l)].' 36 Experiments were performed also with a crude preparation of the squalene cyclase from the procaryote Acetobacter pasteuriunus.' 37 Thus the natural metabolites diploptene (52a) and diplopterol(53a) were formed when squalene was provided as a substrate.However the 3a- and 30-hydroxy-derivatives (52b) (53b) (52c) and (53c) of these triterpenes were formed when (3RS)-squalene 2,3-epoxide was incubated with the cell- free extract.' 37 The bacterium Methylococcus cupsulutus is unique among procaryotes in that it forms the sterol 4~-methylcholest-8( 14)-en-3p-01 as well as hopane derivatives. Diploptene (52a) and diplopterol (53a) were formed when squalene was supplied as the substrate to a cell-free extract from the 0rgani~m.I~~ Alternatively lanosterol (23a) and the unnatural 3-epi-lano- sterol were formed when racemic squalene 2,3-epoxide was supplied to the cell-free extract. It was shown that 3-epi- lanosterol arose from cyclization of the unnatural (3R)-squalene 2,3-epoxide.This is the first demonstration of 2,3-epoxysqua- lene lanosterol-cyclase activity in a procaryote though the cyclase is clearly less specific than normal in its choice of substrate. It was shown also that M. cupsulutus is capable of epoxidizing squalene. 38 The triterpenoid hosenkol-A (54) has been isolated from seeds of the ornamental plant Impatiens balsaminu.' 39 Hosen-kol-A is the first naturally occurring representative of the baccharane class of triterpenoid and in terms of its carbon skeleton represents a biogenetic 'missing link' between squalene 2,3-epoxide and the lupane and shionane triterpenoids. (48)a; R = NMe b; R = NEt2 C; R = hEt3 R' (49)a;R1= R2=H,R3=R4=R5= Me b; R'= D R2= H,R3=R4=f$= Me c; R1= OH R2= H,R3= R4= R5= Me d;R1= H,R2=OH,R3=Rb=R5=Me e; R' = R2=H R3= Me,R4= R5= CD3 f ; R1= OH,R2= H R3= Me,R4= R5= CD3 g;R' = H,R2= OH R3= R5 = CD ,R4= Me R (50)a; R = Me b; R = CD c;R =CD3;2,3-epoxide (511 (53)a;RLR~=H (52)a ; R' = R2= H b; R'= H R2= OH b;R'=H,R2=OH C; RLOH,R*= H C; RLOH H ,~2= HO (54) 5 The Formation of Sterols in Vertebrates 5.1 The Biosynthesis of Cholesterol It is generally recognized that the first stage in the nuclear transformation of lanosterol (23a) or 24,25-dihydrolanosterol (55)is the oxidative removal of the 14a-methyl group as formic acid.This process requires NADPH and molecular oxygen is dependent on cytochrome P-450 and in the case of the dihydrolanosterol(55) it proceeds uiathe diol intermediate (56) the A8(9)-carbaldehyde (57) and the A8(9),15compound (58) (Scheme S).' It has now been shown that only the initial oxidation of 24,25-dihydrolanosterol to furnish the diol (56) is dependent on cytochrome P-450 in rat liver homogenates; the subsequent conversions of compounds (56) and (57) into cholesterol (2b) are unaffected by carbon monoxide.I4O Compounds (56) and (57) both inhibited the demethylation of 24,25-dihydrolanosterol in rat liver homogenates ;I 4o the same compounds and their A7 analogues (59a) and (59b) also H (55) (56) li (58 1 Reagents i NADPH 0 Scheme 8 H H (59)a; R = CH,OH b; R = CHO c;R = H H NATURAL PRODUCT REPORTS 1985 inhibited the demethylation of lanosterol in cultured hamster lung cells and strongly inhibited the incorporation of acetate into sterols in a variety of cultures of mammalian cells.l4I The main effect in the latter case was a decrease in activity of the HMG-CoA reductase. A substance that was identified tenta- tively as the carbaldehyde (57)or its A7 analogue (59b) has been isolated from cultured human lymphocytes. 14* It was reported earlier that the unnatural 14a-methyl-A7- sterol (60a) was efficiently metabolized to cholesterol by rat liver homogenates under aerobic conditions. The A7-sterol (60b) its A8(9)analogue (61 b) the dienols (62) and (63) and the A8(14)-~tero1 Under (64a) are among the metabolites of (~OZL).'~~ anaerobic conditions the A'-sterols (60a) and (60c) are equilibrated with their analogues (61a) and (61c) respectively by liver microsomes.54 The A8(I4)-sterol (64a) which is known to be metabolized to cholesterol in the presence of oxygen and NADPH was formed in an NAD+-dependent reaction sequence when the diol(6Oc) was incubated with liver microsomes under anaerobic conditions. This observation is apparently inconsistent with the mixed-function oxidase pathway that has been reported for the natural precursor (56) (see above). More surprisingly other workers have isolated the A8(14)-3P,7a-diol (64b) following the incubation of the carbalde- hyde (60d) with liver microsomes. It was suggested that the diol (64b) was the first intermediate to be formed following the oxidative elimination of formic acid.Is6 The relevance of these observations to the demethylation of lanosterol is unclear; it seems likely that the mechanisms that are involved in the enzymic removal of the 14a-methyl of compound (60a) differ fundamentally from those for the natural substrates lanosterol and 24,25-dihydrolanosterol.The intermediate steps in the biosynthesis of cholesterol from lanosterol are summarized in Scheme 9 in which the steps of the sequence from 24,25-dihydrolanosterol are depicted. The A8 pathway is shown because this is believed to be the major route. However studies with A7 substrates remain popular as discussed below. The stepwise oxidation of the 4a-methyl group of (65) to furnish the carboxylic acid (66) requires molecular oxygen and either NADH or NADPH.It has now been shown that these two coenzymes utilize different routes of electron transport during the oxidative demethylation of the 4,4-dimethyl-A7-sterol (59c); similar observations were made during the oxidative deformylation of the A'-ketone (67) by rat liver microsomes. During the oxidative demethylation of H (61) a; R = Me (6010;R = Me b;R = H b;R=H c;R = CH,OH C; R = CHZOH d;R = CHO d; R = CHO H (64)a;R = H H (62) (631 b;R=OH NATURAL PRODUCT REPORTS 1985 -~ D. M. HARRISON (55) Reagents i NADPH O2 or NADH 02;ii NAD+; iii NADPH Scheme 9 H HO HO,C@ R CHOH R-R (67) (68) (69)a;R = Me (59c) a high concentration of NADH inhibits the NAD+-b; R = C02H (70)a;R = Me dependent oxidative decarboxylation of the intermediate b;R =H carboxylic acid (68) which consequently accumulates.48 Conflicting results have been reported concerning the dependence on cytochrome b of the cyanide-sensitive 4-H methylsterol oxidase of rat 50 Gaylor and his colleagues have isolated a microsomal fraction that contained 4-methylsterol oxidase and cytochrome-b reductase activities but which was free of cytochrome b,. The ability of this fraction to oxidize 4a-methyl-5a-cholest-7-en-3P-ol(69a) to the carboxy- lic acid (69b) was restored when cytochrome 6 was added. Evidence for the obligatory role of cytochrome b in intact HO microsomes was furnished by the observation that their 4a- iA methylsterol oxidase activity was inhibited with antibodies R that had been raised to cytochrome b5.49 Conversely Maitra et af. have isolated a cyanide-sensitive oxygenase that contained (71)a; R = CH,CN non-haem iron from rat liver microsomes; the ability of this b; R = CH(0H)CN protein to oxidize the 4a-methyl of 4,4-dimethylzymosterol (70a) was reconstituted with a labile electron-transport system C; R = COCN in the presence of oxygen and NADH or NADPH.lS0 No dependence on cytochrome 6 could be demonstrated though H involvement of the latter was not completely excluded. It was suggested that a hitherto unrecognized redox protein may be involved. Genetic evidence has been cited in support of the contention that at least one common component exists for the enzyme systems that demethylate 4,4-dimethyl- and 4a-methyl- sterols.The 4a-cyanomethyl-sterol (7 1a) is an irreversible inhibitor of microsomal4-methylsterol oxidase. 52 It was suggested that inhibition occurs owing to the stepwise oxidation of the 4- I;r substituent of (71a) to form the cyanohydrin (71b) and then the R acylnitrile (7 lc) which irreversibly acylates a nucleophilic (72) a; R = Me group at the active site of the oxidase. b;R=H NATURAL PRODUCT REPORTS 1985 (75) (76) The keto-steroid reductase that catalyses the NADPH-dependent reduction of the A7-ketones (72a) and (72b) to the sterols (69a) and (73) respectively has been isolated from liver microsomes.153 The properties of the soluble enzyme were similar to those of the microsome-bound enzyme but only the latter was stimulated by cytosolic 'Z-protein' (fatty-acid-binding protein).A sterol-carrier protein SCP2 that activates the microsomal oxidative demethylation of the 4,4-dimethylsterol (65) to furnish C2 sterols has been isolated from rat 1i~er.I~~ Protein SCPz also activated the microsomal conversion of cholesta- 5,7-dien-30-01 (74) into cholesterol but had no effect on the microsome-catalysed conversion of squalene into lanosterol. The final steps in the biosynthesis of cholesterol are summarized in Scheme 10. The cyanide-sensitive mixed- function oxidase (desaturase) system that catalyses the oxida- tion of the A'-sterol (73) to the 5,7-diene (74) has been isolated from rat liver microsomes.On purification a fraction was isolated that contained an oxidase phospholipid and cyto- chrome-b reductase (E.C. 1.6.2.2). The desaturase activity was reconstituted if cytochrome b5was added to this fraction in the presence of NADH.I5 It was suggested that the A7-sterol As-desaturase and the 4-methylsterol oxidase system may involve the same terminal oxidase. This suggestion was made on the basis of the dependence of both activities on the presence of cytochrome bs149*5s (but cf ref. 150) and the failure to achieve any resolution of those activities in this study. 55 Details of the large-scale isolation and purification of 'squalene and sterol carrier protein' (SCP) have been de- scribed.156 It was reported that this protein constitutes 8% of the soluble proteins of rat liver; SCP was assayed by its stimulatory effect on the activity of membrane-bound A7-sterol As-desaturase.It was suggested that SCP may be identical to the fatty-acid-binding protein (Z protein) that was described previously. Other workers have confirmed that the microsomal A7-sterol A5-desaturase is stimulated by SCP but have shown H H (77) H b;R = Et that SCP does not act by conveying the substrate from the aqueous medium to the microsomes.157 It was suggested that SCP may function in the intra-membrane translocation of the substrate (73). Human leucocytes have a low ability to biosynthesize It has now been shown that this is in part due to a deficiency in sterol-carrier proteins.' 59 Human granulocytes were shown to contain lanosterol synthase but to lack both the squalene mono-oxygenase and the enzymes that are required for the conversion of lanosterol into cholesterol.6o The 5,8-diene (75) has been isolated together with other steroids from the livers of pregnant rats that were fed the drug AY-9944. 61 The biosynthetic relevance of this observation is not yet clear. The unnatural 5,7,22-triene (76) was reduced to cholesta- 5,22-dien-30-01(77) by a rat liver homogenate. 62 The isomeric triene (78) is a potent inhibitor of the biosynthesis of cholesterol in cultured mammalian cells.163 In contrast neither the 5,7-diene (74) nor the A7v9(I1) isomer showed any inhibitory activity. The triene (78) was esterified by rat liver homogenates but was not metabolized to cholestero1.1b4 It is known that many steroids that possess oxygen functions at C-15 can serve as inhibitors of the biosynthesis of chole~terol.'~~ Amongst these the enone (79) is a potent hypocholesterolemic agent ; 66 the biosynthesis of cholesterol is blocked by this inhibitor at some post-mevalonate stage.16' The enone (79) is metabolized efficiently to cholesterol in rat liver homogenates. 68 As noted previously the 14a-hydroxymethyl-steroid (56) is a natural inhibitor of the biosynthesis of cholesterol. 14a-Hydroxymethyl-steroids some of which possess a 15a-hydroxyl group are potent inhibitors of the biosynthesis of sterols in mouse liver cells.169 A major site of inhibition is the enzyme HMG-CoA reductase (NADPH).The triols (80a) and (81) show additional sites of inhibition in the post-lanosterol phase of the biosynthesis of cholesterol in cell-free rat liver preparations.170 Thus the A7 compound (80a) inhibits the NATURAL PRODUCT REPORTS 1985 -D. M. HARRISON demethylation of lanosterol which consequently accumulates [together with 24,25-dihydrolanosterol (55)]when mevalonate is supplied as substrate; in contrast the A6 isomer (81) inhibits the sterol A8-A7-isomerase and causes the accumulation of 5a-cholest-8-en-3P-o1(61 b) under the same conditions.' 70 The 14a- ethylsterol (80b) and the derived 3-ketone are extremely potent inhibitors of the synthesis of sterols in mouse L cells.'71 The former compound acts rapidly by suppressing the metabolism of lanosterol and 24,25-dihydrolanosterol;the HMG-CoA reductase activity is also suppressed but more slowly.172 The epimeric 15-hydroxy-ketones (82a) and (82b) which both contain the unnatural C~S-C/Dring junction suppress HMG-CoA reductase activity in animal ~e1ls.l~~ The 15a- hydroxy-compound (82a) but not its epimer (82b) was metabolized efficiently to cholesterol via the 8,14-diene (63).' 74 Other steroidal inhibitors of the biosynthesis of cholesterol that have been reported include a 3or-chloro-l5-oxo-steroid,~ 75 a 1401-hydroxy-A7-sterol,'76 9a-hydro~ylanostanes,~ 77 and 9a- hydroxy- or 9a-fl~oro-A~(~~)-I5-one derivatives.' 78 All of the above types of inhibitor act at least in part by suppressing the activity of the HMG-CoA reductase.The demethylation of lanosterol by hepatoma cells in culture was inhibited by 7a- and 70- hydrox yc holesterols. I Several analogues of lanosterol that possessed modified side- chains significantly inhibited the biosynthesis of cholesterol from lanosterol in rat liver homogenates.IS0 One such in hi bitor 24,25-di hydro-27-norlanosterol was metabolized to 27-norcholesterol in 6.5% yield.' * Analogues of lanosterol with additional oxygen functionality in the side-chain or the nucleus were tested also; amongst these compounds the 7-0x0- derivative (83) was an extremely potent inhibitor.'*' Choles- terol analogues with modified side-chains have also been tested as inhibitors of the biosynthesis of cholesterol. 83 25-Hydroxycholesterol (2a) cholestane-3P,5a,6P-triol(84) and mevalonolactone all suppressed the H MG-CoA reductase activity rapidly in rat hepatocyte cultures.In addition the steroidal compounds inhibited the demethylation of lano- ster01.I~~ Other relevant studies of inhibition have been reported. Is5 The absolute rate of biosynthesis of cholesterol can be estimated by the uptake of radioactive tracer from tritiated water and from 14C-iabelled precursors'86 and also by measurement of the rate of formation of desmosterol in the presence of triparanol. 87 Methods that utilize the precipita- tion of sterols with digitonin tend to over-estimate the rate of biosynthesis of cholesterol. 88 The diurnal variation in concentrations of squalene and of methylsterols in human plasma lipoproteins has been studied.Is9 A method for the separation of precursors of cholesterol based on h.p.l.c.has been described.Ig0 The resonances due to the 25bro-25R)- and 25(pro-25S)- methyl groups in the I3C n.m.r. spectrum of cholesterol were assigned earlier with the aid of cholesterol that had been enriched biosynthetically by a feeding experiment with [4 Me-'3C2]mevalonate.' The assignments were based on the known stereochemistry of reduction of the AZ4precursor of cholesterol. These conclusions have now been confirmed independently by a non-biosynthetic study.I9l Comprehensive reviews have been published on the biosyn- thesis of cholesterol," on the regulation of the formation of cholesterol in the liver,I9' and on the demethylation steps in the biosynthesis of steroids.93 Shorter accounts describe aspects of the stereochemistry of the biosynthesis of sterolsIg4 and the enzymes of the biosynthesis of cholesterol. 195 5.2. The Biosynthesis of Steroidal Hormones The steroidal hormones are derived from pregnenolone (85a) which is formed (together with 4-methylpentanal) by oxidative cleavage of the side-chain of cholesterol (2b). This process requires molecular oxygen and NADPH and is catalysed by a cytochrome-P-450-dependent mixed-function oxidase [choles- terol mono-oxygenase (side-chain-cleaving) (E.C. 1.14.15.6)]. Earlier studies had suggested that the oxidative cleavage of cholesterol involves the transient formation of (22R)-22-hydroxycholesterol (86a) and the (22R)-20,22-dihydroxy-com-pound (86b).However the corresponding (22s)-compounds were also substrates of the same oxidase system. It has now been demonstrated (by g.c.-m.s. studies) that the (22R)- compounds (86a) and (86b) are the only hydroxylated deriva- tives of cholesterol that are formed during the aerobic incubation of cholesterol and NADPH with adrenal mitochon- dria. 96 Furthermore these (22R)-compounds were the only major oxidized sterols that were bound to the purified adrenal cytochrome P-450 that was active in the reaction to cleave the side-chain [cytochrome P-45OsCc]. 97 It was reported also that the triol (86b) accumulated when (22R)-22-hydroxycholesterol was incubated with adrenal mitochondria under conditions (pH 7.8) that suppressed the C-C bond-cleavage reaction.198 It is concluded that the diol (86a) and the triol (86b) are genuine intermediates in the biosynthesis of pregnenolone.(81) (82) a; R1=H R2=OH (83) b; R1= OH,R2= H R3 H H (86) a; R' = R2= HI R3= OH (85)a ; R = H b ; R' = R3= OH,R2=H b;R=OH c;R'= H,R2R3= 0 NATURAL PRODUCT REPORTS 1985 Cholesterol that was labelled stereospecifically with deuter- ium 196 or tritiumt99 has been employed to demonstrate that the hydroxylation at C-22 proceeds with retention of configura- tion.196,t99 It was shown also that no 22-0x0-intermediate is involved in the conversion of cholesterol into pregnenolone that is catalysed by adrenal mitochondria. 99 The acetylene (87) and related compounds inactivate cytochrome P-45OSCC irreversibly in the presence of NADPH and oxygen.Inactivation of the enzyme may occur via reaction (88) with an intermediate oxirene [e.g.(88)].*0° The hydroxylated amines (89a) and (89b) are competitive inhibitors of cyto- (87) chrome P-45OsCc; it is believed that the nucleus of the H inhibitors is bound at the same site that binds the nucleus of cholesterol while the amino-group in the side-chain co-ordinates to the iron atom of haern.*Ot 22-Oxocholesterol(86c) rNH2 is a potent competitive inhibitor of cytochrome P-450sCc.202 Further studies have been reported on the substrate specificity of cytochrome P-450sCc.203*204 The [22-14C]-cyclo- propyl-derivative (90a) was converted into [carboxy-l 4C]cyclo-(89) a as shown (90) a; R = H propanecarboxylic acid by adrenal mitochondria.?04 Retention b; 5ar. ,6-dihydro b;R=OH of the intact cyclopropyl ring in the latter product was taken as evidence that oxidative cleavage of the intermediate trio1 (90b) or of the natural intermediate (86b) does not involve the generation of a radical centre at C-22.’04 Cytochrome P-45OScCof adrenal mitochondria requires an electron-transport system that includes adrenodoxin for enzy-mic activity. The mutually facilitated binding of cholesterol and adrenodoxin to the cytochrome in phospholipid vesicles has been studied.’OS An important series of papers describe the structural organization of the oxidase system.206 Other reports (91) (92)a R = H concern the primary structure of adrenal cytochrome P-b:R =OH 450scC,207the interaction of the latter with Triton X-100,208 the 0 binding of oxygen and carbon monoxide to the cytochrome in 0 the presence of sterol substrates,2o9 the inhibition of cleavage of the side-chain of cholesterol by antibodies that have been raised to cytochrome P-450sCC,z O the side-chain-cleaving enzyme system of human placenta,’ I and the purification of cytochrome P-45OScC of bovine corpora lutea.’ Cytosolic modulators of cytochrome P-450scc have been isolated and (93) described.” A key step in the biosynthesis of the steroidal hormones is the 0 oxidation of pregnenolone (85a) to furnish the 3-0x0-As-steroid (94)a;R = H (91) which is then isomerized to progesterone (92a).These two b; R =‘H steps are catalysed by the NAD+-dependent enzyme 3(or 17)p-hydroxysteroid dehydrogenase (E.C.1. I. 1.51) and by steroid A- R isomerase (E.C. 5.3.3. l) respectively. In a similar sequence dehydroepiandrosterone (93) is converted into the 3-0x0-As- (95a). steroid (94a) and thence into androst-4-ene-3,17-dione RH Both pregnenolone and dehydroepiandrosterone serve as substrates for the enzyme 3P-hydroxy-A5-steroid dehydrogen- (95) a R = H ase (E.C. 1.1.1.145). The subcellular distribution of the 3(or 17)P-hydroxysteroid dehydrogenase of Gallus domesticus’ and b; R =‘H (96)a:R = COMe the substrate specificity of a dehydrogenase of human placenta2I5 have been studied. b;R=OH It is generally accepted that the dehydrogenase (E.C. 1.I. 1.51) and the isomerase (E.C. 5.3.3.1) are separate enzymes.The lack of significant inhibition of the isomerase activity by the steroidal substrates of the dehydrogenase is in accord with this interpretation.’ The oxidation of dehydroepiandroster- ..t one (93) by mammalian dehydrogenase preparations was X -Enz irreversibly inhibited in a time-dependent manner by incuba- tion of the crude dehydrogenase with the 5,lO-seco-steroids (97) a; R = COMe (96a) or (96b); inactivation of the dehydrogenase must involve b R =OH 0&(98) the conversion of these inhibitors into their 3-0x0-derivatives OH (97a) and (97b) respectively followed by Michael addition to the conjugated enone system of these products.2t7 Other mechanism-based irreversible inactivators of the dehydrogen- ase have been described./ Further studies have been reported on the mechanism of the enzyme-catalysed conversion of 3-0x0-As-steroids into their 3- o~o-A~-isomers.~~It is known that the 4P-proton of the 9-221 steroid (94a) becomes in part the 6P-proton of the product (99) NATURAL PRODUCT REPORTS 1985 -~D. M. HARRISON androst-4-ene-3,17-dione (95a) when the reaction is catalysed by the isomerase of the bacterium Pseudornonas testosteroni. A re-investigation of this observation has revealed that the deprotonation at C-4 is not completely stereospecific in this organism. [4-'4C]Androst-5-ene-3,17-diol (98) was metabolized to an- drost-4-ene-3,17-dione (95a) by a microsomal enzyme prepara- tion from human placentae ;the intermediate testosterone (99) could be trapped by addition of unlabelled material.220 Double- labelling experiments with the ' "C-labelled and specifically tritiated precursor revealed that the 40-proton of (98) is lost during this conversion while the 4a-proton is retained at C-4 of androst-4-ene-3,17-dione. The 6-proton of (98) was retained also in the androstenedione (95a) at the 6a-position.These results are summarized in Scheme 11 and are similar to those reported earlier for the isomerase of P. testosteroni (but see ref. 219) except for the lack of detectable intramolecular migration of the 4P-proton of the substrate.220 In contrast in the isomerization that is catalysed by bovine adrenal microsomes some 9% of the 4P-deuterium label of (94b) was retained at the 6P-position of the product [as (95b)].221 This observation is consistent with the involvement of a single basic group in both the enzyme-catalysed deprotonation at C-4 and the reprotona- tion at C-6.The adrenal steroid A-isomerase is inactivated by incubation with the acetylenic seco-steroid (100). Presumably the isomeric allene (97a) is generated at the active site of the enzyme which is then inactivated by Michael addition to the conjugated enone moiety.*** Studies have been conducted also on the inactiva- tion of the isomerase of species of the genus Pseudornonu~.~*~ Studies continue on the details of the biosynthetic conversion of progesterone into androst-4-ene-3,17-dione (95a). A possible intermediate in this process is 17a-hydroxyprogesterone (92b).A cytochrome P-450 (mol. wt. 59 000) that was isolated from microsomes of pig testes catalysed the NADPH-dependent oxidation of progesterone to its 17a-hydroxy-derivative and to androst-4-ene-3,17-dione when supplemented with a flavopro- tein reductase; it was suggested that the same active site is responsible for both activities.224 Another cytochrome P-450 (mol. wt 52 000) was isolated from pig adrenal microsomes; its 17a-hydroxylase and 17-20 bond-cleavage (lyase) activity were both reconstituted with NADPH-cytochrome P-450 reduc- ta~e.~~~ Other studies have shown that the electron-transport system that is associated with the testicular 17-20-lyase is more complex than is implied in the latter study and in particular that cytochrome b5 is an essential ~omponent.*~~,**~ It has been suggested that the N AD+-dependent oxidation-isomeri- zation of pregnenolone to progesterone (see above) is coupled to the NADPH-dependent cleavage of the side-chain of 17a-hydroxyprogesterone with electron transfer occurring via cytochrome b5.227 The proposed role of 17a-hydroxyprogester- one in the conversion of progesterone into androst-4-ene-3,17- dione has been challenged on the basis of the relative rates of 5 39 conversion of the former two compounds to the latter in enzyme preparations from rat testes.228 A study has been reported on the metabolism of [17a- 2H]pregnenolone (85a) by microsomes from boar testes under an atmosphere of [ '*0]0xygen.~~~ Amongst the labelled products that were isolated are 17a-[ 17-hydroxy-1 *O]hydroxy- pregnenolone (85b) [ 17-1*O]dehydroepiandrosterone (93) [ 17-2H]androsta-5 16-dien-3P-01 (1 0 1) and (surprisingly) [ 17P-*H 17-hydroxy-' 80]androst-5-ene-3P 17a-diol (102).The pres- ence of the deuterium label in the latter two compounds reveals that they arise by loss of the side-chain without prior 17a- hydr~xylation.~~~ A recent suggestion for the biosynthesis of (101) is not consistent with the above 0bservations.~3~ The biosynthesis of oestrogens from androgens has been the subject of intensive mechanistic study during the period under review.231-236 The process involves oxidative loss of C-19 as formic acid from a derivative of androst-4-en-3-one concomi- tant with the aromatization of ring A and is catalysed by the placental enzyme system 'aromatase'.Testosterone (99) was converted into 17P-oestradiol(103) by human placental microsomes in 10% yield. Oxygen- 18- labelling experiments revealed that the product retained over 50% of the carbonyl oxygen atom of the precursor; it was concluded that no Schiff-base intermediate is involved in the aromatization reaction.231 Further feeding experiments with specifically deuteriated androst-4-ene-3,17-dione(95a) re-vealed that the 4- and 6P-protons of the latter are quantitatively retained while the 6a-proton is substantially retained during the aromatization reaction.232 These results preclude the intermediacy of a 3,5-dienol. A deuterium isotope effect of 3.2 was observed during the conversion of [ 19-*H3]testosterone into oestradiol by placental microsomes so confirming that hydroxylation at C-19 is the rate-determining step.232 Earlier results had shown that the aromatase-catalysed oxidation of androst-4-ene-3,17-dione (95a) involves the follow- ing three steps each of which utilizes one mole equivalent of NADPH and molecular oxygen (i) hydroxylation at C-19 to afford the primary alcohol (104); (ii) oxidation of the latter to furnish (presumably) the geminal diol (1 09 which eliminates water to yield the aldehyde (106); and (iii) a further oxidation reaction that results in the release of formic acid and the formation of oestrone (107) as summarized in Scheme 12.It was known also that the 19(pro-l9R)-hydrogen of (104) is removed during the second oxidation step.The latter observa- tion enabled Caspi et a/. to investigate the stereochemistry of the initial hydroxylation reaction [step (i)] by supplying the stereospecifically labelled substrates (I 9R)-[ 19-2H1 W3H and (19S)-[ 19-*H ,19-3H,]-3P-hydroxyandrost-5-en-l 7-ones (93) to human placental micro some^.^^^ This elegant study depended on the existence of an isotope effect (c$ ref. 232) in the hydroxylation of the labelled substrate that discriminated against loss of the heavier isotopes of hydrogen and involved measuring the ratio of the tritium that is released as water to Scheme 11 vo OH H (1001 (101) NATURAL PRODUCT REPORTS 1985 that which is released as formic acid when each of the two as a transient intermediate only in the biosynthesis of oestrone labelled substrates was metabolized.The result that was precludes investigation as to whether the second hydroxylation obtained i.e.that hydroxylation of the intermediate androst-4- ene-3,17-dione (95a) occurred with retention of configuration is consistent with other studies on the biological hydroxylation of methyl groups.233 The occurrence of the geminal diol (105) (95a) bi 0 A + 0& m H-C,* //O H20A 0 + OH <H-e o& / (107) (106) Reagents i NADPH Ol; ii NADPH 0:; iii NADPH 0; Scheme 12 II / (108) 0Li!P aromatase 1 (109) occurs with retention or inversion of stereochemistry. Reten- tion of configuration is assumed in Scheme 12.Time-course studies have confirmed that the aldehyde (106) is a true intermediate in the conversion of (104) into oestrone (107) by human placental micro some^.^^^ The fate of the labelled oxygen of the (19S)-[19-2Hl ,180]-alcohol (104) and the [19-2H1 80]-aldehyde (106) during the aromatase-catalysed reaction sequence has been studied. Complementary experi- ments were performed by incubating the deuteriated substrates with placental microsomes in the presence of an atmosphere of [I80]oxygen. The major conclusions were that the 19-oxygen atom of (104) is retained in the aldehyde (1 06) and in the formic acid that is subsequently released that the newly introduced oxygen atom of the geminal diol (105) is lost as water on its conversion into the aldehyde (106) and that the second oxygen atom of formic acid derives from the oxygen molecule that was utilized in the third step of the reaction sequence.These important conclusions are summarized in Scheme 12. Finally it was shown that a plausible intermediate in the aromatization sequence namely the formate ester (108) is not metabolized to oestrone by placental microsomes. 234 Fishman et al. had earlier isolated the 2P-hydroxy-aldehyde (109) as a labile intermediate in the aromatase-catalysed reaction. Oestrone was formed spontaneously from (109) in aqueous solution at pH 7.1. It has now been shown by the same workers that the spontaneous aromatization of (109) involves stereospecific loss of the lp-prot~n.~~~ Since it has been established that the 10-and 2P-protons of androgens are lost during the aromatase-catalysed sequence the present results are consistent with the suggestion that step (iii) of that sequence involves 2B-hvdroxvlation of the aldehyde (106) with retention of configurati*on followed by non-catalysed fragmentation of (109) to furnish oestrone and formic Taking account of the results of Akhtar et al.,234 it now seems probable that the terminal step in the biosynthesis of oestrone occurs via the reaction sequence that is summarized in Scheme 13.Results of studies on the inhibition by the alcohol (104) and by the aldehyde (106) of the enzyme-catalysed conversion of 4C-labelled androst-4-ene-3,17-dione(95a) into oestrone sug- gest that steps (i) and (ii) of the reaction sequence occur at the same active site while 2P-hydroxylation of (106) occurs at a separate enzyme site236 (cf ref.234). An X-ray-crystallographic study has been performed in order to determine the probable 1 1 Scheme 13 OH 0& / (110) a; R' = R*= H b; R' = OH,R2= H (113) (114) a;R'= F R2= H C; R1R2=0 b; R'= F R2=OH d; R' = H,R2= OH C; RW=o NATURAL PRODUCT REPORTS 1985 D. M. HARRISON ~ conformation that is involved in the oxidation of alcohol (104) by ar~matase.'~' The acetylene (1 10a) and the (19S)-19-hydroxyacetylene (1 lob) both caused irreversible inactivation of aromatase in a time-dependent manner when they were incubated with human placental microsomes in the presence of NADPH and oxygen.238 It was suggested that these artificial substrates were both oxidized by the enzyme which is then inactivated by Michael addition to the product ynone (1 10c).In keeping with this suggestion the synthetic ynone (1 1Oc) caused inactivation of aromatase in the absence of NADPH while the (19R)-ynol (1 IOd) which possesses the wrong stereochemistry for enzymic conversion into (1 lOc) is merely a competitive inhibitor of the enzyme. Other workers have shown that the allene (I 1 I) inactivates aromatase irre~ersibly.'~~ It was suggested that inactivation involved the reaction of the enzyme with an intermediate allene oxide (112) and also that an oxirene intermediate could explain the results that were obtained with the inhibitor (1 10a).Other mechanism-based inhibitors of aromatase include the methylthio-steroid (1 13)'j0 and 19,19- difluoroandrost-4-ene-3,17-dione(1 14a).241 Inhibition by the latter compound presumably involves prior oxidation to the difluoro-alcohol (I 14b) which would eliminate hydrogen fluoride to form the acyl fluoride (1 14c) at the active site of the enzyme.*jl Several other derivatives of androst-4-ene-3,17- dione modified at C-19 have been tested as inhibitors of the aromatase system.242 4-Hydroxyandrost-4-ene-3,17-dione (1 15a) and the acetate (115b) are both potent inhibitors of the biosynthesis of oestrogens. It has been shown that they act as suicide inhibitors A of ar~matase.'~~ new mechanistic sequence has been proposed for the aromatase-catalysed transformation based on these and other studies of the inhibition of the enzyme system; however the mechanism that has been suggested does not account adequately for the stereospecificity of loss of the 1P-proton of the natural substrate during aromatization.244 17a- Ethynyl-19-nortestosterone,which is an ingredient of contra- ceptive pills is a suicide inhibitor of ar~matase.'~~ Short reviews have been published on the biochemical mechanism of the aromatization sequence2s6 and on the relevance of the biosynthesis of oestrogens to the pathology of breast tumours.247 The formation of the biologically active metabolite 17P-hydroxy-5a-androstan-3-one (1 16a) from testosterone is cata- lysed by the NADPH-dependent enzyme cholestenone 5a-reductase (E.C.1.3.1.22). Nuclear and microsomal versions of this enzyme have been solubilized. Considerable medical interest attaches to studies directed at the inactivation of this enzyme. The diazopregnane (1 17) and related diazo-com- pounds act as irreversible inactivators of the enzyme presum- ably owing to enzymic protonation at C-4 which generates a powerful alkylating agent at the active site of the enzyme.149 The enzyme is irreversibly inactivated also by (1 18) presum- ably owing to Michael addition to the conjugated dienone moiety. 50 Studies with micro-organisms as well as with mammalian systems have established that several hydroxysteroid oxidore- ductases exhibit dual activity. Thus the 20a-hydroxysteroid dehydrogenase (E.C.1. I. 1.149) from bovine erythrocytes catalyses the NADPH-dependent reduction of progesterone to the 2011-alcohol (1 19) but also catalyses the reduction of 17P- hydroxy-5a-androstan-3-one(1 16a) to the 3P-hydroxy-steroid (1 16b).251 The 20a-hydroxysteroid dehydrogenase has been purified also from human erythrocytes.252 The enzyme oestradiol 17P-dehydrogenase (E.C. 1.1. I .62) isolated from human placentae catalyses the reduction of oestrone to 17P- oestradiol (103) and also the reduction of progesterone to the 20a-hydroxy-steroid (1 19).253 Intensive studies with irrevers- ible inactivators of the enzyme have established that the 17p- and 20a-activities are destroyed at the same rate in agreement with the postulate that a single active site of the enzyme is involved.253,254 Oestradiol 17P-dehydrogenase has been 541 0 OH OR (115) a;R = H b;R=Ac (OHH4\Y Me -H CH2 (117) (118) (120)a; R' = R~= H b; R1= H ,R2= OH c;R1= R2=OH 0 (121) a; R'= RZ= H ,~3= OH b;R'= H,R2=R3=OH C; R'= R3= OH R2 = H d;R1=R3=H,Rz=OH isolated also from liver of guinea-pig'55 and chicken.'56 Other dehydrogenases that display dual activity are the 3(or 17)~-hydroxysteroid dehydrogenase of rabbit kidneyz5' and the (R)-20-hydroxysteroid dehydrogenase (E.C. 1.1.1.53) of the bacterium Streptomyes hydrogenans. 258 An early step in the biosynthesis of corticosteroids is the oxidation of progesterone or 1701-hydroxyprogesterone to furnish the 2 1-hydroxy-derivatives (I 20a) and (120b) respec- tively.A cytochrome P-450 (mol. wt. 47 000) has been isolated from bovine adrenal microsomes; its 2 1-hydroxylase activity was reconstituted with NADPH-cytochrome P-450 reduc- ta~e.'~~ Studies have been reported also on the 21-hydroxylase of pig adrenal microsornes260 and of rabbit liver micro-somes.261 Cytosolic activators of the 2 1-hydroxylase system have been studied.262 The adrenal cytochrome P-450 that catalyses 1lp-hydroxyla-tion of steroids has been in~estigated.'~~ Hydroxylation of 18- hydroxy-1 1-deoxycorticosterone (121a) with a reconstituted 1 1P-hydroxylase system furnished the 1I@-hydroxy-steroid (121 b) together with the new 19-hydroxylated metabolite (121c).264 NATURAL PRODUCT REPORTS 1985 The biosynthesis of aldosterone (1 22) from corticosterone (121d) by a mitochondria1 preparation from adrenal glands has been studied.26s The intermediate 18-hydroxycorticosterone (121 b) was converted into aldosterone only in the presence of molecular oxygen and NADPH and this step was blocked by carbon monoxide and by other inhibitors of cytochromes P-450.It was concluded that oxidation of (121 b) involved further hydroxylation at C-18 to furnish a geminal diol (Scheme 14) rather than a dehydrogenase-mediated reaction.26s,266 The carboxylic acid (123a) is a major metabolite of corticosterone in the mouse.267 Similarly progesterone was oxidized to the carboxylic acid (1 23b) and also degraded to the androst-4-en-3-onecarboxylicacid (1 24) by liver micro-somes.26s The latter carboxylic acid was identified as a metabolite of 2 1-hydroxyprogesterone also.269 Further discussion has occurred on the possible existence of biosynthetic routes to steroidal hormones that do not in- volve squalene and cholesterol as intermediate^.^^^."^ The conversion of 20-hydroxy-23,24,25,26,27-pentanor-[7-3H]cholesterol (1 25) into cortisol (1 ~OC) in 0.002% radiochemical yield by the guinea-pig was cited as evidence for a biosynthetic route via a pentanorsqualene; for comparison it was shown that pregnenolone (85a) is metabo- lized to cortisol by the same organism in 0.007% yield.271 0 (121d) (121b) I Hq H\ .o \ - c (122b) (122a) Scheme 14 However it seems more likely that the former conversion is simply a reflection of the known lack of specificity of the enzyme system that cleaves the side-chain of cholesterol.5.3. The Biosynthesis of Bile Acids The rate-limiting step in the biosynthesis of the bile acids is the hydroxylation of cholesterol to furnish 7a-hydroxycholestero1 (126).272Cholesterol 7a-mono-oxygenase (E.C. 1.14.13.17) is a cytochrome-P-450-dependent mixed-function oxidase with a short half-life,273 and is subject to rapid modulation of activity via a reversible phosphorylation-dephosphorylationcycle.274 In contrast to HMG-CoA reductase (NADPH) the active form of the enzyme is pho~phorylated.~~~~~~-~~~ The loss of activity of the enzyme system in vitro in the presence of divalent metal ions has been attributed to activation of an endogenous microsomal phosphatase which inactivates the hydroxylase by catalysing its depho~phorylation.~~~ The regulation of the activity of the hydroxylase by ATP-Mg2+ is ~H-dependent.~~~ The next step in the biosynthesis of bile acids is the oxidation of 7a-hydroxycholestero1 to give the conjugated ketone (1 27a).The oxidoreductase [cholest-5-ene-3P,7a-diol 3P-dehydrogen-ase (E.C. 1.1.1.181)] that catalyses this reaction has been isolated from rabbit liver microsomes and purified; the enzyme utilizes NAD+ as cofa~tor.~~~ The major pathway to cholic acid (128a) in rat liver is initiated by hydroxylation of the enone (127a) to furnish the 7a 12a-dihydroxy-enone (1 27b) followed by the conversion (1 27b) -+ (1 29a) -+ (1 30a) -+ (1 30b) -+ (1 28a).Alternatively direct reduction of the enone (127a) leads to chenodeoxycholic acid (128b) by the route (129b) + (13Oc) + (130d) -+ (128b). The sequence of events in human liver is less certain. Precursors (1 26) (1 27a) (1 29c) and (1 29b) were each converted efficiently into both chenodeoxycholic acid and cholic acid while the trio1 (129d) and the tetraol (129a) furnished only the latter acid.279,2so The most selective precursor of (1 28b) was the diol (127c) which furnished the former acid and cholic acid in a ratio of 7 1.280 It was suggested that multiple pathways are involved in the biosynthesis of bile acids in human liver. The trihydroxycholestanoic acid (1 30a) is converted into cholic acid by rat liver peroxisomes.28 The intermediate tetrahydroxy- compound (I 30b) was isolated also. Studies have been reported on the influence of the stereochemistry at C-25 on the conversion of 3a,7a 12a- trihydroxy-5P-cholestan-26-oicacid (1 30a) into cholic acid in rat liver282 and in human liver.283 In both organisms the (25R)-cholestanoic acid and its (25S)-isomer were each converted efficiently (ca 90%) into cholic acid. Thus the (124) (123)a; R1= R2= OH,R3= H H b;R1= H,R2R3= 0 H (125) H0" (127) a; R1= R2= H H b; R'= OH,R2= H (128)a; R = OH (126) c;R'= H,R2=OH b;R=H NATURAL PRODUCT REPORTS. 1985 D. M. HARRISON ~ H H Y2 HO” HO” H H (129) a; R’ = R2 = OH b; R’ = H,R2 = OH (130) a ; R’ = OH R2= H C; R’= R2= H b; R’ = R2= OH d; R’ = OH,R2= H C; R’= RZ= H R d; R’= H,RZ=OH H H HO” (132) HO H (131)a; R = H b;R=OH HO HO (134) configuration at C-25 is not criti~al.~~~,~~~ The stereospecifi- city of purified mitochondria1 cholestanetriol26-mono-oxygen-ase (E.C.1.14.13.15) has been studied in a reconstituted system.284 This enzyme system oxidized the trio1 (129d) to a single tetraol (129a) which was identified as the (25R)-isomer by direct comparison with authentic material.284 The trihy- droxycholestanoic acid (130a) (isolated from human bile) was identified as the (25R)-isomer in a similar manner,285 in confirmation of an earlier less satisfactory experiment.286 The enzyme cholestanetetraol 26-dehydrogenase (E.C. 1.1.1.161) which catalyses the oxidation of (129a) to the C-26 aldehyde is a major isozyme of the alcohol dehydrogenase (E.C.1.1.1.1) of human liver.287 The hydroxylations at C-7 C-12 and C-26 that occur during the conversion of cholesterol into bile acids are catalysed by cytochrome-P-450-dependent mixed-function oxidase systems. Considerable progress has been made in separating purifying and studying the cytochrome P-450 proteins that are associated with each hydroxylation step. In particular studies have been reported on the isolation of the components and the reconstitu- tion of the activities of the 7a-hydroxylase system of human liver microsomes,288 the 7a-hydro~ylase~~~ and 26-hydroxylase systems290 of rat liver and the 7a- 12a- and 25-hydroxylase systems of rabbit liver.291 The activity of the 7cr-hydroxylase system is selectively stimulated by thi~ls,~~~ and also by a cytosolic protein that was isolated from rat liver.293 A cytosolic protein which stimulates and another that inhibits the 12a- hydroxylase system have been isolated from rabbit liver.294 The natural occurrence of 25-hydroxy-5~-cholestanesand their conversion into bile acids in enzyme preparations from liver suggested earlier the possibility of an alternative (25- hydroxy) route to bile acids.In parallel experiments 3a,7a 12a,26-tetrahydroxy-5~-cholestane (1 29a) was converted (133) H H efficiently (84%) into cholic acid by humans while the 3cr,7a 12a,25-tetrahydroxy-isomer (1 3 1 a) furnished cholic acid in only 15% yield.It was concluded that the 25-hydroxy pathway to cholic acid is of only minor significance.295 The pentahydroxy-5P-cholestane(1 31 b) which is an estab- lished intermediate in the 25-hydroxy route was converted into the 24-ketone (132) by a rat liver homogenate that was supplemented with NAD+.296It was suggested that the ketone is probably an intermediate in the conversion of (131b) into cholic acid. It is known that the hydroxylation at C-25 of the trihydroxy-5P-cholestane(1 29d) involves a cytochrome-P-450- dependent enzyme system; it has now been shown by using immunological techniques that cytochrome b5 is also a functional component of the hydroxylase system.297 25-Hydroxycholesterol is incorporated into bile acids in relatively poor yield (10-2073; thus preliminary hydroxylation of cholesterol at C-25 is precluded as a significant pathway.298 The rare inherited disease cerebrotendinous xanthomatosis is characterized by defective biosynthesis of bile acids and by the accumulation of cholesterol cholestanol and bile alcohols.Subjects that suffer from this disease lack the 26-hydroxylase that is necessary for the normal biosynthesis of cholic acid.299 Furthermore the capacity to hydroxylate 3a,7a 12~,25-tetra- hydroxy-5P-cholestane (1 3 la) at C-24 is impaired.300 Other relevant studies have been reported.301 6 Triterpenoids and Steroids in Higher Plants Algae and Fungi 6.1 The Biosynthesis of Triterpenoids Seedlings of Cucurnis satiuus converted the 2-3H-labelled triterpene (133) into cucurbitacin C (134) in 0.075% yield; neither parkeol (1 35) nor cycloartenol (1 36) was incorporated significantly into cucurbita~ins.~~~ The role of triterpenoid (133) as a precursor of the cucurbitacins was further substan- tiated by its formation in 1.3% radiochemical yield together with cycloartenol and other triterpenoids when [3-3H]squalene 2,3-epoxide was incubated with a microsomal enzyme prepara- tion from Cucurbita maxima.303 Direct formation of (1 33) from squalene 2,3-epoxide can be rationalized as one of several alternative modes of collapse of the putative enzyme-bound intermediate (137) as summarized in Scheme 15.The biosynthesis of steroids and triterpenoids in tissue cultures of C. maxima has been studied.304 The biosynthesis of pentacyclic triterpenes from mevalonate and squalene has been demonstrated in the soybean (Glycine max) and in alfalfa (Medicago sati~a).~~~ Isotopically labelled L-leucine and L-valine both furnished labelled squalene and p-amyrin when supplied to Pisurn sati~um.~~~ Degradation revealed a uniform distribution of radioactivity in the isopentenyl-diphosphate-derived and in the dimethylallyl-diphosphate-derived portions of the labelled squalene; it was concluded that the site of biosynthesis of triterpenoids differs from that for monoterpen~ids.~~~ The biosynthesis of glyco-(22a) + (25) H I4 (137) Scheme 15 (138) a;R = Et b;R=Me H (139) a ; R’ = HIR2= Me b; R’ = Me,R2= H c ; R’ = CH,OH ,R2= H d; R’ = CHO R2= H NATURAL PRODUCT REPORTS 1985 sides of oleanolic acid in Calendula oficinalis has been studied.307 Acetate and mevalonate can both be utilized in the biosynthesis of cycloartenol and lanosterol in the latex of Euphorbia lathyri~.~~~ 6.2 The Biosynthesis of Phytosterols The course of biosynthesis of sterols in cell cultures of bramble (Rubus fruticosus) is altered dramatically and specifically by exposure to several antifungal agents.309 31 The major steroids of untreated cultures were the As-sterols p-sitosterol (1 38a) (70%) campesterol (1 38b) (1 473 and isofucosterol (139a) (12%).After four weeks growth in the presence of the systemic fungicide tridemorph bramble cells no longer produced As-sterols owing to specific blocking of the key enzyme that isomerizes cycloeucalenol to furnish the A8-sterol obtusifoliol.As a result cyclo-steroids such as 24-methylene- cycloartanol (140) cycloeucalenol (1 41) and the pollinastanol derivatives (142a-c) constituted over 80% of the sterols that were present.309 In contrast if .the same bramble culture was adapted to growth in the presence of fenarimol it furnished unusually high yields of 14a-methyl-As-sterols such as obtusifo- liol (143a) its 4-demethyl analogue (143b) and the respective homologues (144a) and (144b).310 The natural azasterol A25822B (150) is known to block the nuclear reduction of steroidal 8,14-dienes in fungi (cf Section 6.3). Bramble cells responded to this agent in a similar manner and the sterols (145) and (146a-c) acc~mulated.~~ This observation provides powerful additional confirmation .for the postulated intermediacy of A8 14-stero1s in the biosynthesis of phytoster- 01s.Finally the drug AY-9944 blocked the isomerization of the sterol (147a) to 24-methylenelophenol (149) leading to the accumulation of As-sterols such as (147a) (147b) and the homologues (148a-~).~” Some of these inhibitors of the biosynthesis of steroids appear to act in a significantly different manner on fungi (see Section 6.3). These studies of the inhibition of some biosynthetic pathways are summarized in Scheme 16; broken arrows signify transformations that are not observed in the absence of the specific inhibitor. It has been suggested that the known sequence of nuclear demethylation in higher plants (see Scheme 16) reflects in part the steric inaccessibility of the 14a-methyl of cycloartenol.This steric crowding is relieved following the isomerization of cycloeucalenol (141) to obtusifoliol (143a).31 A microsomal enzyme system from maize embryos was able to isomerize 24,28-di hydrocycloeucalenol (I 51a) to 24,28-dihydro-obtusifo- liol; neither the 40-epimer (151 b) of the former steroid nor the homologue (151c) was a substrate for the i~omerase.~~~ The ketone (151d) was reported as a powerful inhibitor of the cycloeucalenol-obtusifoliol isomerase of maize seedlings. The sterol content of the Senita cactus (Lophocereus schottii) is unusual in that the plant lacks As-sterols and contains steroidal 8,14-dienes and 4a-methyl-A7-sterols as major com- ponents.It was suggested that the alkaloids of the Senita cactus might be active in inhibiting the normal biosynthesis of steroids,316 but this suggestion has now been withdrawn following the observation of normal steroid profiles for fungi that were growing on the cactus.317 Feeding studies with 4C-labelled acetate revealed that cell suspension cultures of carrot (Daucuscarota) were most active in the biosynthesis of phytosterols at the end of the growth phase.318 Removal of the auxin 2,4-D from the culture medium markedly reduced the rate of incorporation of 4C-labelled mevalonate into steroid^.^ * Mevalonate is incorporated into sterols by a cell-free extract of Dioscorea~orib~nda~~O and the biosynthesis of sterols from I4CO2in leaves of a mulberry has been studied.321 In the green alga Chlamydumonas reinhardtii the rate of biosynthesis of sterols was highest during the light period of a regular cycle of 12 hours of light and 12 hours of darkness.The biosynthesis of steroids in higher plant~~f~~~ (particu-larly in the S~lanaceae~~~) has been reviewed. NATURAL PRODUCT REPORTS 1985 -D. M. HARRISON ..& J. ,R (142)a R = H b. R = Me c. R = H.(24R)-24.28-dihydro H O W H OI H W IH (143)a.R = Me (144)a.R = Me b,R=H b,R=H (146)a.R = Me b.R=H c 9 z H (24R)-LL 18-olhyaro &+& G H H HO I A I (147)a.R Me HO I I AH b.R = H (148)a. R = Me b,R=H c.R = H,(ZLR)-24.28-dlhydro 'H (149) Inhibitors i tridemorph; ii tenarimol; iii A25822B; iv AY-9944 Scheme 16 6.3 The Biosynthesis of Sterols in Fungi A form of cytochrome P-450 is involved in the 14a-demethylation of lanosterol in a semi-anaerobically grown culture of the yeast Sacchuroniyces ceretisiur.Evidence has now been reported for the presence of this cytochrome P-450 in microsomes of aerobically grown yeast.In particular 14~-demethylation of lanosterol by yeast microsomes was found to I HO R1 R2 (151)~;R1= H,R2= Me,R3= H b;R1= Me,R2= H,R3= H2 C; R1=R2= Me,R3= H2 d; R1= H.R2=Me R3= 0 (152) HO H (153) require molecular oxygen and NADPH was sensitive to carbon monoxide and was inhibited by antibodies that had been raised to yeast cytochrome P-450.325 Several commercial fungicides owe their antifungal activity (at least in part) to interference with the biosynthesis of ergosterol (152) by blocking the 14a-demethylation of lano-or by blocking the reduction of the intermediate steroidal 8,14-diene.327 The fungicide buthiobate inhibited the 14a-demethylation of lanosterol in a reconstituted enzyme system from Succhuroniyces cerecisiue by binding to the cytochrome P-450 The lack of this specific binding behaviour was used to demonstrate that a mutant of the same yeast that was blocked at the same point in the biosynthesis of ergosterol produced a chemically altered cytochrome P-450.The biosynthesis of ergosterol in Ustilugo niujdis is inhibited by tridem~rph~~~ (150);331in or by the 15-aza-~-homo-steroid each case the 8.14-diene (153) accumulated.An ergosterol- deficient mutant of U. niuyciis produced 24-methylene-24,25- dihydrolanosterol (154) obtusifoliol (1434 and 14a-methyl- fecosterol (143b) as the major steroids.33' These same steroids accumulated at the expense of ergosterol when the wild-type NATURAL PRODUCT REPORTS 1985 H HI OH (1 54) (155) H (157) fungus was treated with the inhibitors fenarimol etaconazole or miconazole. Oxidative removal of the 4-methyl groups of 4,4-dimethyl- zymosterol (70a) in yeast involves a cyanide-sensitive enzyme system. It has been suggested that the cyanide-sensitive component is the terminal oxidase that acts directly on the substrate (70a).It has also been shown that cytochrome h is an essential component of the 4-demethylating system in yeast microsomal preparations.333 Studies with a yeast mutant have confirmed that 4P-methyl-steroids cannot be demeth~lated.~~" An acetone powder extract of Succharomyces cereuisiue has been described which equilibrates zymosterol(70b) with its A7-isomer. Either sterol can act as a substrate in the transmethyla- tion reaction (involving S-adenosylme t hion ine) to furnish fecosterol (147b) or episterol (1 55) respectively. Fecosterol is subsequently isomerized to epister01.~~~ This isomerization is blocked in S. cerevisiue by the drug AY-9944; the reduction in the amounts of esters of ergosterol which results from applying this drug to a culture is matched by a dramatic increase in the formation of esters of feco~terol.~~~ The final steps in the biosynthesis of ergosterol (152) i.e.reduction of the double-bond of episterol (155) and formation of the As and A2? double-bonds occur in several competing sequences in a cell-free enzyme system from Succharomyces cere~isiae.~~' Introduction of the As double-bond requires molecular oxygen-337 and NADPH (or NADH) and so is catalysed by a mixed-function oxida~e.~~* The oxidase is inhibited by cyanide ions but not by carbon monoxide and resembles the fatty-acyl-CoA desaturase It is generally considered that the mechanism of action of the sterol 5,6-desaturase does not involve the intermediate formation of 5-or 6-hydroxy-compounds. Neverthless a mutant of this yeast whose biosynthesis of sterols was blocked at the step of As -+A7 isomerization accumulated significant quantities of the 3P,6a- diol (156a).339 The 14a-methyl homologue (156b) of the latter diol was isolated together with other 14a-methyl-steroids (cf ref.332) when the biosynthesis of ergosterol in Ustilugu maydis was blocked by the fungicide etac~nazole.~"~~ It is known that introduction of a As double-bond into a A8-sterol can occur in Succhuromyces cereuisiue. 34 I The biosynthesis of ergosterol from 22,23-dihydroergosterol has been studied using microsomes from Saccharomyces cerevisiue. The 22,23-desaturase system requires NADPH and molecular oxygen and is inhibited by carbon monoxide but not by cyanide. These and other data suggest the involvement of a mono-oxygenase system that contains a cytochrome P-450.34' Studies with mutants of this yeast and the non-inhibition of the desaturase by buthiobate suggested that the cytochrome P-450 that is involved is structurally distinct from that which is involved in the 14a-demethylation of lano~terol.~"~ All steps in (15610; R = H b; R = Me the biosynthesis of ergosterol from zymosterol are mediated by enzymes that are located in the mic~osomes.~~~ The biosynthesis of sterols de now by Succharomyces cerevisiue is prevented by strictly anaerobic growth conditions especially in the presence of the lanosterol synthase inhibitor 2,3-dihydro-2,3-iminosqualene.Under these conditions the organism retained the ability to perform the transmethylation reaction on exogenously supplied ~I~~-steroIs and to reduce the A24(28)double-bond of 24-methylene-ster01s.~~~ Thus both desmosterol(157) and 24-methylenecholesterol(l58)furnished 22,23-dihydrobrassicasterol(1 59) but reduction of the A24(28) double-bond of fucosterol (139b) did not occur.22,23-Dihydrobrassicasterol but not 24a-methyl- or 24-ethyl-sterols satisfied the sterol requirement of the organism.346 Other studies have been reported on the sterol requirement of anaerobically grown S. cere~isiue~~~ and of sterol auxotrophic mutants of the same yeast.3sX A detailed study has been reported on the time-course of incorporation of [2-' 5C]mevalonate into the sterols and sterol esters of Neuruspora crass^.^^^ The first step in the biosynthesis of ergosterol in this organism is the conversion of lanosterol into 24-methylene-24,25-dihydrolanosterol(154); subsequent steps involve parallel transformations on the latter and on 24P-methyl-lanosterol.The 14C-labelled intermediate (1 47a) has been isolated from N. cru~su.~~~ It is generally accepted that cycloartenol is the starting triterpene for the biosynthesis of sterols in photosynthetic plants and algae while lanosterol performs the equivalent role in animals and in the few fungi that have been investigated hitherto. Cold-trap experiments with the Basidiomycete Uro-myces phuseoli have now established that lanosterol (and not cycloartenol) is formed from I4C-labelled squalene. Further- more only the former triterpenoid was metabolized in a cell- free enzyme extract which is in agreement with the prevailing view.3s1 Cultures of Saprolegniu jt.ru.u that had been treated with tridemorph furnished A8-sterols which included zymosterol (70b) fecosterol (147b) and stigmasta-8,24(28)-dien-3P-01.~~' Cultures that were grown in the presence of triarimol furnished only lanosterol.These results suggest that lanosterol is the starting point for the biosynthesis of sterols in this species also. Selected species of the order Peronosporales that are unable to epoxidize squalene and which therefore do not synthesize sterols were able to metabolize exogenously supplied cycloar- tenol to lanosterol. Some organisms also synthesized fucosterol ergosterol and cholesterol from exogenously supplied cycloar- tenol and so retain vestigial abilities to synthesize sterols.3s3 The sterol compositions of Huliphthoros miIfOrdensis and Atkinsiellu duhiu which are fungal parasites of marine NATURAL PRODUCT REPORTS.1985 D. M. HARRISON ~ ~ I I (b)Iii * / H /Me Me Me Reagents i S-adenosylmethionine (SAM); ii SAM Enz-X-Scheme 17 crustaceans have been investigated.354 A sterol auxotroph of the former species was capable of metabolizing cholesterol to fucosterol; a strain that did not require lipids produced both fucosterol (1 39b) and 24-methylenecholesterol (1 58).354 The biosynthesis of cholesterol from lanosterol in Botrytis cinerea has been demonstrated.355 6.4 Alkylation of the Sterol Side-chain It was reported (in a preliminary communication) that the 25(pro-24E)-methyl of labelled lanosterol (23a) becomes the 25(pro-25R)-methyl of isofucosterol (139a) during the biosyn- thesis of the latter in seedlings of Pinuspinea.' A full account of this work has been published.356 It is known that the 24-proton of the precursor is retained at C-25 of isofucosterol.The present results are consistent with the two extreme mechanistic descriptions that are summarized as routes (a) and (b) in Scheme 17.356 The labelling patterns of the phytosterols isofucosterol (139a) 24-methylenecholesterol (1 58) 0-sitosterol (1 38a) stig- masterol (1 60) clionasterol (161) and a-spinasterol (1 62) were recorded by 3C n.m.r. following the feeding of [I 3C2]acetate to cell cultures of several plant species.In each case it was deduced from the observed labelling pattern (Table 1) that the 25(pro-25R)-methyl of the phytosterol was derived predomi- nantly from C-2 of meval~nate.~~~ This is remarkable in view of the different biosynthetic routes that have been proposed for the 24a- and 24P-alkyl-sterols (see below). Furthermore the opposite result was reported for the 25-methyls of ergosterol (152) that had been biosynthesized from [2-' 3C]mevalonate by the mould Claviceps paspali.' In the present the labelling pattern for the 25-methyls of isofucosterol in Physalis A (163) H (162) (164) (1 66) (165) deuterium atoms (as expected) while the cyclolaudenol (1 66a) retained three deuterium atoms in confirmation of earlier studies.In each case the deuterium label was shown (by mass spectroscopy) to be present in the side-chain presumably at C- 28.361A plausible interpretation of these results is summarized in Scheme 18. It remains to be seen whether more than one transmethylase is involved in the biosynthesis of these three steroids. peruoiana was assumed on the basis of the result that was reported earlier for the same sterol in Pinus ~inea;~~~ the resonances for the diastereotopic 25-methyls of clionasterol and 0-sitosterol were assigned following the recording of a 13C n.m.r. spectrum from the diastereoisomeric mixture that resulted from catalytic deuteriation of the biosynthetically 3C-labelled isofucosterol.357 Carbon-1 3 n.m.r.assignments have been reported for several phyto~terols.~~~ 24-Meth~l-A*~-steroids have gained prominence recently as constituents of maize (Zea mays).359.360 In principle the steroid cyclosadol (165) could arise from cycloartenol (1 36) directly by a transmethylation reaction involving S-adenosyl- methionine (SAM) or indirectly by isomerization of 24-methylenecycloartanol (140). The results that were obtained from time-course studies on the biosynthesis of cyclosadol and of 24-methylenecycloartanol from [Me-'4C]SAM were consis- tent with the former suggestion.360 Compelling evidence for the direct formation of cyclosadol was gained from studies with an enzyme extract from maize shoots.361 It was shown that the [2H3]cyclosadol (165a) was formed when [Me-2H3]SAM and cycloartenol were supplied to the enzyme system; the major metabolite i.e.24-methylenecycloartanol (140a) retained two The major sterols of maize namely p-sitosterol (138a) and stigmasterol (160) belong to the 24a series. In contrast 24- methylcholesterol occurs as a mixture of 24~- and 24P-epimers [(159) and (138b) respectively]. It has been suggested that the biosynthesis of the 24a-compound (campesterol) involves isomerization of 24-methylenecholesterol (1 58) to the A24(25)-isomer followed by stereospecific reduction of the latter [route (a)in Scheme 191. Two possibilities have been discussed for the biosynthesis of 24P-methyl-sterols namely reduction of a 240- meth~l-A~~-steroid [route (b)] or stereospecific reduction of a 24-methyl-A23-steroid [route (c)].~~O The 24-proton of cycloar- tenol which originates from the 4(pro-4R)-proton of mevalon- ate would be lost in route (a)but retained in routes (6) and (c) of Scheme 19.Circumstantial evidence for the biosynthesis of phytosterols oia route (a)and either route (6) or route (c) was obtained by supplying (4R)-[2-l 4C,4-3H Imevalonate to maize shoots which furnished 24-methylcholesterol consisting of -25% of the 24a-compound (138b) and -75% of the 24P- epimer (159).362 The 3H:14C atomic ratio of the epimeric mixture was 2.82 :5 which is in agreement with the predicted ratios of 2 :5 for the 24a-compound 3 :5 for the 24P-compound and ca 2.75 :5 for the epimeric mixture that was examined.362 548 NATURAL PRODUCT REPORTS.1985 Table 1 The incorporation of [I3C,]acetate into phytosterols in cell cultures of higher plants (ref. 357). Sterol Labelling pattern of side-chain Species / lsofucosterol (1 39a) Physalis peruciuna yJfH 24-Methylenecholesterol (1 58) P. peruviana P-Sitosterol (I 38a) / P. peruciana Dioscorea tokoro isodon japonicus Stigmasterol (1 60) P. peruuiunu D. tokoro Bup/eurumJa/cutum Clionasterol ( 161 ) B. jalcatum a-Spinasterol (1 62) B. jalcatum * * H CD (166a) [ -H+CC-23)I H* (1650) Scheme 18 Bryonia dioica (Cucurbitaceae) is unusual in that both 24a- The biosynthesis of poriferasterol(l67a) has been studied in and 24P-phytosterols are found in seeds and seedlings while two species of alga.In a species of the genus Trebouxia the (168) was incorporated efficiently (ca 25%) into only 24a-sterols are found in the mature plant. Time-course As~zz~z5-sterol studies have been reported on the incorporation of [2-poriferasterol both in uiuo and in a cell-free h~mogenate.~~~ On *C]acetate into phytosterols in this plant.363 Tritium-labelled the basis of this and earlier work it was suggested that the cyclolaudenol(l66) served as a precursor for both a-spinasterol biosynthesis of poriferasterol in this organism follows the route (162) and the 240-sterols (163) and (164) in seedlings of (169) -+ (168) -+ (167a). By contrast in the chrysophyte alga Cucurbita maxima.364 These observations are surprising since a Ochromonas rralhamensis the biosynthesis of poriferasterol 24-methylene-sterol is an obligatory intermediate in the involves reduction of a 24-ethylidene-steroid such as (1 39a) or formation of 24-ethyl-sterols.(1 39b). The stereochemistry of this reduction step has been NATURAL PRODUCT REPORTS 1985 -D. M. HARRISON 549 H* &H* AH* vdv I Scheme 19 / (167) a; R1= R2= H b; R'= 0 R*= H C; R' = H R2= D (169) d; R' = R2= R2 I I (170)a; R1=H,R2= D (171) b; R1= D,R2= H determined as follows.366 A sample of poriferasterol was isolated following a feeding experiment with [Me-'H3]meth- ionine and shown (by mass spectrometry) to contain solely the 'H0- 'H - 'H3- and 'H,-labelled species (167a-d). The labelled poriferasterol was shown to have exclusively the (28s) configuration by comparison of the 'H n.m.r.spectrum of its 22,23-dihydro-derivativewith the 'H n.m.r. spectra of the model ['H,]sterols (170a) and (170b) for which the configura- tions are known. The highly stereospecific labelling of poriferasteroi in this experiment suggests that a 24-ethylidene- steroid of defined configuration is the natural precursor.366 It has been confirmed that 0. malhamensis is capable of introducing a methyl or ethyl group at C-24 of cholesterol to furnish brassicasterol (1 71) and poriferasterol respectively.367 The biosynthesis of the fascinating steroid dinosterol (1 72a) in the marine dinoflagellate Crypthecodinium cohnii has been Feeding experiments with [Me-'H,]rnethionine furnished deuteriated dinosterol that was shown by mass spectrometry to contain the 'H2- ?H3- and 'H,-labelled species (1 72b-d).A new sterol which was tentatively identified as a 4cr,24-dimethylcholestanol (I 73a) incorporated two deuterium atoms to produce (173b) in the same feeding ~HRZ, ;.A (173)a; R = H I HO Ik b;R=D (172) a;R' = R2= H (174) a; R = H b;R = Me H Ye experiment.368 Several plausible transmethylation sequences are consistent with these data. 25-Azacycloartanol (1 74a) at low concentrations (0.5-1 .O pmol dmV3) strongly inhibits the cycloartenol 24-methyl- transferase of maize in and of bramble cell cul- ture~.~~"*~~~ In the latter case steroids with C side-chains i.e. cycloartenol (1 36) desmosterol (1 57) and cholesterol (2b) which constituted less than 1% of the sterol content in control cultures attained 53% of the sterol content in treated cells.370 Most probably the ammonium cation (175) is the active inhibitor which functions by mimicking the normal carbo- cation intermediate (1 76) in the transmethylation reaction NATURAL PRODUCT REPORTS 1985 between S-adenosylmethionine and cycloartenol.In agreement with this interpretation the 24-methyl-25-aza-derivative (1 74b) was yet more efficient as an inhibitor of the transmethy- lation reaction in a microsomal enzyme preparation from maize and in a culture of bramble cells.371 The extensive use of mutants and of aza-sterols in the investigation of the biosynthesis of ergosterol in yeasts has been reviewed briefly.372 A study has been reported on the inhibition by aza-sterols of the transmethylase reaction in sterol mutants of the yeast Saccharomyces cerevi~iae.~’~ One signifi- cant result was the accumulation of cholesta-5,7,24-trien-3P-ol CH3-CO2 H CD3 C02H (177) to the extent of 28% when the erg 5 mutant (which is blocked at the step of 22,23-desaturation) was treated with 25- azacholesterol (1 78) or with 25-azacholestanol at a concen-tration of 1 pmol dm-3.The major sterol (62%) in both treated cultures was zymosterol (70b).373 Several structural analogues of S-adenosylhomocysteine have been shown to function as inhibitors of the transmethylation reaction of sterol biosynthe- sis in yeast.374 The detergent Triton X-100 inhibits the Az4-sterol methyltransferase (E.C.2.1. I .41) of a cell-free enzyme system of Saccharomyces cerevisiae. The methyltransferase from uredospores of Uromyces phaseoli has been solubilized and purified 155-f0ld.~,~ The best sterol substrate was zymosterol; however the product i.e. 24-methylene-24,25-dihydrozymosterol (1 47b) undergoes a sec- ond transmethylation reaction with S-adenosylmethionine albeit at a much lower rate. The two transmethylase activities increased at the same rate during the purification of the enzyme which suggests that a single enzyme or enzyme complex may be involved. 6.5 Further Metabolism of Steroids [U-3H]Lanosterol (23a) furnished viridin (1 79) in 21% radio-chemical yield when supplied to the mould Trichoderma viride.Several more-highly oxidized precursors showed significantly lower incorporations into ~iridin.~,’ The biosyntheses of the closely related metabolites wortmannin (1 80) and demethoxy- viridin (181) have been studied in the fungi Penicillium wortmannii3 and Nodulisporium hinnuleum 379 respecti vely . Both metabolites showed labelling patterns from [I 3C,]acetate that were consistent with their derivation oia the degradation of lanosterol or of a lanosterol-like triterpene (Scheme 20). In each case the labelling pattern also revealed that the carbon substituent at C-4 is derived from the methyl of mevalonate in contrast to the case of the mould metabolite fusidic These conclusions were confirmed in respect of demethoxy- viridin by further feeding experiments with labelled mevalonates.374 Considerable interest attaches to the nature of the immediate steroid precursor of these metabolites and to the mechanism of removal of the steroid side-chain.In this connection it is significant that (3S)-3,4-dimethylpentanol and related unsatu- 0 (180) Scheme 20 OH I moOH ROUL (182)a;R = H b; R = Me2CHC0 c; R = EtCO D (181) OH I (183)a; R = H b; R = MeZCHCO C; R = EtCO ambisexualis] is unique in that a group of steroidal hormones the oogoniols play a key role in sexual reproduction. It has been shown that oogoniol (1 82a) oogoniol-l (1 82b) and oogoniol-2 (1 82c) each co-occur with their probable precursors [the 24,28-didehydro-derivatives(1 83a) (1 83b) and (1 83c) respectively] and that these dehydro-compounds (rather than the oogoniols) may be the true hormones.381 It was shown also that the oogoniols possess the (24R) stereochemi~try.~~~.~~~ Fucosterol (139b) which is the putative precursor of the oogoniols was incorporated into oogoniols in A.heterosexualis albeit in low yields.383 In contrast 29-hydroxyfucosterol(l39c) and the derived aldehyde (139d) were both incorporated efficiently into oogoniol its dehydro-derivative (I 83a) and into other oogoniols. Cold-trap experiments established that 29-hydroxyfucosterol is an intermediate in the incorporation of both [3-3H]fucosterol and the [23,23,25-3H3]-aldehyde (1 39d) into oogoniols; this is in keeping with the suggestion that the alcohol and the aldehyde are reversibly interconverted in these rated compounds have been isolated from N.hinn~leum.~~~ species of the genus A~hlya.~*~ Moreover when [2-14C]mevalonate was supplied to the fungus demethoxyviridin and the C7 alcohols had relative molar activities of approximately 3 :2. This ratio is consistent with a common origin of demethoxyviridin and the C alcohols via cleavage at the 20-22 bond of a 4,24-dimethyl-~teroid.~~~ The water mould Achlya [as its hermaphroditic form A. heterosexualis or as its male (E87) or female (734) strain of A. [ 3C2]Acetate was incorporated into the steroidal sapogenins diosgenin (184) yamogenin (I 85) tokorogenin (1 86) and neotokorogenin (1 87) by tissue cultures of Dioscorea tokoro with the labelling patterns shown.384 These results are consistent with the biosynthetic route that is summarized in Scheme 21 in which an early step is reduction of cycloartenol (or another AZ4-steroid precursor) at the 25si face.384 NATURAL PRODUCT REPORTS 1985 -D.M. HARRISON 55 1 HO 7c\ Hu--I -(1 36) Scheme 21 [4-l 4C]Progesterone (92a) was incorporated into cardenolide glycosides by leaves but not by rhizomes of Convulluriu majali~.~~~ [1-l 3C]Acetate was incorporated significantly only into non-squalene-derived portions of cardenolides in the milkweed Asclepias curassavica. 386 The biosynthesis of ecdysteroids in higher plants is dis- cussed for convenience in Section 7.1. The biosynthesis of steroidal alkaloids has been reviewed comprehensi~ely~~~ and will not be treated in this Report.7 Triterpenoids and Steroids in Invertebrates 7.1. Insects It is known that many insects are incapable of biosynthesis of steroids de novo. In the case of some phytophagous insects the cholesterol that is required for cell membrane functions and for the synthesis of hormones is acquired by the dealkylation (at C- 24) of dietary phytosterols. The degradative route that is utilized is illustrated in Scheme 22 for the conversion of p-sitosterol (1 38a) into cholesterol (2b). The intermediates isofucosterol (139a) or fucosterol (139b) could be formed (in principle) by hydroxylation of p-sitosterol at C-24 or at C-28 followed by dehydration. These possibilities are precluded by the following observations.A mixture of [4-l 4C]-P-sitosterol and the diastereoisomeric [28-3H]-diols (189) was fed to larvae of Tenebrio molitor in the presence of both unlabelled fucosterol and isofuc~sterol.~~~ The 3H :14C ratios in the recovered fucosterol and isofucosterol revealed that p-sitosterol is ten times more efficient than the diol mixture (189) as a precursor of these compounds. The (24RS)-24-hydroxy-steroid(190) was incorporated as efficiently as p-sitosterol into both fucosterol and isofucosterol but could not be detected by cold-trap experiments when [4-l 4C]-P-sitosterol alone was fed to the larvae. It was concluded that the conversion of 0-sitosterol into fucosterol and isofucosterol involves a dehydrogenation reac- tion rather than a hydroxylation-dehydrationsequence.388 The diastereoisomeric epoxides (1 88) of fucosterol were utilized to equal extents by T.molitor as sources of cholesterol.389 However the (24R728S)-24,28-epoxide of isofucosterol was metabolized to cholesterol by the same species some twelve times more efficiently than the (24S,28R) diastereoisomer and may be the natural precursor.389 The assignments of configura- tion to the two 24,28-epoxides of fucosterol have been revised.390 [23,23,25-3H3]-24-Methylenecholesterol(l58) and the diaster- eoisomeric mixture of epoxides (191) both served as efficient precursors of cholesterol in larvae of T. m~litor.~~~ Thus c28 phytosterols also can satisfy the sterol requirement of this insect.The cholesterol that was formed following the feeding of [23,23,25-3H3]-24-methylenecholesterol was degraded chemi- cally to locate the tritium label; the result established that the 25-hydrogen of the precursor (1 58) had migrated to C-24 in the product cholesterol (Scheme 23a) as had been observed previously for the metabolism of p-sito~terol.~~~ Similar results were obtained for the larvae of the silkworm (Bombyx mori); thus [23,23,25-2 H J-24-methylenec holesterol NATURAL PRODUCT REPORTS 1985 (138a) (139a) (139b) \J f--& (157) Scheme 22 (190) (158) was metabolized to [23,23,24-2H3]desmosterol(157) and [23,23,24-2H 3]c holesterol. 393 Furthermore the same organism was able to demethylate the diastereoisomeric [23,23,25-*H3]- 24-methylcholesterols to furnish [23,23,24-2H3]cholesterol,pre-sumably as shown in Scheme 23b.Location of the label in this study was performed by mass spectrometry. Silkworm larvae that were reared on mulberry leaves contained both isofucosterol (1 39a) and fucosterol (139b) neither of which was present in the diet.394 However while the two diastereoisomeric 24,28-epoxides of fucosterol were iso- lated from the silkworm larvae the corresponding epoxides of isofucosterol were absent.395 Moreover it was shown that the latter two epoxides do not support the growth and development of silkworm larvae while the fucosterol epoxides were both metabolized to desmosterol and cholesterol by a cell- free enzyme system.396 Silkworms that have been reared in the absence of dietary phytosterols require cholesterol or appropri- ate alternatives for their normal growth and development; the permissible variations in structure of the ~ide-chain~~~ and of the nucleus398 of sterol supplements have been described.It has been shown that suitable 29-fluoro-phytosterols can function as insect-activated selective poisons owing to the degradation of the side-chain to furnish (presumably) fluoro- H (158) I H* H* (157) (2b) Throughout a ; H*=3H b; H*=*H Scheme 23 honeybee Apis mellifera,40 and Dysdercus Jasciatus4°2 all lack the ability to convert phytosterols into cholesterol. Several studies that are relevant to the mechanism of formation of the cis A/B ring junction of the ecdysteroids have acetaldehyde and thence fluoroacetate and flu~rocitrate.~~~ The earlier claim that the [~CX-~H]-A~,~- been rep~rted.~O~-~O~ The epimeric As* 2-fluoro-sterols (1 92) displayed significantly greater toxicity toward larvae of the tobacco hornworm (Manduca sexta) than their 22,23-dihydro-derivatives;it was suggested that this difference was due to the anticipated greater ease of dehydrogenation of the former epimers to furnish the fluoro-triene (193) and thence fluoroacetaldehyde and cholesta- 5,22,24-trien-3P-ol (194) (Scheme 24).The ability to dealkylate dietary phytosterols is not ubiqui- tous among phytophagous insects. In this regard it was reported that the beetle Trogoderma gran~rium,~OO larvae of the steroid (195) was incorporated into 20-hydroxyecdysone (196a) in Calliphoru stygia has been The apparent incorporation of tritium into (196a) may be due to the enzymic oxidation of the putative precursor to the 3-oxo-derivative with compartmentalized transfer of tritium to a natural precursor.Moreover the incorporation (0.004%) of the [la,2a-3H,]-4,7-dienone (1 97) into 20-hydroxyecdysone in the same species was cowidered to be insignificant in comparison with the incorporation (0.07%) that was observed for [la,2cx-3H2]cholestero1.403 Thus it is unlikely that A4-steroids are NATURAL PRODUCT REPORTS 1985 -D. M. HARRISON CH CH F CHCHzF 'FC H 2CHd s2 L (193) Scheme 24 (195) OH RI *OH 0 (196) a; R = OH b;R= H (1 97) (198) a; R = H b;R=OH involved in the biosynthesis of ecdysteroids though the probable existence of preferred sequences of steps and possible differences between species render a general conclusion unsafe (see below).The [3a-3H]-5a-enones (198a) and (198b) were each metabo- lized to their 25-hydroxy- and 22,25-dihydroxy-derivativesby the prothoracic glands of Manduca sextu.J04 In neither case was hydroxylation at C-2 observed nor were SP-metabolites formed. A cis A/B ring junction may be a prerequisite for 2p- hydroxylation. I "h (196a) f--fl Hi OH Scheme 25 Samples of [4-14C]cholesterol in which the 3a- 4a-,or 4p- protons were labelled with tritium were each incorporated into 20-hydroxyecdysone in the fern Polypodium uulgare without change in the 3H:14C ratio.405 Partial degradation of the labelled samples of 20-hydroxyecdysone revealed that the 5p-proton originated (259%) as the 4P-proton of the precursor and that neither the 3a- nor the 5p-proton of the product was derived from either the 3a- or the 4a-proton of cholesterol.These results are consistent with the biosynthesis of 20- hydroxyecdysone in this fern occurring uia the 5a,6a-epoxide of cholesterol or of 7,8-didehydrocholesterol,as summarized in Scheme 25.40s In similar feeding experiments with mature females of the locust Schistocercu gregaria the 4a-proton of cholesterol was retained completely in samples of ecdysone (196b) and of 2-deoxyecdysone that were isolated from eggs or ovaries while the 3a- and 4P-protons of the precursor were completely lost in the These results are consistent with the intermediacy of a A4-3-oxo-steroid in the biosynthesis of ecdysteroids in the locust (but cj ref.403). It is unfortunate that these important studie~~~~."~~ utilized degradation proce- dures that were not completely satisfactory for the unequivocal location of tritium label in the metabolites that were being investigated. It has been demonstrated convincingly that hydroxylation at C-22 of cholesterol occurs with retention of configuration during the biosynthesis of ecdysone in S. gregaria .jo7 Tritium-labelled ecdysone (196b) is metabolized to 20-hydroxyecdysone (196a) to further hydroxylated ecdysones and to highly polar products in Surcophaga bullata,s08 Calliphora ui~ina,"~~ and Tenebrio molitor.41 O The metabolic products in the cabbage white butterfly (Pieris brassicae) include carboxylic acids that have been derived from oxidation of ecdysone and of 20-hydroxyecdysone at C-26."' Other studies have been reported on the accumulation of 2-deoxy- ecdysteroids in the ovaries of the silkworm (Bombyx rnori)"I2 and on the control of the biosynthesis of 20-hydroxyecdysone in tissue cultures of the plant Trianthema portulacastrum.4' The biosynthesis of ecdysteroids'Y."'"."' and the metabo- lism of sterols in insects4' have been reviewed.7.2 Other Invertebrates Marine invertebrates continue to provide novel and biosynthe- tically intriguing steroid^.^' Recent isolations include the 4,27- dinor-steroid (199) from the sponge Teichaxinella morchell~,~ the cyclopropanes 23H-isocalysterol (200) and 23,24-dihydro- calysterol(201) from the sponge Calyx nice~ensis,~~ mutasterol (202) and pulchrasterol (203) from the deep-sea sponges Xestospongia mum4 and Aciculites pul~hra,~’~ respectively and papakusterol (204) from an unidentified gorgonian of the genus Acantkagorgi~.~~ It was suggested that the side-chain of 27-nor-sterols [e.g.(199)] might derive biosynthetically from papa kusterol .4 * A recurring question concerns whether sterols of marine invertebrates are biosynthesized entirely by the animal or whether dietary sources or their biosynthesis by symbiotic algae play a major or a minor role. Zooxanthellae that were isolated from several gorgonians contained 4a-me thyl-sterols which did not possess the highly alkylated side-chains that are characteristic of the sterols of their hosts.42* On the other hand zooxanthellae that were isolated from the sea anemone Aiptasiu pulchella synthesized both gorgosterol (205) and 23-demethyl- gorgosterol (206) in the absence of the Furthermore the biosynthesis of dinosterol (172a) by the dinoflagellate Crypthecodiniurn cohnii may be relevant to the occurrence of gorgosterol in host invertebrate^.^^^ Marine phytoplankton provides potential dietary sources of steroids; in this connec- tion mevalonate was incorporated into cholesterol and its A22-24-methyl derivative by the phytoplankton Pseudoisochrysis purado~a.~~~ The sponge Axinella polypoides contains a unique series of 19-nor-steroids that are typified by compounds (207a-c) and (208a-c).[4-14C]Cholesterol but not 5a-cholestan-30-01 was incorporated into the 19-nor-sterols ; double-labelling experi- ments established that the 40-proton and the 7-protons of cholesterol are retained in the biosynthesis of these compounds from cholesterol.425 However the 3a-proton of cholesterol is partially lost in this process. Thus either there is no 3-0x0- intermediate in the loss of C-19 or tritium is partially re-introduced after oxidation at C-3 via a compartmentalized The biosynthesis of the 30-hydroxymethyl-~-nor-steroid (209) by the sponge Axinella uerrucosu is known to involve loss of the 3a- and 4P-protons of cholesterol with extrusion of C-3.It has now been demonstrated that cholest-4- en-3-one (210) is an intermediate in the biosynthetic [ 1,2-3Hz]Cholesterol was metabolized to pregnenolone (85a) and progesterone (92a) by ovaries of the starfish Asterias rub en^.^^' Progesterone428 and androst-4-ene-3,17-dione (95a)429 were metabolized to other steroidal hormones in the same species. The sterol composition of wild oysters (Crassostrea virginica) differed from that of their dietary algae. The results were consistent with the conversion of dietary phytosterols into cholesterol or with the selective concentration of dietary cholesterol or with the biosynthesis of cholesterol de ~OOO.~~O Both acetate and mevalonate were incorporated into choles- terol 24-methylenecholesterol (1 58) and fucosterol (1 39b) in the same species in support of the latter e~planation.~~’ It was shown also that the freshwater mussel Anodonta cygnea was capable of the biosynthesis of 24-alkyl-sterols from cholesterol and from methi~nine.~~’ NATURAL PRODUCT REPORTS 1985 (201) (202) (203) H (205) ‘.R H b;R = Me (207)a;R = H c;R = Et b; R = Me c;R = Et HO (199) (210) NATURAL PRODUCT REPORTS 1985 -D. M. HARRISON 555 The biosynthesis and metabolism of marine sterols has been H. Mabuchi R. Takeda and H. Okamoto Biochem. Biophys. Res. re vie wed. 43 Commun. 1982 108 240. All photosynthetic plants that have been examined are 26 M. Sinensky and J. Logel J. Biol. Chem. 1983 258 8547.capable of the biosynthesis of steroids. It has now been shown 27 P. A. Edwards S. F. Lan and A. M. Fogelman J. Biol. Chem. 1983 258 10 219. that the photosynthetic bacteria Rhodopseudomonas sphaeroides 28 T. Kita M. S. Brown and J. L. Goldstein J.Clin. hest. 1980,66, and Chromutium vinosum do not biosynthesize Thus 1094. there is no fundamental link between photosynthetic activity 29 J. Ryan E. C. Hardeman A. Endo and R. D. Simoni J. Biul. and the capacity to synthesize sterols. It was further shown that Chem. 1981 256 6762; D. J. Chin K. L. Luskey R. G. W. the bacterium Escherichiu coli does not biosynthesize sterols Anderson J. R. Faust J. L. Goldstein and M. S. Brown Proc. when grown under aerobic conditions contrary to an earlier Natl. Acad. Sci. USA 1982 79 1185.30 J. R. Faust K. L. Luskey D. J. Chin J. L. Goldstein and M. S. Brown Proc. Natl. Acad. Sci. USA 1982 79 5205. 31 B. Middleton A. Middleton A. Miciak D. A. White and D. Bell, 8 References Biochem. 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Bohlin U. Sjostrand C. Djerassi and B. W. Sullivan J. Chem. SOC. Perkin Trans. I 1981 1023. 418 L. N. Li H. Li R. W. Lang T. Itoh D. Sica and C. Djerassi J. Am. Chem. SOC.,1982 104 6726. 419 L. N. Li U. Sjostrand and C. Djerassi J. Am. Chem. Soc. 1981 103 115. 420 B. V. Crist X. Li P. R. Bergquist and C. Djerassi J. Org. Chem. 1983 48 4472. 421 C. Bonini R. B. Kinnel M. Li P. J. Scheuer and C. Djerassi Tetrahedron Lett. 1983 24 277. 422 W. C. M. C. Kokke W. Fenical L. Bohlin and C. Djerassi Comp. Biochem. Physiol. B 1981 68 281. 423 N. W. Withers W. C. M. C. Kokke W. Fenical and C. Djerassi Proc. Natl. Acad. Sci. USA 1982 79 3764.424 D. S. Lin A. M. Ilias W. E. Connor R. S. Caldwell H. T. Cory and G. D. Daves Lipids 1982 17 818. 425 L. Minale D. Persico and G. Sodano Experientia 1979,35 296. 426 A. de Stefan0 and G. Sodano Experientia 1980 36 630. 427 H. J. N. Schoenmakers Comp. Biochem. Physiol. B 1979,63,179. 428 H. J. N. Schoenmakers and P. A. Voogt Gen. Comp. Endocrinol. 1980 41 408. 429 H. J. N. Schoenmakers and P. A. Voogt Gen. Comp. Endocrinol. 1981 45 242. 430 C. J. Berenberg and G. W. Patterson Lipids 1981 16 276. 431 S. Teshima and G. W. Patterson Lipids 1981 16 234. 432 S. Popov I. Stoilov N. Marekov G. Kovachev and S. Andreev Lipids 1981 16 663. 433 L. J. Goad Pure Appl. Chem. 1981 53 837. 434 W. R. Nes J. H. Adler C. Frasinel W. D. Nes M. Young and J.M. Joseph Phytochemistry 1980 19 1439.

ISSN:0265-0568    

DOI:10.1039/NP9850200525

出版商:RSC    

年代:1985

数据来源: RSC

摘要:

The Biosynthesis of Porphyrins ChlOrOphylk and Vitamin B, F. J. Leeper University Chemical Laboratory Lensfield Road Cambridge CB2 I E W Reviewing the literature published during 1984 (Continuing the coverage of literature in Natural Product Reports 1985 Vol. 2 p. 19) ~~ ~ ~ 1 The Biosynthesis of Haem 1.1 6-Aminolaevulinic Acid Synthase [5-Aminolaevulinate Syn t hase] 1.2 The Synthesis of 6-Aminolaevulinic Acid in Plants 1.3 6-Aminolaevulinic Acid Dehydratase [Porp hobilinogen Synthase] 1.4 Porphobilinogen Deaminase [Hydroxymethylbilane Syn t hase] 1.5 Uroporphyrinogen-111 Synthase [Cosynthetase] 1.6 Uroporphyrinogen Decarboxylase [U roporp hyrinogen-I I I Carboxy-lyase] 1.7 Protoporphyrinogen Oxidase 1.8 Ferrochelatase 1.9 Cytochromes 1.10 Bile Pigments 1.10.1 Biliverdin 1.10.2 Bilirubin I.10.3 Bile Pigments of Plants 2 The Biosynthesis of Chlorophylls 2.1 Steps to Protochlorophyllide 2.2 The Reduction of Ring D 2.3 Reduction of the Vinyl Group and Other Possible Variations in the Pathway 2.4 Esterification of Chlorophyllide a 2.5 Other Chlorophylls 3 The Pathway to Vitamin B12 3.1 From Uro'gen 111 to Cobyrinic Acid 3.2 Factor F-430 3.3 The Biosynthesis of the Nucleotide Loop 4 Miscellaneous Topics 4.1 Physical Methods 4.2 Geoporphyrins 4.3 The Synthesis of Tetrapyrroles 4.4 Medical Aspects 5 References This review follows the pattern of the previous one' but includes additional sections devoted to bile pigments (Section 1.10)and to the synthesis of compounds that are related to the biosynthesis of tetrapyrroles (Section 4.3).Thus Section I deals with the biosynthesis of porphyrins. The overall pathway remains unchanged (Scheme I) and the majority of work in this area is in the enzymology of the various steps. Section 2 describes advances in our knowledge of the biosynthesis of chlorophylls while Section 3 describes work on the pathway to vitamin BIZ. Finally Section 4 contains details of various related topics including spectroscopy and the synthesis of tetrapyrroles. 1 The Biosynthesis of Haem 1.1 6-Aminolaevulinic Acid Synthase [5-Aminolaevulinate Synthase (E.C.2.3.1.37)l The 'Shemin' pathway to 6-aminolaevulinic acid (ALA) (I) which involves the condensation of glycine and succinyl-CoA is well established and the major recent contributions have been in the enzymology of this reaction. The transport of pre- ALA synthase into the mitochondria in chicken embryo livers is accompanied by a decrease in molecular weight from 74 000 to 68 000 and is inhibited by haemin.' The cytosolic form is active and has similar kinetic properties3 to the mitochondria1 form except that it has a higher isoelectric point indicating that the portion that is cut off has a high proportion of basic residues. Both forms dimeri~e,~ and the structure of the dimer has been observed4 by electron microscopy; Pb'+ ions have been found to activate the native mitochondrial form of the en~yme.~ Previous workers who had observed inhibition by Pb2+ were probably working with a proteolytically degraded form.The ALA synthase from mitochondria of Saccharomyces cerevisiae has been purified to homogeneity and its properties have been studied ;6 unusually for a pyridoxal-dependent enzyme it is inhibited by thiol-directed reagents. The enzyme from chicken embryos has been cloned' and the sequences of bases in some of the corresponding DNA fragments have been determined and compared with the amino-acid sequences that have been determined for parts of the enzyme. The largest section of DNA that was cloned was 2800 base-pairs long and probably contains the full cDNA sequence.In addition the enzyme from Saccharomyces cerevisiae has also been cloned by two group^.^.^ The insertion of plasmids that contain the gene into yeast mutants that were deficient in the enzyme restored the ALA-synthesizing ability to a level that was up to sixteen times8 that of the wild-type organism. 1.2 The Synthesis of 6-Aminolaevulinic Acid in Plants The main route to ALA in plants is known to be from glutamic acid (2) and not by the Shemin pathway. Further confirmation of this has been obtained by the incorporation of [I-14C]glutamic acid into ALA or tetrapyrroles in Spirulina platensis,lo Cyanidiurn culicarium,' and cell cultures of peanut (Arachis hypoguea). The major work on the pathway from glutamate to ALA is by Gough Kannangara and co-workers.They have confirmed that glutamate I-semialdehyde (4) which was synthesized as its diethyi acetal is a precursor of ALA in preparations from barley (Hordeurn vulgare).' Furthermore they have been able to fractionate the enzymes from barleyIJ and from Chlamydo-rnonus reinhurdtiils to a certain extent by affinity chromato- graphy. The fraction which bound to blue Sepharose contained an enzyme which given Mg'+ and ATP was able to convert glutamate into an ester with RNA (3). The required RNA was found in a fraction which bound to a haem-containing Sepharose. I4 The blue Sepharose fraction also contained a NADPH-dependent dehydrogenase which could reduce the glutamyl-RNA to the semialdehyde (4).The fraction which bound to neither resin contained the aminotransferase activity for conversion of (4) into ALA (I).There is further evidence that 4,5-dioxovaleric acid (DOVA) (5) is not a free intermediate in the conversion of the semialdehyde (4) into ALA. First it has been claimed that using an improved method of assay DOVA is not normally detectable;16-1 secondly a further example has been report- NATURAL PRODUCT REPORTS 1985 CO,H H H H H H HO ALA PEG 1-Hydroxymethylbilane foZH C02H C02H C02H C02H Protoporphyrin Copro'gen Ill Uro'gen 111 IM2+ Haem Vitamin 812 pathway Chlorophyll path way Scheme I NHZ I H02C H02C-co2 -RNA (2) (3) NADPH 1 Scheme 2 0 H02C&CHO edIa in which a DOVA transaminase (purified from Chlorella regularis) is more active as an alanine-glyoxylate transaminase.DOVA may nevertheless be an enzyme-bound intermediate between (4) and ALA. It was previously reported that in Euglena gracilis the C pathway from glutamate is used for the synthesis of chloro- phylls and haems in the chloroplasts but the Shemin pathway is used for the synthesis of haems in the mitochondria. The same workers have now reported I that in Cyanidium caldarium the Shemin pathway is not used at all and [ I-'JC]glutamate is still the precursor of protohaem and haem u even under conditions when no chlorophyll or phycobilin is formed. Similarly [U-lJC]glutamate (and not glycine) is the precursor of the haem of a peroxidase that is excreted by a cell culture of peanut (Arachis hypogueu).lzOn the other hand tentative evidence has been claimed for the operation of the Shemin pathway for the formation of mitochondria1 haem in higher plants.16. l9 In one reportz0 it has now been suggested that the C pathway operates in animals also. [ 1 TIGlutamate was incorporated into the haem in duck's blood with approximately the same efficiency as [1 ,4-IJCz]succinate.A limited degrada- tion of the haem showed that the incorporation was not random. Two recent reviews'6+z1 have covered the work on the biosynthesis of ALA up to 1983. 1.3 6-Aminolaevulinic Acid Dehydratase [Porphobilinogen Synthase (E.C. 4.2.1.24)l Dimerization of ALA (I) to give porphobilinogen (PBG) (6) is catalysed by ALA dehydratase.The enzyme from bovine liver is an octamer which can bind up to eight Znz+ or Cdz+ ions but which only requires four ions for maximal activity. The importance of thiol groups has again been demonstrated in recent papers. Three thiol groups per subunit were found to react with MeSS02Me.?? The modified enzyme had lost its NATURAL PRODUCT REPORTS 1985 -F. J. LEEPER Zn?+ and was inactive but could be re-activated by reduction of the S-S linkages in the presence of a suitable metal ion and this was a convenient method for replacement of the zinc by another metal. Another groupz3 has observed a single n.m.r. signal at 79 p.p.m. for 13Cd2+ that is bound to the enzyme. This signal did not change when the substrate i.e. ALA was added which implies that the ions were not bound close to the substrate.On the other hand inhibition of ALA dehydratase by 2-bromo-3-(5-imidazolyl)propionate(which specifically al- kylates thiols that are co-ordinated to Zn’+) was prevented by the presence of ALA,Z5 suggesting that the substrate does bind close to the metal ion. The ALA dehydratase from radish has been purified by affinity chromatography on Sepharose to which ALA had been attached.25 It was found that the enzyme only bound to the resin if the ALA was attached by its amino-group and not if it was attached by the carboxyl group. 1.4 Porphobilinogen Deaminase (E.C. 4.3.1.8) (Hydroxymethyl-bilane SynthaseJ Four molecules of PBG (6) are condensed by the enzyme porphobilinogen deaminase to give the 1-hydroxymethylbilane (7).It is known that the first molecule of PBG binds covalently C02H PBG deaminase -Ho2c+NH Enz -X/ C02H \ Uro‘gen I11 (8) CO2H \ to the enzyme with the release of one molecule of ammonia and this provides ring A of (7) and thence ring A of uro’gen I11 (8) as shown in Scheme 3. Recent attempts to elucidate the mechanism of the process have concentrated on establishing the identity of the enzymic group to which the PBG becomes attached. It has been demonstrated2h for the deaminase from Euglena gracilis that the active site contains an essential lysine residue that has the unusually low pK; of 6.7. The amino-group of this lysine residue reacts reversibly with pyridoxal phos- phate and inhibition is made irreversible by reduction of the imine linkage with NaBH,.Prior binding of PBG protects the enzyme from inhibition and prevents the binding of just one molecule of pyridoxal phosphate. This lysine residue must be a likely candidate for the point of attachment of the first molecule of PBG. A different conclusion was reached by Scott and co-workers” on the basis of a 3H n.m.r. study on the porphobilinogen-PBG deaminase complex. [2,6,6,11,11-3H ,]Porphobilinogen was synthesized enzymically from [3,3,5,5-3H,]ALA (Scheme 4).The activity of this PBG was quoted as 132 Ci mmol-’ ,which corresponds to more than 4.5 atoms of 3H per molecule (however the 3H n.m.r. spectrum suggests a slightly lower level of tritiation especially at C-1 1).After the tritiated PBG was bound to the deaminase from COZH HOzC’ H02C C02H 1 cosynthetase (non -enzymic) CO;!H (7) CO H Uro’gen I (9) Scheme 3 564 NATURAL PRODUCT REPORTS 1985 C02H T 2 NH2 Scheme 4 H02C/ \CO2H Rhodopseudomonas sphaeroides the 3H n.m.r. spectrum consis-ted of very broad peaks a peak at 6 6.18 p.p.m. was reasonably assigned to 3H at C-2 of the bound PBG; a broad hump from 6 1.5 to 6 4.5p.p.m. was interpreted as consisting of two peaks at 6 2.48 and 6 3.28 p.p.m. which were assigned to 3H at C-6 and at C-1I of the bound molecule of PBG respectively. The latter chemical shift was claimed to be consistent with a -S-CHT-pyrrole group indicating that PBG is attached to a cysteine residue.However in view of the breadth of the peaks this conclusion must be considered speculative. Nevertheless the technique of 3H n.m.r. might prove useful in the study of enzyme-substrate interactions in future though it necessarily involves very high levels of radioactivity. The kinetics of the reaction of rat porphobilinogen deamin-ase with PBG have been studied by three groups.28 30 Inhibition was observed with all types of bi-and ter-valent metal ions (hard intermediate and soft),'9 and also by the linear tetrapyrrole bilirubin and its ditaurine conjugate.30Part of the gene for PBG deaminase from rat spleen has been cloned in Escherichia ~oli;~ four recombinant plasmids were obtained containing sequences of DNA of up to 1280 base-pairs which hybridize to the mRNA (which contains 1800 bases) of rat PBG deaminase.The cloned DNA also hybridizes to the corresponding human gene and should be useful for its isolation. 1.5 Uroporphyrinogen-111Synthase (E.C. 4.2.1.75) [Cosynthetase) Whereas the hydroxymethylbilane (7) cyclizes non-enzymically to give uro'gen I (9) cyclization occurs very rapidly in the presence of cosynthetase with inversion of ring D to give uro'gen 111 (8). In one paper3' it has been claimed that cosynthetase from rat contains a folate cofactor which is responsible for the rearrangement and furthermore that PBG deaminase free of cosynthetase can be induced to form uro'gen I11 by the presence of folates. This claim is questioned in a very recent paper by Hart and Batter~by,~~ who point out that the level of folate that was detected (14-50 ng per mg of protein) corresponds at most to 10-3 mole of folate per mole of cosynthetase.Furthermore no folate could be detected spectrophotometrically in the cosynthetase of Euglena gracilis nor did folates have any effect on the PBG deaminase of the same organism. f HO2C CO2H "wl2)R = CH-LHz (13) R = Et (14) R = H 1.6 Uroporphyrinogen Decarboxylase (E.C. 4.1.1.37) [Uropor-phyrinogen-111 Carboxy-lyasel This single enzyme is responsible for the decarboxylation of all four acetate side-chains of uro'gen 111(8) to give coproporphyr-inogen 111 (10). The sequence of the decarboxylations has been taken to be on the acetate groups of rings D A B and c respectively on the basis of the structures of intermediates that were isolated from the faeces of porphyric rats.However the identification (by h.p.1.c.) of other intermediates in the urine of normal and porphyric human patients has now been de-s~ribed,~~,~~ and it is suggested that different sequences of the decarboxylations are possible. Uro'gen I is also decarboxylated by the same enzyme but in this case the order of decarboxyla-tions is known to be random. It is generally found that uro'gen I is not as good a substrate as uro'gen 111 and the main observable differences are in the rates of the first and last decarbo~ylations.~~.~~ The enzyme is inhibited in vivo by chloro-and bromo-aromatics including tetrachlorodibenzo-p-dioxin (TCDD)36*38 and polychlorinated biphenyls (PCB'S).~~ TCDD was not an inhibitor in vitro and thus it is probably a metabolite of it which is the inhibitor.38The P-448 isoenzyme of cytochrome P-450 has been implicated in the conversion of 3,4,3',4'-tetrachlorobiphenyl into the true inhibitor.39 Deficiency of uroporphyrinogen decarboxylase in humans leads to porphyria cutanea tarda which is the commonest type of porphyria.The decarboxylase of human erythrocytes has been purified to homogeneity.s0 There appear to be no significant differences between the enzymes from human erythrocytes and liver.41Portions of the DNA that codes for the rat liver decarboxylase have been cloned.s2 As with PBG deaminase (Section 1.4) the cloned DNA hybridizes with the corresponding human gene and this should permit its isolation in studies of porphyria.1.7 Protoporphyrinogen Oxidase (E.C. 1.3.3.4) No new chemistry has been reported on the conversion of copro'gen I11 into protoporphyrinogen (1 1) [protoporphyrino-gen 1x1. The enzyme which oxidizes the latter porphyrinogen to protoporphyrin (1 2 ;X = H H) has been compared in various organism^.^^ The proto'gen oxidase from plants and mammals can be solubilized using detergent but the bacterial enzyme cannot. The full paper on the stereochemistry of the conversion of (1 1s)-[11-3HI]PBG into protoporphyrin has a~peared."~ The overall result (retention of 3H at C-10 only) is the combination of the individual stereochemistries of PBG deaminase cosynthetase and proto'gen oxidase ;further results are needed to determine any of these stereochemistries on its own.1.8 Ferrochelatase (E.C. 4.9.1.1) Kinetic studies on the insertion of Fe2+(and other metal ions) into protoporphyrin by ferrochelatase from human liver,45 NATURAL PRODUCT REPORTS 1985 -F. J. LEEPER chicken erythrocytes,46 and bovine liver47 have been reported. For the most part these confirm previous findings that (i) Zn2+ can be a better substratejS even than Fez+ but other metals are inhibitors;j6 (ii) meso- and deutero-porphyrin (( 13; X = H,H) and (14; X = H,H)] are both better substrates than protopor- phyrin;46 (iii) thiol-directed reagents are inhibitor^.^^ It was ~ho~n~~.~~ that Fe2+ but not protoporphyrin protects the enzyme from thiol-directed reagents and it was suggested that the Fe2+ is in fact co-ordinated by two thiol groups because arsenite was also found to inhibit the enzyme.An interesting observation,48 therefore is that Fe2+ can be inserted non-enzymically into protoporphyrin under mild conditions in the presence of a fatty acid (preferably unsaturated) and a thiol (e.g. dithiothreitol). 1.9 Cytochromes The structure of the prosthetic groups of cytochrome-c oxidase (E.C. 1.9.3.1) i.e. haem a (18) has been confirmed by the synthesis of the dimethyl ester (17) of porphyrin a [cytopor-phyrin] which is the iron-free deri~ative."~ Coupling of the side-chain to the porphyrin nucleus was achieved by the reaction of the porphyrin acid chloride (1 5) with the magne- sium chelate (16) as shown in Scheme 5.It was shown that the dimethyl ester of naturally derived porphyrin a was identical to synthetic (1 7) but could readily be distinguished from a closely related isomer of it. It has been shown that the insertion of iron precedes the binding of the pigment to apocytochrome-c oxidase.50 Thus griseofulvin which is an inhibitor of ferrochelatase inhibits the biosynthesis of haem a but does not inhibit the binding of haem a to the protein to give the active cytochrome. The magnetic circular dichroism (m.c.d.) spectrum of an oxidized bisimidazole complex of haem a is very similar to the spectrum of the cytochrome u component of cytochrome-c oxidase indicating that the imidazole moieties of two histidine molecules provide the extra ligands in the natural system.51 In cytochrome c the covalent attachment of the haem to the peptide through thioether links to the vinyl side-chains is catalysed by another enzyme cytochrome-c synthetase.A peptide that contains the first 25 N-terminal amino-acid residues of the apocytochrome was synthesizeds2 (by the solid- phase method of Merrifield) and it was found that the cytochrome-c synthetase of mitochondria of the yeast Saccharo-myces cereuisiae was able to attach the haem covalently to this peptide uiu the cysteine residues at positions 14 and 17 (albeit with reduced efficiency compared to the complete apo-cytoc hrome). The cytochrome oxidase of Escherichiu coli and other bacteria contains a green haem now called haem d which has a chlorin chromophore.The structure (19) has been suggested but not definitely established. In particular the reduction of ring D (rather than of any of the other rings) was assumed purely by analogy to the chlorophylls. Smith and Lai 53 have synthesized (19; R = Et) from chlorophyll a and also by reduction of mesohaemin with sodium in isoamyl alcohol. The latter route gave a mixture of the four isomeric chlorins reduced in each of the four rings which were separated by h.p.1.c. (as their metal- free methyl esters) and identified by an n.0.e. study in the 'H n.m.r. The u.v.-visible spectra of the four haems were very R' c several steps (17) X = H,H; R2= Me (18) X = Fen;R2= H Scheme 5 HO R I H HOZ C C02H (19) R = Et ,CH=CH* or CH(0H)Me similar showing that the natural haem d could in fact be reduced in any of the four rings.A similar green haem is present in the nitrite reductases of organisms such as Pseudomonas aeruginosa and Paracoccus denitrijcans and has been termed haem d,. However the structure (20) that has been proposed for this haem is very different from that of haem d and the alternative name acrylochlorin has been suggested. The structure is based on H n.m.r. data of the iron-free chlorin tetramethyl estersJ and is supported by an n.0.e. study.ss However the alkylation of both P-positions of one pyrrolic ring is most unusual and further confirmation of the structure by other means would be valuable. A green haem from the catalase of Neurospora crassa may be similar because it has been reporteds6 to have four carboxyl groups and cannot be oxidized to a porphyrin (as would be expected for a C-methylated chlorin).1.10 Bile Pigments 1.10.1 Biliverdin In humans the haem in the bloodstream has a lifetime of about 120 days and is then degraded and excreted. The first degradation step is oxidative ring-opening of protohaem (1 2; X = Fe) at C-5 to give biliverdin (21) [biliverdin IXa] by the enzyme haem oxygenase (decyclizing) [E.C. 1.14.99.31. The mechanism shows a strong similarity to the reaction in uitro of protohaemin (12; X = FeCl) with oxygen coupled with a reducing agent which gives a mixture of the four possible biliverdins IX the other products being carbon monoxide and hydrogen peroxide.It has been shown using the enzyme that had been purified from bovine spleen,s7 that the two new oxygen atoms both come from O, but by using a mixture of I8O2and I6O2 it was clear that they arise from different molecules of 0' because the mass spectrum of the biliverdin showed the statistical mixture of M [M+ 21 and [M+ 41 peaks. The same result has been obtained several times with the non-enzymic reaction as well as with the enzymic one in uico and in uifro.s* In addition it has been shown that no isotopically scrambled 160-180 or Hl6O-' 80H is produced.s* The first intermediate in the reaction that is catalysed by haem oxygenase is the meso-hydroxylated porphyrin (Scheme 6). A second intermediate of which the structure is as yet OH loz Scheme 6 NATURAL PRODUCT REPORTS.1985 unknown has been characteri~ed.~~ This intermediate absorbs light of wavelength 688 nm and can be purified by reversed- phase h.p.1.c. The overall reaction from protohaem to biliverdin requires three moles of dioxygen as well as some NADPH whose reducing power is delivered uia the flavopro- tein of a cytochrome P-450 reductase. The conversion of the intermediate that absorbs at 688 nm into the Fe3+ complex of biliverdin still has the requirement for this reducing system.s9 A mechanism that has been suggesteds8 for the overall reaction is shown in Scheme 6. The non-enzymic oxidative cleavage of iron porphyrinates has been used to synthesize the four isomers of urobiliverdin III,60 e.g.(22) and coprobiliverdin 111,61 e.g. (23) (as well as SN-labelled material6') from the corresponding porphyrins (which were synthesized enzymically). The four isomers that were produced in each case could be separated by chromatography. In contrast to haems porphyrins do not usually undergo ring- cleavage. For example protoporphyrin undergoes self-sensi- tized photo-oxidation mainly by reaction at the vinyl side- chains. However in biological membranes or in an oil-in-water microemulsion that contained oxidizable amino acids it has been found63 that rapid destruction of the chromophore occurs presumably by initial ring-opening to a biliverdin-type compound. I. 10.2 Bilirubin Biliverdin is rapidly reduced in the mammalian body at the central meso-position (C-lo) to give bilirubin (25).The specificity of three forms of the NADPH-requiring biliverdin reductase (E.C. 1.3.1.24) from rat liver for the isomers biliverdin XIIIa XIIIP and XIIIy (formed by cleavage of the corresponding isomer of haem at C-5 C-10 and C-15 respectively) has been The major form reduces the XIIIct isomer much more rapidly than the other isomers. The rates of reduction of the isomers of biliverdin XI11 were very similar to those that had been found previously for the normal type IX isomers. Bilirubin is normally excreted as a more water-soluble glucuronide. If the levels of bilirubin in the bloodstream are too high as can happen in some diseases or in new-born babies brain damage can occur.In these cases it is known that phototherapy can help to reduce the levels of bilirubin. Considerable interest has therefore been shown in the photoisomerization of the double-bonds of bilirubin6s*66 and the photocyclization of the vinyl side-chain~.~~."~ After excretion into the gut the metabolism of bilirubin is continued by bacteria (Scheme 7). The reactions which occur are mostly reductions. The first double-bonds to be reduced are probably those of the vinyl side-chains followed by the 4-5 and 15-16 double-bonds giving urobilinogen (26). Further reduc- tions of the double-bonds in rings A and D give the half- stercobilinogens [2,3(or 17,18)-didehydro-10,23-dihydrosterco-bilins] e.g. (27) and stercobilinogen (10,23-dihydrostercobilin) (28).These bilinogens are often re-oxidized at C-10 during isolation to give the corresponding bilin e.g. stercobilin (32). Gottschall and Plieningerh9 have synthesized samples of the stercobilins and didehydrostercobilins and related compounds that have the natural relative stereochemistry. Thus the reduction of the pyrromethenone (29) with sodium amalgam gave a mixture of the isomers (30) and (31) which could be separated by reversed-phase h.p.l.~.~O Using this reaction followed by the coupling of two suitable halves a variety of stercobilins didehydrostercobilins and urobilins were synthe- sizedb9 (Scheme 8). 1A0.3 Bile Pigments of' Plants In other organisms the oxidative cleavage of haem is not merely a degradative reaction.For example biliverdin IXa is found as a green pigment in the shells of avian eggs and the NATURAL PRODUCT REPORTS 1985 F. J. LEEPER __I_) H02C C02H H02C C02H H (21) R’ = Me,R2= CH-CH;! (25) (22) R’ = CHzCOzH R2 = CH2CHzCOzH (23) R’= Me,R2= CH2CH2COZH (24) R’ = Me,R2 = Et H02 C C02H (28) Scheme 7 __I_) NH NH / </ CO2H C02H f f HOzC H02C HOZC (29) (30) C02H (32) Scheme 8 NATURAL PRODUCT REPORTS 1985 I I 0 4-I -Enz -Enz HO;! C CO;! H H02C C02H f (33) R = Et (35) R = Et H02C CO ;!H (34) R = CH-CH2 (36)R= CH-CH;! (39) b I f HOZC C02H (37) corresponding y-isomer is a pigment in butterflies. However the most widespread use of bile pigments is in algae and plants.In algae protein-bound bile pigments are present in large quantities and function as light-harvesters. Phycocyanin (33) and phycoerythrin (37) both have the pigments covalently bound by a thioether link to a cysteine residue in the same manner as the haem is bound in cytochrome c. The pigments can be released from the protein by elimination of the thiol in which case the phycocyanobilin or phycoerythrobilin is obtained with an exocyclic double-bond7 as in (38). The stereochemistry at C-2 of phycocyanin (33) is thought to be R based on the degradation of the molecule to the imide corresponding to (38). Rapoport and co-workers7' have synthesized several model 2,3-dihydrobilidiones of both cis and trans configuration in ring A and they found that the H-2- H-3 coupling constant is consistently smaller for the trans (ca 5.0 Hz) than for the cis (ca 8.1 Hz) isomer.The observed coupling constant for one of the three molecules of bile pigment from C-phycocyanin of Synechococcus sp.6301 showed that the substituents in ring A are trans and so if C-2 is in the R configuration the configuration at C-3 must be R also.71 The presence of three molecules of bile pigment attached at different points along the peptide chains of each molecule of C- phycocyanin could allow different stereochemistries though this seems unlikely. The stereochemistry at C-3' has been assumed to be R also on the basis that oxidative elimination of the sulphur (which was assumed to be antiperiplanar) leads to the E double-bond as in (38).However Gossauer7? has shown that elimination across the C-3-C-3I bond is reversible and the more stable E configuration of the double-bond arises from either diastereo- isomer of a model compound. Therefore the stereochemistry at C-3' is still uncertain. It would seem probable that the molecules of bile pigment of phycocyanin (33) are derived from biliverdin (21) by reduction of two double-bonds and the formation of a thioether. This has been confirmed73 by detecting that I4C-labelled biliverdin is incorporated into the phycocyanin in the red alga Cyanidium caldarium. The existence of a haem oxygenase to produce the I I 0 y biliverdin was further confirmed74 by the observation that I4C- labelled mesohaem (1 3; X = Fe) was oxidatively cleaved to mesobiliverdin (24) but that this was not incorporated into the phycocyanin.Smith and Pande~~~ have developed a new synthesis of biliverdin (21) and of its derivatives in which one or other of the vinyl side-chains has been reduced to ethyl. These compounds if synthesized with an isotopic label could be tested as biosynthetic intermediates. A synthesis of biliverdin IXy (pterobilin) has also been reported76 in which the final step was oxidative ring-opening of a 15-hydroxyhae- min in air. Gossauer has reviewed synthetic methods77 in one of a collection of papers on bile pigments.78 The B-phycoerythrin of Porphyridium cruentum has been found to contain two molecules of the bile pigment (37) attached to the GI chain three to the p chain and four to the y chain.After digestion with trypsin five peptides (having bilins attached) were isolated arising from the major GI and p chains. 79 The amino-acid sequences of the peptides were determined; for four of them there was a considerable homology with sequences at the sites of attachment of bilins of other biliproteins. The fifth bilin molecule however is unique in that it is attached by two thioether links to two cysteine residues that are separated by ten residues. A study (by secondary-ion mass spectrometry and n.m.r.80) of this doubly attached bilin after it had been further degraded by thermoly- sin (which left a total of ten amino-acid residues) indicated that the structure was (39) with the second linkage being to the vinyl group at C-18.Comparisons81.82 of the n.m.r. spectra of all four singly linked bilins individually have shown that they all have the same structure (37) with the linkage to C-3I and a trans configuration in ring A. Circular dichroism spectra further showed that the configuration at C-16 is R in each case. In contrast to algae higher plants contain only a very small amount of bile pigments as they are not used for light- harvesting. However phytochrome (34) is an extremely important biliprotein in plants as it is the controlling factor for a number of light-dependent processes (e.g. differentiation). The inactive form (Pr),which occurs in the absence of light absorbs red light (h= 660 nm); this causes an isomerization to the active form (36) (P,).The active form however absorbs light in the far-red region (h=730nm) which causes it to isomerize back to the inactive form. Riidiger and co-workers have studied both forms by IH n.m.r. spectroscopy after proteolytic digestion of the peptide. The spectra were com- pared with similar ones from phycocyanin which can also be made to undergo the same isomerization. These showed that NATURAL PRODUCT REPORTS 1985 -F. J. LEEPER the P form (34) and natural phycocyanin (33) have the all-Z onf figuration,^^ whereas the P form (36) and the isomerized phycocyanin (35) have the C-15-C-16 double-bond in the E c~nfiguration.~~ Differences in reactivities of the Pr and P forms have also been noted.8s Reviews on all aspects of the action of phytochrome are collected in Volume 16 of the 'Encyclopedia of Plant Physiology'.86 The importance of phytochrome in the plant Kingdom has prompted a number of studies notably by Falk and co-workers on the properties of model systems.These include the basicity,87 stereochemistry,8s n.m.r. spectra,89 and reactionsg0 of various 2,3-dihydrobilindiones. Resonance Raman and circular dichroism studies have also been rep~rted.~' A review by Glazer,92 covering biochemical methods relating to the cysteine-bound bilins has appeared. 2 The Biosynthesis of Chlorophylls The broad outlines of the pathway to chlorophyll a that is shown in Scheme 9 are generally accepted for its biosynthesis in all organisms.As reported in the previous review,' there is considerable evidence especially from Rebeiz and co-workers that reduction of the vinyl group at C-8 can occur at either an early or a late stage. Rebeiz et a/.have reviewed their own work on the biosynthesis of chlorophyll^^^ and two other reviews of the field have also appeared.1h*" 2.1 Steps to Protochlorophyllide The magnesium chelatase activity which inserts Mg2+ into protoporphyrin to give magnesium protoporphyrinate (40) is apparently located on the chloroplast envelope,94 as it is inhibited in a preparation of intact chloroplasts by p-chloro- mercuribenzenesulphonate which does not penetrate membranes. The methyltransferase which methylates the propionate at C-13 of (40) to give (41) has been purified from Rhodopseudo- rnonas sphaeroides by affinity chromatography using S-adenosyl-L-homocysteine that was supported on agar~se.~~ The kinetics show that the magnesium protoporphyrinate must bind to the enzyme before the S-adenosylmethionine if the reaction is to occur.In contrast to the magnesium chelatase the oxidative cyclase activity which converts the magnesium protoporphyrinate methyl ester (41) into the divinyl protochlorophyliide (44) Protoporphyrin appears to be located within the chloroplast as it is not greatly inhibited in intact chloroplasts by p-chloromercuribenzene- sulphonate but it is inhibited by p-chloromercuribenzoate (which does penetrate membranes).95 There are at least two enzymes involved in the oxidative cyclization and they can be separated by high-speed ~entrifugation.~~ The activity can be restored if the two fractions are mixed together and supplied with NAD(P)H.Two groups have reported the conversion of precursors such as glutamate ALA and protoporphyrin into protochlorophyl- lide in intact isolated chloroplast^;^^.^^ in one case,98 a rate of conversion was achieved that was greater than that in uivo. 2.2 The Reduction of Ring D The enzyme protochlorophyllide reductase (E.C. 1.3.1.33) forms a ternary complex with protochlorophyllide and NADPH ;the complex absorbs light of wavelength 650 nm but hydrogen is only transferred if the system is illuminated. It has been found"' that a purified membrane preparation of the enzyme can also form a ternary complex with protochlorophyl- lide and NADP+ absorbing at 642 nm which is not photo- active.Protochlorophyllide in which the magnesium has been replaced by zinc is also reduced by the enzyme.' Several studiesIo0 Io3 have been reported on the changes in spectro- scopic properties of the photo-active complex that follow its exposure to very short pulses (down to picoseconds) of light from a laser but these changes are not readily interpreted in terms of the chemistry that is involved. Two reviewsIo4 on the action of light on the production of chlorophyll(ide)s include discussions of these spectroscopic changes. Light has been shown to have a further effect on this enzyme at the nucleic acid level.'0s Strangely irradiation with red light causes a reduction in the level of mRNA that codes for the reductase which was measured by its hybridization with cloned sequences of complementary DNA; this effect is reversed by far-red illumination and so is probably phyto- chrome-mediated (see Section 1.10.3).Exactly the opposite effect is seen with the light-harvesting chlorophyll a/b protein -levels of both mRNA and protein are increased if the system is illuminated with red light. A further phytochrome-mediated effect controls the synthesis of ALA (]),?I which is shut off when protochlorophyllide accumulates in the dark. When the tissues are illuminated and the protochl6rophyllide level drops there is a lag in the resumption of synthesis of ALA while the R3 I I R' (40)R' = CH-CH, R2= H Pr ot oc hlorophyllides ChlorophyIl(ide)s a,R3= Me (411 R' = CH-CH, R2= Me (44)R'= CH-CH and b R3= CHO (42)R' = Et R2= H (45)R'= Et (46)R' = CH-CHZ ,R2 = H (43) R' = Et R2 = Me (47)R'= Et R2= H (48)R1 = CH-CH;! ,R2= phytyl (49)R' = Et ,R2= phytyl Scheme 9 relevant enzymes are synthesized de now.This lag can however be abolished by pre-illumination with red light. If the control point of synthesis of ALA is by-passed by adding exogenous ALA protochlorophyllide continues to accumulate and reaches excessive levels. This is the basis for the action of ALA as a herbicide.Io6 In contrast it appears that ALA is not taken up by Euglena gracilis as [4-I4C]ALA was not incorporated intact into chlorophyll a judging by the results of a specific degradation of ring B of the pigment to determine the location of the labelled atoms.Io7 When the levels of protochlorophyllide drop in vim the reductase activity also drops as a specific protease degrades it.This effect has now been observed in vitro also;lo8 the protease which has been found to be membrane-bound does not degrade reductase molecules to which a protochlorophyllide molecule is bound. The claim by Adamson' that barley that has grown in the light contains a light-independent protochlorophyllide reduc- tase whereas dark-grown barley does not has been extended to include lo However a different group' ' adminis-tered ''C-labelled ALA to barley shoots that had previously been illuminated for various lengths of time and then returned to the dark but they found no evidence for any incorporation into anything beyond protochlorophyllide.2.3 Reduction of the Vinyl Group and Other Possible Variations in the Pathway Rebeiz and co-workers have previously proposed that the biosynthesis of chlorophyll a can follow any of four parallel routes.' These are the monovinyl and divinyl routes each of which is subdivided into a monoester route (as described above) and a diester route (in which the carboxyl at C-17 is esterified with various unspecified alcohols). This suggested scheme has now been expanded to a six-branched pathwayg3 by the inclusion of routes in which the carboxyl at C-17 is esterified and that at C-13 is not; however the evidence for this is as yet minimal.The evidence that the divinyl protochlorophyllide and chlorophyllide a derivatives (44) and (46a) occur naturally has been strengthened considerably by reports' of the confirma- tion of their structurgs by n.m.r. and mass spectroscopy. Nuclear magnetic resonance has also been used to show that one vinyl group of the protochlorophyllide that was isolated from etiolated (dark-grown) tissues had been reduced. A detailed analysis of the Fast Atom Bombardment (FAB) mass spectra of chlorophyll b (49b) and its divinyl derivative (48b) has shown'I4 that this is a reliable method for distinguishing them and has allowed confirmation of the structure of (48b) which had been isolated from a mutant of maize and previously identified by u.v.-visible absorption and fluorescence studies only.The existence of monovinyl and divinyl pathways seems to have been established but the point at which they diverge remains in doubt. Rebeiz et al.93 consider that the divergence may be as far back as copro'gen I11 (lo) but they have not identified any component that contains only one vinyl group before the magnesium 81,82-dihydroprotoporphyrinate (42). It has been pointed out'' that an esterase which hydrolyses the methyl ester (41) to the acid (40) is known to exist and thus (42) may arise by hydrolysis of (43). Therefore it was concluded that the most likely point at which the two paths separate is the monomethyl ester (41) because the reduction of the vinyl group of this substrate had been directly observed some years ago.' According to Rebeiz et the two pathways remain separate until the stage of the didehydrochlorophyllide a (46a) when reduction of the vinyl group to give (47a) is again possible.The relative importance of the two pathways depends on the type of plant and the illumination conditions. For example in dicotyledonous plants (e.g. cucumber) that have grown in a normal day/night cycle it was found that the divinyl route is NATURAL PRODUCT REPORTS 1985 almost exclusively followed in the daytime and the monovinyl route only becomes functional (to a small extent) at night. In monocotyledonous plants on the other hand the monovinyl route is functional during the light part of the cycle also. The existence of fully esterified versions of the intermediates on the biosynthetic pathway to chlorophyll a (e.g.protochloro-phyll) has been confirmed by workers other than Rebeiz et al. but the nature of the esterifying group on the carboxyl at C-17 is not agreed; Shioi and Sasalo2 have identified phytyl esters and their dehydro-forms (including geranylgeranyl) whereas Re- beiz claim~~~ that the esterifying alcohols are different from phytol (by g.c. and m.s. evidence) but cannot identify any of them. There is also disagreement over whether or not the fully esterified protochlorophylls can serve as precursors of chlorophylls. 2.4 Esterification of Chlorophyllide (I Evidence that was quoted in the previous review' indicated that geranylgeranyl diphosphate (50) is involved in the conversion of chlorophyllide a (47a) into chlorophyll a (49a) either by initial formation of a geranylgeranyl ester followed by reduction to the phytyl ester or by reduction of geranylgeranyl diphosphate to phytyl diphosphate (51) followed by the formation of a phytyl ester.The incorporation of radiolabelled geranylgeranyl diphosphate (50) and of the monophosphate as well as of phytyl diphosphate (51) into chlorophylls a and b in cell cultures of tobacco (Nicotiunu tabacum) has been report- ed ' ' s and it was found that the saturating concentration of (5I) was rather lower than that of the geranylgeranyl phosphates. Other groups however have found that phytol itself can be used for the esterification. Shlyk et ~1."~ were able to esterify exogenous chlorophyllides with exogenous phytol using either a suspension of chloroplasts or the supernatant after cells had been mechanically disintegrated and Daniel1 and Rebei~~~ achieved the same result by using intact etiochloroplasts.The latter workers observed a requirement for ATP and Mg'+ and so the conversion of phytol into itsdiphosphate (51) seems quite probable. 2.5 Other Chlorophylls An enzyme chlorophyll oxidase has been identified in barley I which oxidizes chlorophyll Q to the 13'-hydroxy- compound (52). A new pigment termed chlorophyll RC 1 has been isolated from photosystem 1 of Scenedesmus obliquus' and Spirulinu geitleri'I9 and has been shown by both groups (using n,m.r. mass spectroscopy and other techniques) to contain a chlorine atom at C-20 as well as the hydroxyl in ring E i.e.to be (53). Rebeiz et~l.~~ have tentatively proposed that ring E of a chlorophyll which they have identified spectroscopically may be a lactone as (54). It is well known that oxidation of ring E can occur non-enzymatically in air (see e.g. ref. 114). A chlorin that is produced by a mutant of Rhociopseudomonas sphueroides has been demonstratedI'O to be a derivative of chlorophyllide a in which the vinyl group at C-3 has been hydrated to a hydroxyethyl group (55). The positions of the 00 geranylgeranyl d iphosphate (50) 00 phytyl diphosphate (51) NATURAL PRODUCT REPORTS 1985 F. J. LEEPER 571 I I RO (52) X = H (55) (56) (53) x = CI (54) X = H ;ring E =* HO C02Me R' (59) R=COMe (60)R = Et (57) R'= CH-CH (58)R' = COMe side-chains were unambiguously established using H nuclear Overhauser effects.It is very likely that this is an intermediate in the biosynthesis of bacteriochlorophyll a (56; R = phytyl). The bacteriochlorophyll a fraction from Rh. sphaeroides and Chromatium uinosum consists mainly of the phytyl ester but 4% of the pigment is a mixture of the geranylgeranyl ester and di- and tetra-hydro-forms of it.' 21 The highest levels of these unsaturated esters occurred at the beginning of the production of bacteriochlorophyll a and so it was concluded that they are the normal intermediates on the path to the phytyl ester (as in the case of chlorophylls a and b).A new bacteriochlorophyll designated gGg from Heliobac-terium chlorum has been described."' Its structure (57),' t3 is related to bacteriochlorophyll b (58; R = phytyl) except that C- 3 retains a vinyl group; the esterifying group R' is geranylger- anyl. In a study of the photo-oxidation of bacteriochlorophyll b (58; RZ= phytyl),' 24 it was found that the major site of reaction is the exocyclic double-bond at C-8 which migrates into the ring leaving an oxidized side-chain e.g. (59); in the absence of oxygen light still causes the migration but an ethyl side-chain results (60). Similar photo-oxidation of (57; R2 =geranyl-geranyl) was also observed.' 23 A description of the reversed-phase h.p.1.c. separation of the various bacteriochlorophylls c (6I) from Prosthecochloris aestuarii has been published.'25 Six bands were obtained in all two pairs of which were diastereoisomers that differed only in the absolute configuration of the hydroxyethyl side-chain on C- 3.When the substituent at C-8 was ethyl the configuration of the hydroxyethyl group was exclusively R but increasing proportions of the S-isomers were present if the larger n-propyl and isobutyl substituents were at C-8. '0 farnesyl (61) R' = Et ,Pr",Bu' or neopentyl R2= Me or Et 3 The Pathway to Vitamin B, 3.1 From Uro'gen 111 to Cobyrinic Acid It has been established that the first three steps in the biosynthesis of vitamin 9 (cyanocobalamin) (70) from uro'gen I11 (8) are methylations at C-2 C-7 and C-20 respectively which give a dihydro-form of Factor I11 (65) -probably (63) (see Scheme 10).After this the order of the steps to the next known intermediate i.e. cobyrinic acid (66) is not clear. They include five methylations decarboxylation of the side-chain at C-12 insertion of cobalt ring-contraction and extrusion of C-20 and its attached methyl group as acetic acid. An experiment to reveal the order of the five methylations by Uzar and Battersby was reported in the last review.' In this experiment the cell-free system from Clostridium tetanomor- phum was incubated with sirohydrochlorin (64) and a defi- ciency of S-adenosylmethionine (SAM) for a few hours and then with an excess of [Me-'3C]SAM to complete the conversion into cobyrinic acid (66).The material that was derived from this incubation therefore had more I3C in the methyl groups that had been introduced later in the biosynthe- tic sequence. Nuclear magnetic resonance spectroscopy re-vealed that the methyl group at C-17 contained the least I3C and was consequently the first to be introduced. This experiment has now been repeated using Propionibac-terium shermanii (Propionibacterium jreudenreichii su bsp. sher-manii) by Scott Muller and co-workers;' 26 both they and Uzar and Battersby"' have also performed the inverse experiment in which incubation with a deficiency of [Me-I3C]SAM is followed by an excess of unlabelled SAM. Detailed analyses of the n.m.r. spectra revealed the order of the first 572 NATURAL PRODUCT REPORTS 1985 C02H three of the methylations after (63) in one case I 16 and all five in \ the other case.I1’ The order is C-17 C-12 C-I C-15 and then C-5.The agreed observation that methylation at C-12 precedes CO2H that at C-1 was quite unexpected as it is difficult to formulate plausible mechanisms for a route which has this sequence of events. For example Scott and Miiller 1h simply propose that after methylation at C-12 the C-20-C-I double-bond is hydrated and then the hydrogen at C-1 is replaced by a methyl group. No mechanisms were given. Uzar and Battersby”’ have suggested a pathway as shown in Scheme 11. In this Scheme the attack of the nucleophilic oxygen at C-20 H accompanies the electrophilic attack of the methyl group from SAM on C-1.A novel suggestion is that the acetate side-chain on C-19 provides the nucleophile thus forming a lactone ring which is opened up in the ring-contraction step. This proposal might be tested by an 180-labelling study. This Scheme is consistent with all results to date on the biosynthesis. For example it was found ?* that the hydrogen atoms at C-18 and 3 x SAM C02 H \ Y C02H Ho2cy?Y$ .CO H r R CO2H C02H C02 H COzH (62) R =H (64)R = H (63) R = Me (65) R = Me C02H - HO’ (70)R’= R2= Me (71) R’= H,RL OH Scheme 10 ~ NATURAL PRODUCT REPORTS 1985 F. J. LEEPER C02 H CO;! H C02 H HO (63) 'Me+' HO2C H02C CO2H R = CH2C02H;X = H,H R = Me;X = H,H R = Me ; X = Co' 'Me+'1 C OzH C02 H -0H HO2C C02 H HOzC C02 H H /C02H COzH I-MeC02H 1 -H02C C02 H H02C C02 H (66) Scheme II C-19 come from the medium if the enzyme system is incubated for the cobalt-inserting enzyme an increase in the incorpora- with I3C-labelled uro'gen 111 (8) in D20.Further results that tion of s7C~2+ would have been expected. are in accord with the Scheme are discussed below. Nussbaumer and Arigoni 30 have tested the norcobyrinic Possible final steps in the path to cobyrinic acid have been acids (68) and (69)' as possible immediate precursors to tested by two groups. Podschun and Miiller1'9 have prepared cobyrinic acid (66). However they were not able to detect any hydrogenobyrinic acid (cobalt-free cobyrinic acid) (67) from incorporation of I4C-labelled SAM into cobyrinic acid in the the corresponding cobalt-free amides that are produced by Rh.presence of either nor-compound. Possibly the enzyme system sphaeroides if it grows on a medium that is devoid of cobalt. was not working well as the incorporation of ALA was very Using the cell-free extract from P. shermunii they found that low also. the incorporation of 57C02+into cobyrinic acid being made Both of these experiments would have been more compelling from endogenous precursors was reduced by the presence of if the isotopic label had been in the compound that was being (67). If the hydrogenobyrinic acid (67) had been the substrate tested; nevertheless they do provide evidence that neither methylation nor insertion of cobalt is the final step in the pathway and this is why the final step in Scheme 11 has been shown as the deacetylation at C-19.3.2 Factor F-430 Factor F-430 is a nickel-containing coenzyme that is found in methanogenic bacteria and which is involved in the reduction of the methyl-sulphur bond of methylcoenzyme M (72) to give methane and the thiol. Its structure (73) has been con-firmed13'.132 to be the penta-acid corresponding to the penta- ester structure (74) already determined for the methylated cofactor (F-430M). It seems probable that F-430 is derived from the intermediate in the vitamin Bl pathway dihydrosiro- l~~ hydrochlorin (62). Bykhovskii et ~1. have detected at least six pigments that are excreted by methanogenic bacteria and which are similar to sirohydrochlorin (64).It is possible that these are related to precursors of F-430. Eschenmoser and co-workers have synthesized I 34 a nickel complex (75) which contains the chromophore of F-430 and have found it to have similar spectroscopic magnetic electrochemical and co-ordination properties to the coenzyme. The X-ray crystal structure 35 of the isothiocyanato-nickel complex (75; X= NCS) showed that the nickel is six-co- ordinate in the crystal in contrast to nickel(1r) complexes of corrins which are four-co-ordinate. This difference is due to the larger size of the central hole in the hydroporphinoids compared to corrinoids which means that the electrophilicity of the small nickel(I1) ion cannot be satisfied by the four nitrogen atoms of the macrocycle.If no fifth or sixth ligands are available considerable ruffling of the macrocycle occurs to allow the nitrogen atoms to approach the nickel ion more CO2R \ NATURAL PRODUCT REPORTS 1985 closely. No doubt this axial reactivity of the nickel ion in F-430 is important in the mechanism of its biological action. 3.3. The Biosynthesis of the Nucleotide Loop Vitamin B (70) includes a dimethylbenzimidazole group as an internal ligand to the cobalt ion but another naturally occurring corrin (7 1) (confusingly also referred to as Factor 111) has a 5-hydroxybenzimidazole group. The biosynthesis of this in Methanosarcincr barkeri136 has been studied and it was found that 5-hydroxy[2-'~C]benzimidazole was incorporated into (7 1) whereas [2-'4C]benzimidazole itself was not.Glycine that was labelled with IJC in either carbon atom was incorporated into the 5-hydroxybenzimidazole portion and [I-' 3C]glycine spe- cifically labelled C-3a. In contrast [1-13C]glycine labelled C-1Oa of the deazaflavin (76) (also confusingly called Factor F-420) indicating a different biosynthesis. In this organism glycine is not a precursor of the corrin nucleus but [U-l"C]-glutamate is. 4 Miscellaneous Topics 4.1 Physical Methods Separation of the natural tetrapyrroles remains an area of interest because of the clinical applications; as a result several papers have dealt with the t.l.c.I3' and reversed-phase h.p.l.~.~"-~~-'~~ IJ0 of porphyrins. This field has been re-viewed.'"' Also the t.1.c.'"' and n0rma1I-l~ and reversed- phase'"" IJ6h.p.1.c.of chlorophylls have been described. The separation of porphyrins and metalloporphyrins by capillary g.c. and the application of g.c.-m.s. has been 0 II -LCO2R CN C02R (75) F-430 (73)R = H F-430M (74)R = Me Et (or Me) 0 (76) (80) R'= Et ; R2R3= [CH2]2 (77)R' = R2= C02H (81) R' R2 = [CH2]4 ; R3= H (78)R' = H ,R2= C02H (82) R1 R2,R3 or R' ,R2R3 = [CH213 ,Et (79)R'= R2= H (83)R1 R2= CHMeCH2 ; R3= H (84) R' R2= CH2CH2CHMe ;R3= H NATURAL PRODUCT REPORTS 1985 -F. J. LEEPER reviewed. 47 Chemical Ionization (CI) mass spectroscopy has been applied to the structure determination of petroporphyr- insIJ8 and the use of Fast Atom Bombardment (FAB)'"."" or Secondary Ion mass spectra in the study of chlorophylls has already been described.In addition the mass spectra of a range of chlorophyll derivatives were obtained by 25 'Cf plasma desorption (also called 252Cf fission-fragment ionization) and have been reported. 14g*1 Studies of n.m.r. spectra of tetrapyrroles have mainly concentrated on ,C. For example the ,C n.m.r. spectra and assignments have been reported for the zinc complexes of all fifteen isomers of protoporphyrin dimethyl ester,' for chlorophyll a and several of its derivatives,15' and for pyropheophytin a (to investigate its protonation). 53 Solid-state I3Cn.m.r. spectra of chlorophyll a and some of its derivatives using cross-polarization and magic-angle spinning have also been reported.' 54 Two-dimensional ,C/' H chemical shift correlation has been used to assign the 'H chemical shifts of 'cobester' [the heptamethyl ester of dicyanocobyrinic acid (66)] in CDC13.1S5 The I3C n.m.r.spectra of aqua- adenosyl- methyl- and carboxymethyl-cobalamin have been interpreted. 56 In the rapidly growing field of the bile pigments there have been several papers which focus on 'H n.m.r. studies of the structure of the linear tetrapyrroles in solution 58 as well as 5771 a 13Cstudys9 and I5N studies.62,159,160 X-Ray crystal structures of linear tetrapyrroles have been reviewed by Sheldrick and other crystal structures that have been published include one of methylcobalamin,162 a series of papers on the monocarboxylic acid which is obtained on hydrolysis of cyanocobalamin 63 an octaethylbacteriochlorin (as a model for the aggregation of bacteriochlorophyll b),164 a meso-tetramethyl-isobacteriochlorin(as a model for siro-haem),16s the F-430 model that was mentioned earlier,135 and vanadyl 132,15-cycloetioporphyrinate111 [80; M = V(O)] from oil shale.66 The results of what is described as the first coherent investigation of the absorption and magnetic circular dich- roism spectra of a series of monosubstituted free-base porphyrins possessing vinyl cyano ethoxycarbonyl acetyl and formyl substituents have been reported,167 and so have the m.c.d. spectra of porphyrins having these substituents adjacent to an unsubstituted position.16s 4.2 Geoporphyrins Several derivatives of chlorophylls have been identified in a Lower Miocene fossil sample by h.p.l.~.'~~ Bonnett et a1.169 have found iron gallium and manganese porphyrinates and metal-free porphyrins in a Turkish lignite.The iron porphyrin- ates included mesohaem (77; M = Fe) as well as a monocarbox- ylated porphyrinate [probably (78; M = Fe)] and etiohaem (79; M = Fe). It seems unlikely that mesohaem could arise from any chlorophyll and it is suggested that it derives from the cytochromes of micro-organisms. In a sample of lignite from Victoria Australia 7004 of the iron porphyrinate fraction consisted of mesohaem. In coals and petroleum it is usual that the side-chains of porphyrins have been degraded to alkyl groups or removed entirely.For example Bonnett and Czechowski 70 report finding a series of gallium porphyrinates in bituminous coal corresponding to etioporphyrin 111 (79; M = H,H) and its lower homologues that have lost up to six methylene groups from the side-chains. Etioporphyrin I11 could plausibly arise from either cytochromes or chlorophylls but many of the metalloporphyrins from oil shales have carbocyclic rings which makes it likely that they derive from chlorophylls. Examples of the types (80) and (81) were mentioned in the previous review' and further examples which have been described are of the types (82),IJ8 (83),17' and (84).172A synthesis of (80) is outlined in the following section. 4.3 The Synthesis of Tetrapyrroles The total synthesis of pyrrole pigments (1973-1980) is the subject of a review by Jackson and Smith in the series 'The Total Synthesis of Natural Products'.' 73 A simple synthesis of ALA (1) has been described which employs a reduction of the succinyl cyanide (85) by zinc to give (86) which can be hydrolysed to ALA.In the preparation of pyrroles as precursors for porphyrins the propionate side-chain is often obtained by oxidative rearrangement of an acetyl-pyrrole using T1( NO,) . An alternative two-step procedure via the chloromethyl ketone has been reported175 which avoids the use of toxic thallium salts and uses silver salts instead. Another commonly used reaction of the side-chain of pyrroles is the conversion of a 2-hydroxyethyl group into a 2-haloethyl using SOCl in pyridine or Ph3P and CBr,.It has now been shown176 that in most cases the reaction proceeds via an ethylenepyrrolonium ion (e.g. Scheme 12) which is observable by 'H n.m.r. When 13C or ?H labels were in the side-chain scrambling of the label was observed. No scrambling was observed in the corresponding reactions of porphyrins. Among the total syntheses of porphyrins that have been described have been ones of methyl-devinyl-I 77 and trideuter- iomethyl-demethyl-protoporphyrins,' 78 for use in n.m.r. stu- dies of reconstituted haemoproteins. Djerassi and co-workers168 have described the syntheses of many of the porphyrins that were used in their work on m.c.d. spectra. Amongst the techniques which they used are a Vilsmeier formylation of dipyrrylmethanes using dimethylformamide and p-nitrobenzoyl chloride,' 79 and the use of a thallium(ii1) complex of a porphyrin that has an unsubstituted 0-position in order to raise its oxidation potential and protect it during the reaction of a vinyl side-chain with OsO,.' 68 Ozonolysis is another method that has been used to cleave vinyl groups to formyl.lso Clezy and co-workerslsl have synthesized a number of polyhydroxy- and polyacetoxy-porphyrins that are related to haematoporphyrin and which were intended for testing as photosensitizers in anti-tumour therapy (see next section).Smith el a1.182 have described a total synthesis of deoxophyl- loerythroetioporphyrin (DPEP) [ 13' 15-cycloetioporphyrin 1111 (92a) and its 172-methoxycarbonyl-derivative(deoxophyllo-erythrin) (92b) (Scheme 13).This involved the synthesis of the 0 0 NHAc Me02C A C N Me02C HO Jx-X Scheme 12 576 NATURAL PRODUCT REPORTS 1985 R (a,R = H ;b R = C02Me) (87) X = Y =H; M=Zn (91) X = C02Me (88) X = H;Y = HgCl ;M = Zn (89) X = Y = HgCl; M = Zn (90)X = H; Y = CH-CHCO2Me;[I M= H,H (92)X = H Scheme 13 C02Me I i Me02C C0,Me (931 mono-unsubstituted zinc porphyrinate (87a) which was mer- curated with Hg(OAc) to give a mixture of mono- and di- mercurated products (88a) and (89a). This mixture reacted with methyl acrylate and LiPdC1 to give the acrylate (90a) from the monomercurated porphyrinate and the ring-cyclized porphyrin (91 a) from the dimercurated porphyrinate. The conversion of (91a) into DPEP (92a) was accomplished by hydrolysis and decarboxylation.The synthesis of the methyl ester (92b) from the zinc porphyrinate (87b) followed the same lines and the structure of (92b) was confirmed by the synthesis of identical material from chlorophyll a. Another type of cyclization of a side-chain of a porphyrin on to a meso-position has been described by Clezy et who converted coproporphyrin I1 tetramethyl ester into coprorho- din I1 trimethyl ester (93) by treatment with oleum. The chemistry of such rhodins was also investigated. The deuteriation of octa-alkyl-porphyrins has been re-investigated by Hickman and Goff.Isa They found that deuteriotoluenesulphonic acid in refluxing o-dichlorobenzene caused exchange at the alkyl groups as well as the meso-positions.Gossauer’8s has described the synthesis of aromatic macro- cycles containing five and six pyrrole rings which he has called pentaphyrins and hexaphyrins. The synthesis of reduced porphyrins is of great relevance to the studies on the biosynthesis of vitamin B ?. Three full papers have been published on the earlier work in Cambridge on the synthesis of C-methylated chlorinsl 86 and isobacteriochlor- ins.187 However as mentioned in the previous review,’ Battersby and co-workers have more recently developed a photochemical ring-closure to give isobacteriochlorins or chlorins which is compatible with the ester groups that are required for a synthesis of the naturally occurring pigments. This approach has been used in a synthesis of model isobacteriochlorins that have a methyl or cyano substituent at C-20,188 and most recently in a synthesis of the octamethyl ester of Factor I which is the aromatized form of the first intermediate beyond uro’gen I11 on the pathway to vitamin B,,.Other key steps in this synthesis (outlined in Scheme 14) were (i) the reaction of the phosphonium salt (94) with a monothioimide; (ii) the removal of the cyano-group of (95) by reduction with Raney nickel followed by a fragmentation giving (96); and (iii) the introduction of the carbon atom that is destined to be C-20 by a sulphur-contraction procedure [(97) to (98)]. The overall yield from the two halves [(98) and (99)] to Factor I octamethyl ester (100) was 26% plus 8% of the epimer at C-3.Eschenmoser has made very important contributions to the synthesis of model systems that are of relevance to the pathway to vitamin Bl z. His synthetic model (75) for Factor F-430 has been described earlier (Section 3.2). The synthesis of (75) (Scheme 15) involved 3J an electrochemical oxidative cycliza- tion of a nickel seco-corrin complex (101) to give (102) which could be reduced (either electrochemically or with zinc in acetic acid) to give (103). The addition of cyanide then completed the synthesis of the model (75). It is interesting to note that under different conditions electrochemical or photochemical cyclo- isomerization of the same seco-corrin (101) gave the corrinoid (104). 90 The results of some studies by Eschenmoser’s group of methylations which model the biosynthetic methylations were detailed in the last review.’ The reaction of Me1 with the magnesium complex of 20-methylpyrrocorphin (1 05) leads chiefly to methylation at C-17 with a small amount of methylation at C-18.191Further investigation of the two further minor productsIg2 has revealed that while one of them is simply an N-methylated derivative the other is the ring-opened product (106) (Scheme 16). The surprising stereochemistry about the double-bonds of this product was indicated by the nuclear Overhauser effects. The product presumably arises by methylation at C-19 which after tautomerization allows the fragmentation with cleavage of the C-19-C-20 bond. It was found that complexation of (106) with nickel(I1) acetate followed by addition of acetic acid led to a practically quantitative cyclization at room temperature to the tetradehy- drocorrinate (1 07).It is clear from biosynthetic results that this overall transformation of a pyrrocorphin into a corrin is not however related to the process that occurs in viuo. Some other of Eschenmoser’s methylations have also turned out to be not quite in accord with the biosynthetic sequence. For example NATURAL PRODUCT REPORTS 1985 F. J. LEEPER 577 ~ CO,Me C02Me Me02c+9+ e VCOPM ___) C02 Me + Ph,P+ NH HN CL-CO@ 0 CO*BU' 0 (94) (95) R = CN (96)R = H C02Me C02Me C02Me Meo2cd C02Me S co;!But (98) (97) C02 Me Me02c.;i C02Me C02Me -N \ Me02C I C02Me C02 Me COzMe (99) CO;! Me ihv C02Me one very interesting result ')I (not mentioned in the previous \ review) is that the dihydropyrrocorphinol(lO8)is methylated at C-17 to give (109) (Scheme 17) but this is not strictly biomimetic because it is known that in riz.0 the methylation of C-I7 precedes those at C-12 and C-1 .4.4 Medical Aspects Recent reviews on medical aspects of tetrapyrroles include one on the biochemical basis of acute p~rphyrias,'~~ two on the biochemistry of vitamin B12,19J.1')s and two by Kessel on photosensitization with porphyrins in the treatment of can-cer.1y6.1y7 It has been reported1'>' that the active component of 'haematoporphyrin derivative' has recently been identified as the dimeric ether.The photosensitizing ability of the chlorin bonellin has been compared with that of mesoporphyrin;I"* the chlorin was found to be ten times more active due to the different absorption properties rather than to different NATURAL PRODUCT REPORTS 1985 CN CIO (102) (75) t CN PF6- 03) Scheme 15 amounts of pigment being taken up by the cells. 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ISSN:0265-0568    

DOI:10.1039/NP9850200561

出版商:RSC    

年代:1985

数据来源: RSC