摘要:

CHEMICAL SOCIETY REVIEWS VOLUME 10, 1981 0 Copyright 1982 LONDON THEROYAL SOCIETY OF CHEMISTRY CONTENTS PAGE TILDEN LECTURE. '/)5-CYCLOPENTADIENYLAND T~-ARENEAS PROTECTING LIGANDS METALCOMPLEXES.TOWARDS PLATINUM By P. M. Maitlis 1 OF THE VIOLOGENS. By C. L. Bird and A. T. Kuhn 49ELECTROCHEMISTRY TILDEN LECTURE. SOMEUSESOF SILICON IN ORGANICCOMPOUNDS SYNTHESIS. By Ian Fleming 83 METHODS POLYCYCLICMODERN ANALYTICAL FOR ENVIRONMENTAL AROMATIC By K. D. Bartle, M. L. Lee, and S. A. Wise 113COMPOUNDS. MELDOLA MEDAL LECTURE. THERELATIONSHIP CARBONYLBETWEEN METAL AND SUPPORTEDCLUSTERS METALCATALYSTS. By J. Evans 159 PHOTOCHEMISTRY OF ARYLHALIDES.By J. Grimshaw and AND PHOTOCYCLIZATION A. P. de Silva 181 SINGLET OXYGEN.By A. A. Gorman and M. A. J. Rodgers 205MOLECULAR CHEMICAL ASPECTS OF TRACE CONSTITUENTS OF THE DIET. PARTI CONTROL OF THEREAAND SURVEILLANCETRACE CONSTITUENTS-IS NEED? By D. G. Lindsay 233 PART I1 SOURCES ADVANCESOF, AND ANALYTICAL IN, TRACEINORGANIC CONSTITUENTSIN FOOD. By C. J. Pickford 245 IN THE ANALYSIS CONSTITUENTSPARTI11 ADVANCES OF TRACE ORGANIC OF THE DIET, WITH PARTICULAR TO MASS SPECTROMETRY. REFERENCE By J. Gilbert and R. Self 255 PART Iv NUTRITIONALCHEMISTRY OF INORGANIC TRACE CONSTITUENTS IN THE DIET. By J. K. Chesters 270 AND BIOGENESIS OF THEPART V SOURCES OF TRACE ORGANIC CONSTITUENTS DIET. By S. A. Slorach 280 CYANOKETENES: AND CYCLOADDITIONS.SYNTHESIS By H. W. Moore and M. D. Gheorghiu 289 ISOTOPIC EXCHANGE AND APPLICATIONS.HYDROGEN IN PURINES-MECHANISMS By J.R. Jones and S. E. Taylor 329 INGOLD LECTURE. How DOES A REACTIONCHOOSEITSMECHANISM?ByW. P. Jencks 345 ARYLIODINE(III) By A. Varvoglis 377DICARBOXYLATES. HAWORTH MEMORIAL LECTURE. STRUCTURALSTUDIESOF POLY-SACCHARIDES. By B. Lindberg 409 METHYL IN STEROID By H. L. Holland 435GROUP REMOVAL BIOSYNTHESIS. CLUSTERS QUESTCENTENARY LECTURE. METAL IN BIOLOGY: FOR A SYNTHETIC OF THE CATALYTIC By R. H. Holm 455REPRESENTATION SITEOF NITROGENASE. CHEMISTRY By L. G. Harrison 491PHYSICAL OF BIOLOGICALMORPHOGENESIS. Chemical Society Reviews Vol 10 No 4 1981 Page HAWQRTH MEMQRIAL LECTURE Structural Studies of Polysaccharides 409By B. Lindberg Methyl Group Removal in Steroid Biosynthesis By H.L. Holland 435 CENTENARY LECTURE Metal Clusters in Biology: Quest for a Synthetic Representation of the Catalytic Site of Nbogenase By R. H. Holm 455 Physical Chemistry of Biological Morphogenesis By L. G. Harrison 491 5291981 Zndexes The Royal Society of ChemistryLondon Chemical Society Reviews EDITORIAL BOARD Professor K. W.Bagnall, B.Sc., Ph.D., D.Sc., C.Chem., F.R.S.C. (Chairman) Professor K. R. Jennings, M.A., D.Phil., C.Chem., F.R.S.C. Professor G. W. Kirby, M.A., Ph.D., Sc.D., F.R.S.E., C.Chem., F.R.S.C. Professor B. L. Shaw. B.Sc., Ph.D., F.R.S. Chemical Society Reviews appears quarterly and comprises approximately 20 articles (ca. 500 pp) per annum. It is intended that each review article shall be of interest to chemists in general, and not merely to those with a specialist interest in the subject under review.The articles range over the whole of chemistry and its interfaces with other disciplines. Although the majority of articles are intended to be specially commissioned, the Society is always prepared to consider offers of articles for publication. In such cases a short synopsis, rather than the completed article, should be sub- mitted to The Managing Editor, Books and Reviews Section, The Royal Society of Chemistry, Burlington House, Piccadilly, London, W1V OBN. Members of the Royal Society of Chemistry may subscribe to Chemical Society Reviews at f10.50 per annum (beginning 1982, f 12.50 per annum); they should place their orders on their Annual Subscription renewal forms in the usual way.1981Annual Subscription price, U.K. 231 .00 (beginning 1982, f36 per annum), Rest of World f33.00 (beginning 1982, 238), U.S.A. $78.00 (beginning 1982, $85) including air speeded delivery. Application to mail at second class postage rate is pending at Jamaica, N.Y. 11431. Change of address and orders with payment in advance to The Royal Society of Chemistry, The Distribution Centre, Blackhorse Road, Letchworth, Herts SG6 1HN England. Air freight and mailing in the U.S. by Publications Expediting Inc., 200 Meacham Avenue, Elmont, New York 11003. All other despatches outside the U.K. by Bulk Airmail, and Accelerated Surface Post outside Europe. Note to subscribers. Regrettably publication of the four issues has still not reverted to the usual quarterly dates. The cause of this is a persisting shortage of articles (the production problems of recent years have been largely overcome) but the setting-up of an Editorial Board should result in an increase in the commissioning of reviews in 1982. 0Copyright reserved by The Royal Society of Chemistry 1982 ISSN 0306412 Published by The Royal Society of Chemistry, Burlington House, London, W1V OBN Printed in England by Eyre & Spottiswoode Ltd, Thanet Press, Margate.

ISSN:0306-0012    

DOI:10.1039/CS98110FP007

出版商:RSC    

年代:1981

数据来源: RSC

ISSN:0306-0012    

DOI:10.1039/CS98110FX013

出版商:RSC    

年代:1981

数据来源: RSC

摘要:

Chemical Society Reviews Vol 10 No 4 1981 HAWORTH MEMORIAL LECTURE Structural Studies of Polysaccharides By B. Lindberg Methyl Group Removal in Steroid Biosynthesis By H. L. Holland 435 CENTENARY LECTURE Metal Clusters in Biology: Quest for a Synthetic Representation of the Catalytic Site of Nitrogenase By R.H. Holm 455 Physical Chemistry of Biological Morphogenesis By L. G. Harrison 49 I 1981 Indexes 529 The Royal Society of Chemistry London

ISSN:0306-0012    

DOI:10.1039/CS98110BX015

出版商:RSC    

年代:1981

数据来源: RSC

摘要:

HAWORTH MEMORIAL LECTURE* Structural Studies of Polysaccharides By B. Lindberg DEPARTMENT OF ORGANIC CHEMISTRY, ARRHENIUS LABORATORY, UNIVERSITY OF STOCKHOLM, S-106 91 STOCKHOLM, SWEDEN 1 Introduction Haworth and his co-workers laid the foundation for polysaccharide chemistry, a branch of carbohydrate chemistry that hardly existed before the Haworth era. Few subjects could therefore be more suitable for a Haworth memorial lecture than polysaccharide chemistry. PoIysaccharides are biopolymers composed of monosaccharide residues and, for several of them, non-carbohydrate substituents. They fulfil different functions in Nature, as construction materials, reserve nutrition, thickeners, and lubricants. Some microbial polysaccharides stimulate the production of antibiotic substances in plants, others show antitumour activity. Some poly- saccharides are antigenic and are responsible for immunological reactions.Structural studies of polysaccharides are needed in order to correlate their structures with their biological and physical properties. Such studies should involve the determination of components, linkages, sequences, anomeric configurations, and conformation. There is a vast number of immunologically active, bacterial polysaccharides, both cell-wall polysaccharides and extracellular polysaccharides. In the late 60s we took up studies of these polysaccharides. We felt, however, that in order to cover even a small part of this field, the methods in structural polysaccharide chemistry had to be improved so that they became more accurate and much faster.They also needed to be scaled down so that milligram quantities, rather than gram quantities, would suffice for a structural determination. This was obviously an opportune time for initiating such studies. The combination gas liquid chromatography-mass spectrometry (g.1.c.-mas.) was better than anything we had before for qualitative and quantitative analysis of complex mixtures, and commercial instruments for g.1.c.-m.s. had become available. Somewhat later, nuclear magnetic resonance (n.m.r.) spectrometers operating in the Fourier transform mode became available and lH and I3C n.m.r. rapidly became valuable tools in structural studies of polysaccharides. In the following paper I shall discuss some aspects of structural polysaccharide chemistry.The examples will be taken from our studies of bacterial poly- saccharides. These polysaccharides have regular structures and are composed of *+Delivered at a RSC Meeting on 19 April 1981 at Heriot-Watt University, Edinburgh. Structural Studies of Polysaccharides oligosaccharide repeating units, and it should therefore be possible to arrive at conclusive structures. Some other polysaccharides have less regular structures, and for these only average structures may be proposed. 2 Component Analysis Some 100 different monosaccharide components and 20 different non-sugar components have been found in polysaccharides, and these numbers are in-creasing. Most sugars and other components are obtained as monomers on acidic hydrolysis, and the qualitative and quantitative analysis is preferably performed by g.1.c.-m.s.of suitable dervatives. The sugars are generally trans- formed into the acetylated alditols or aldononitriles. Stereoisomers give similar mass spectra, but for a known sugar the relative retention time gives further information, which permits identification, except for the absolute configuration. For a new sugar, the mass spectrum gives information on the class to which it belongs. Thus, two new sugars, from a Yersinia pseudotuberculosis lipopoly- saccharide1 and from acell-wall antigen of Eubacreriumsaburreum,2 were identified as 6-deoxyheptoses from the mass spectra of their [l-2H]alditol acetates (I ).3 The primary fragments on electron impact, formed by fission between two acetoxylated carbon atoms, are indicated in struture (1).Fission at the methylene group is insignificant. The sugar from the Y. pseudotuberculosis lipopoly- saccharide was, according to immunochemical arguments, assumed to have the D-manna-configuration, and this was proved by synthesis. The alditol from the E. saburreum sugar (2) on oxidation with less than 1 mole of periodate yielded 4-deoxy-~-eryfhro-pentose(3). As oxidation of the glycol groupings with C. G. Hellerquist, B. Lindberg, K. Samuelsson, and R. R. Brubaker, Acfa Cheni. Scand., 1972, 26, 1389. J. Hoffman, B. Lindberg, J. Lonngren, and T. Hofstad, Carbohydr. Rcs., 1976, 47, 261. N. K. Kochetkov and 0.S. Chizhov, Adv. Curhohydr. Chcm., 1966, 21, 39. Lindberg CHzOH CH20H I IHQCH CHO +I HCOH CHO 104-I 1 HCOH -HCOHL IH OH HCOH I I HCH HCH I I CHzOH CHzOH trans-hydroxyls in alditols occurs most readily,4 the sugar was assumed to have the altro-configuration. It was also identical with 6-deoxy-~-altro-heptose, prepared by an unambiguous route. Uronic acid residues, e.g. (4), are preferably carboxy-reduced before the sugar analysis. The reduction is performed by treatment first with a water-soluble carbodi-imide and then with sodium borohydride.5 When the carboxy-reduction is performed with borodeuteride, the dideuteriated alditol deriving from the uronic acid (5) is easily distinguished from the non-deuteriated analogue, obtained from the corresponding neutral sugar.CHzOH I HCOH CO2H Ho<L+ HO C2H20H -1 HOCH 1 HCOH HO ‘0H I HCOH I C2 OH An acidic sugar from the Klebsiella type 37 capsular polysaccharide was assumed to be a uronic acid. When analysed as outlined above, however, it gave J. C. P. Schwarz, J. Chem. SOC.,1957, 276. R. L. Taylor, .I.E. Shively, and H. E. Conrad. Methods Curhohydr. Chern., 1976, 7, 149. 41I Structural Studies of’ Polysaccharides a mass spectrum that was difficult to interpret. The mass spectrum of the alditol acetate (6) obtained on methylat ion analyses of the carboxy-reduced poly- CHZHOAc I HCOMe 118 i 267 MeOCH 162 338, t /---I-223 HC-0-CH ,Me ,0”47 y~I ,\ &H20Me HCOAc t47 C2H20Me saccharide, however, gave valuable structural information.Analysis of this spec- trum indicated that the sugar was a hexuronic acid, etherified at 0-4with lactic acid. Further studies demonstrated that the sugar was 4-O-[(S)-l-carboxyethyl]-D-glucuronic acid (7).6 U CO2H HO2C HO OH An artefact (Sa) was observed in the sugar analysis of the capsular poly- saccharide from Streptococcus pneumoniae type l2F.7 The mass spectrum of the acetylated component (86) showed, inter aka, that it was cyclic and contained one nitrogen atom and five acetyl groups. It was identified as the fully acetylated 1,5-dideoxy-1,5-imino-~-mannitoland is formed from D-mannosaminuronic acid via its lactone (9).On treatment of (9) with borohydride, reduction at C-1 probably occurs first. The aldehyde group formed on partial reduction at C-6 then reacts with the amino-group and the intermediate pyranoside, with nitrogen in the ring, is reduced to 8a. Previously, sugar and non-sugar components that were degraded during the hydrolysis of a polysaccharide might have been overlooked. Today, when B. Lindberg, B. Lindqvist, J. Lonngren, and W. Nirnmich, Curbohydr. Res., 1976, 49, 41 1. K. Leontein, B. Lindberg, and J. Lonngren, Can. J. Chem., 1981, 59, 2081. 412 Lindberg 0RCHzOR (80, R = H; b, R = Ac) (9) n.m.r. is used as a matter of routine, this should not happen. A good example is the Vibriu chulerae 0-antigen, which on acid hydrolysis yielded only small amounts of sugars.* The 13Cn.m.r.spectrum (Figure 1) showed, however, that w I 1 I I I I I 1 180 160 140 120 100 80 60 4 20 Figure 1 The 13Cn.m.r. spectrum of the Vibrio cholerae 0-antigen it had a simple structure and was composed of CIOrepeating units. Further studies, using n.m.r. and different degradations, showed that the sugar component in the 0-antigen is 4-amino-4-deoxy-~-mannose,N-acylated with S-2,4-di- hydroxybutanoic acid (10).The free amino-sugar is decomposed during hydro- lysis with acid. It could, however, be isolated as the N-acyl derivative by treat- ment with anhydrous hydrogen fluoride, followed by hydrolysis with acid under mild conditions. This treatment, during which glycosidic linkages are cleaved but amide linkages remain intact, has proved to be of general value in studies of polysaccharides containing N-acylamino-sugars.Some sugars occur both as the D-and the L-form in Nature, and determina- L. Kenne, B. Lindberg, P. Unger, T. Holme, and J. Holmgren, Curbohydr. Res., 1979,68 C14. 413 Structural Studies of Polysuccharides tion of their absolute configurations is an essential part of the structural studies. We have devised a simple micro-method for doing this, which involves glyco- sidation with a chiral alcohol [e.g. ( +)-octan-2-01], acetylation, and ~.I.C.~ The products from a D-and an L-sugar are diastereomeric and readily separated on a good column. Thus, the galactan from the snail Helix pomatiu contains both D-and L-galactose, which was confirmed by this method (Figure 2).D D h Li D I 30 60 90 (mind Figure 2 G.1.c. separation of acetylated (+ )-2-octyl galactosides obtained from the hydrolysate of the Helix pomatia galactan (Reproduced by permission from Carbohydr. Res., 1978,62, 361.) K. Leontein. B. Lindberg, and J. Lonngren, Curhohydr. Res., 1978, 62, 359. Lindberg Several bacterial polysaccharides contain cyclic acetals of pyruvic acid. Acetals linked to 0-4 and 0-6 in hexopyranosides are the most common, but acetals linked to vicinal positions have also been observed. The absolute con- figuration at the acetalic carbon had only. been determined for some poly- saccharides, using degradation methods. We therefore synthesized model com- pounds, representative of the most common types, and showed that the isomeric acetals, e.g.(lla) and (1 lb), gave typical n.m.r. spectra. lo Thus, in l3C n.m.r. the equatorial methyl group in (1 la) (the S-acetal) gives a signal at 8 25.5, while the Corresponding signal for (llb) (the R-acetal) occurs at 8 17.7. This large R' OH (110, R = Me and R' = C0,H; h, R = CO,H and R' = Me) difference .allows assignment of the absolute configurations of pyruvic acid acetals with similar stereochemistry in polysaccharides.1l 3 Methylation Analysis Methylation analysis, developed by Haworth and his co-workers, is still the most important method in structural carbohydrate chemistry. It involves methylation of all free hydroxy-groups in the polysaccharides and hydrolysis of the methylated polysaccharide' to a mixture of partially methylated mono-saccharides.The free hydroxy-groups in these mark the positions at which the corresponding sugar residues were substituted in the polysaccharide. Qualitative and quantitative analysis of this mixture therefore gives information on how the different sugar residues are linked. It does not, however, give information on sequences or on anomeric configurations, Even if the principle is the same, the technique has been changed. Previously, several methylation steps were necessary in order to achieve complete methyla- tio:i. On treatment of the polysaccharide, in dimethyl sulphoxide, with sodium methylsulphinylmethanide and subsequently with methyl iodide, as devised by Hakomori,12 complete methylation is now obtained in one step. This method has therefore more or less replaced all other methylation methods. Methylation with methyl trifluoromethane sulphonate in trimethyl phosphate, and with 2,6-di-(t-butyl)-pyridine as proton scavanger, is an alternative when the poly- saccharide contains alkali-labile substituents which should be preserved.13 lo P.J. Garegg, B. Lindberg, and I. Kvarnstrom, Carhohvdr. Res., 1979, 77,71. l1 P. J. Garegg, P.-E. Jansson, B. Lindberg, F. Lindh, J. Lonngren, 1. Kvarnstrom, and W. Nimmich, Carbohydr. Rcs., 1980, 78, 127. S. Hakomori, J. Biochem. (Tokyo), 1964, 55, 205. l3 P. Prehm, Carhokvtlr. Res., 1980, 78,372. 415 Structural Studies of Polysaccharides Haworth and his co-workers separated the methylated sugars, as their methyl glycosides, by distillation and identified them as crystalline derivatives.Consider- able effort and skill was devoted to the synthesis and characterization of all possible methyl ethers of the common monosaccharides. Later, the fractionation was performed by partition chromatography on paper or cellulose columns, carbon column chromatography, or thin-layer chromatography, but the necessity of preparing crystalline derivatives remained. We therefore developed a tech- nique by which the sugars, as their alditol acetates, were analysed by g.l.c.-m.s.l4 The methylation pattern of a component is evident from its mass spectrum. Stereoisomers give almost identical mass spectra but it has always been possible to find columns on which they are separable.All components in a mixture may therefore be identified by g.1.c.-m.s., provided that the sugar composition of the polysaccharide is known. Mass spectra and relative retention times of partially methylated alditol acetates have been compiled.15 The interpretation of the mass spectra is simple. Primary fragments are formed by fission of the alditol chain. When a component contains vicinal meth- oxylated carbons (12), fission between these is preferrzd and either fragment may carry the positive charge. When a methoxylated carbon has acetoxylated neighbours (13). fission between the methoxylated and an acetoxylated carbon R1 R1 R1 1 I Hq-OMe HL-me -e-HC=bMe + -e-HC=OMe HL-OMe _____+ He-OMe I I I R2 R2 R2 R1 R' l I HC-OMe -e-HC=OMe I HC-OAC H&-OAC I t R2 R2 becomes significant and the fragment with the methoxylated carbon carries the positive charge.When a component contains methoxy-groups, fragmentation between acetoxylated carbon atoms becomes insignificant. The primary fragments give secondary fragments, and the formation of these has been studied in detail, using specifically deuteriated derivatives. The most important reactions giving secondary fragments are single or consecutive l4 H. Bjorndal, C. G. Hellerquist, B. Lindberg, and S. Svensson, Angew. Chern., Internat. Ed. Engl., 1970, 9, 610. l5 P.-E. Jansson, L. Kenne, H. Liedgren, B. Lindberg, and J.Lonngren, Chem. Commun. (Stockholm Univ.), 1976, 8. 416 Lindberg eliminations of methanol, acetic acid, and ketene. Eliminations of methanol, ketene, and acetic acid from the two primary fragments of m/z 161 are illustrated in Scheme 1. CHzOAc CH2I HCOMe -*‘OH 80MeI I HC=f)Me HC=OMe -I-m/z 161 m/z 101 + + +HC=OMe HY= OMe H$? OMe HY=&MeICH -AcOH HCOAC -MeOH ~OAC-CH,CO C=O II t-IHCOMe CH20Me -__+ CH2 ------+ICH3 m/z 101 m/z 161 m/z 129 m/z 87 Scheme 1 Acetamido-sugars become, to a considerable extent, N-methylated during the Hakomori methylation. The fission of the derived alditol acetates is governed by the N-methylacetamido-group. Thus, for alditol acetates derived from 2-acetamido-2-deoxysugar residues [e.g.(14)] the primary fragment m/z 158 and the two secondary fragments m/z 116 and m/z 98 predominate. Other fragments are, however, strong enough to admit the identification of the methyla- tion pattern. CHzOAc I 158” 98HC-N HMe -----I ‘Ac -1 16 -1---------I233 MeOC-HIHC-OACI HC-OAC ------I ----‘45 CH2OMe (14) As discussed above, deuterium may be introduced at C-1 in order to get unsymmetrical derivatives. Other modifications in the procedure, such as dideuteriation of C-6, trideuteriomethylation or ethylation give derivatives, the fragmentations of which are quite analogous to those discussed above. As the methylation analysis has become so simple, it is also used in connection with different specific degradations of polysaccharides, performed in order to deter- mine sequences of the sugar residues (see below).417 Structural Studies of Polysaccharides Table 1 Methylation analysis of varianose Methylated Sugar Mole % Aa Bb 2,3,4,6-GlcC 14 19 2,3,4,6-Man -2 2,3,5,6-Gal --5 3,4,6-Man -6 3,5,6-Gal -20 2,3,6-Gal Major 28 2,3,5-G a1 -4 2,4-Man -2 3,5-Gal -14 Tri-OMe-Hex,n.i.d 14 -a Haworth et a/., 1935. b Jansson et a/., 1980. 2,3,4,6-Glc=2,3,4,6-tetra-O-rnethyl-~-glucose, etc. d not identified. In 1935, Haworth, Raistrick, and Stacey reported on the methylation analysis of the fungal polysaccharide varianose.'6 They methylated 50 g of polysaccharide and identified two main components as crystalline derivatives (Table 1).One of these components was 2,3,4,6-tetra-O-methyl-~-glucose,and this was the first accurate estimation of end groups in a polysaccharide by methylation analysis. In 1980 we repeated this analysis, starting from 1 mg of poly~accharide.1~ The analysis was completed in two days and four major and five minor components were identified. Another difference is that while the former analysis required considerable experimental skill, the latter mainly required access to some rather advanced equipment. 4 Sequence Analysis Different degradation techniques are used for sequence analysis of polysac- charides. There is no standard method but each polysaccharide presents its own problems. The oldest method is partial acid-hydrolysis and analysis of the oligosaccharides formed.The most commonly used method is the Smith degra- dation, devised by Fred Smith, a former co-worker of Haworth. There are further methods based upon @-elimination and upon deamination of amino- sugar residues. The different methods have been sumrnarized.l8 A. Partial Hydrolysis with Acid.-Some polysaccharides contain linkages that are especially sensitive to acidic hydrolysis, the most common being the furanosidic linkages, e.g. that of the /b-galactofuranosyl group (1 5). Others contain linkages that are only slowly hydrolysed. Thus 2-amino-2-deoxy- glycosides are almost completely resistant, due to the inductive effect of the protonated amino-group [as in ( I6)]. Uronidic linkages in polysaccharides l6 W. N. Haworth, H.Raistrick, and M. Stacey, Biochem. J., 1935, 29, 2668. l7 P.-E. Jansson and B. Lindberg, Carbohydr. Res., 1980, 82, 97. l8 B. Lindberg, J. Lonngren, and S. Svensson, Adv. Carbohydr. Chem Biochcm.. 1975.31, 185. 418 Lindberg [as in (1 7)] are also more resistant to acid hydrolysis than most other glycosidic / CHzOH H linkages. Polysaccharides containing linkages of either type and also 'normal' glycosidic linkages therefore give good yields of oligosaccharides on partial hydrolysis. For other polysaccharides the yields of individual oligosaccharides on partial hydrolysis are low, but isolation and indentification of oligosaccharides is nevertheless a common method in structural polysaccharide chemistry. An alternative to hydrolysis is acetolysis, and the relative rates by which different glycosidic linkages are cleaved differ for the two methods, Pyranosidic (1 -6)-linkages are thus cleaved more readily than other types of pyranosidic linkages on acetolysis. Acetolysis has been a valuable tool in structural studies of fungal a-D-mannans (Scheme 2, in which the mannosyl residues are assumed to be fully acetylated).6)a-~-Manp(l-+6)a-~-Manp(1--+ r' f 1 1 a-D-Manp a-D-Manp ;1a-D-Manp H+ .Ac,O,AcOH D-Man D-Man 7 11 1 a-D-Manp a-D-Manpr" 1 a-D-Manp Scheme 2 The relative rates of cleavage of different types of glycosidic linkages also seems t.:, differ for acidic hydrolysis and treatment with liquid hydrogen fluoride, but the practical consequences of this remain to be explored.' Structural Studies of Polysaccharides If the polysaccharide is methylated before acidic hydrolysis and the oligo- saccharides are reduced and realkylated, using trideuteriomethyl or ethyl iodide, more structural information is obtained.Thus, the isolation of the alkylated disaccharide alditol(l8) demonstrates that the polysaccharide contains the disaccharide element (19), but also that the non-reducing galactosyl group in (19) was linked to the 3-position in the polysaccharide, that the reducing residue was furanosidic, and that both residues were chain residues and not I2H3COCH IHCOMe ICHzOMe branching residues.lg The technique has been further developed by Albersheim and his co-workers,20 who separate the methylated, reduced, and re-ethylated oligosaccharides formed by h.p.1 .c.and characterize them. In favourable cases, when the relative rates of hydrolysis for the different types of glycosidic linkages involved do not differ too much, the complete polysaccharide structure may be determined by this method. B. Smith Degradation.-Substances containing vicinal hydroxy-groups, such as (20), are oxidized by periodate with the formation of carbonyl compounds, the simplest being formaldehyde and formic acid. Several of the methods that have been used in structural polysaccharide chemistry are based upon periodate oxidation, the most important being the specific degradation devised by F. Smith.21 In this degradation, the periodate-oxidized polysaccharide is reduced with borohydride to a ‘polyalcohol’.The acetal linkages in the modified, non- lS B. Lindberg, J. Lonngren, and W. Nimmich, Curbohydr. Res., 1972, 23, 47. 2o B. S. Valent, A. G. Darvill, M. McNeil, B. K. Robertsen, and P. Albersheim, Curbohydr. Res., 1980, 79, 165. 21 I. J. Goldstein, G. W. Hay, B. A. Lewis, and F. Smith, Methods Curbohydr. Chem., 1965, 5, 361. 420 Lindberg R RIHCOH kHO 2 104-+IHCOH HCOzHI + CHzOH HCHO HO CHzOH CHzOH 0 -<HzL CHzOH CHzOH H', Hz0 HO CH20H CHzOH I HOCH %'LO- THI HO OH CHzOH 421 Structural Studies of Polysaccharides cyclic residues are much more sensitive to acid hydrolysis than the glycosidic linkages of the intact sugar residues, and can be selectively cleaved under mild conditions.Characterization of the product, which may be polymeric or consist of low-molecular weight glycosides, often gives considerable structural informa- tion. Smith degradation of a galactoglucan containing the structural element (21) thus gives 2-~-/i?-~-ga~actopyranosyb~-erythritol(23) via the polyalcohol (22). The identification of this substance demonstrates that the polysaccharide contains /?-D-galactopyranosyl residues resistant to periodate oxidation (probably because they are substituted in the 3-position) and linked to 0-4 of D-glucopyranosyl residues. The glycoside (23) was actually isolated after Smith degradation of the capsular polysaccharide, composed of octasaccharide repeating units (24), from Rhizobium meliloti.zz Four of the sugar residues in this unit are parts of the linear backbone.From the isolation of (23), however, it could not be decided if the /?-D-galactopyranosyl residue in this backbone was linked to 0-4 of the branch- ing p-D-glucopyranosyl residue or to 0-4 of one of the chain fbglucopyranosyl residues. In order to decide between those possibilities, the ‘polyalcohol’ was methylated before the mild hydrolysis and the product ethylated. In the glucoside (25), the ethyl groups mark the positions that were substituted in the original CHzOEt Me0 CHzOMe I EtOCH -I CH EtOm0OMe I CHzOMe polysaccharide, namely the 3-position in the p-D-galactopyranosyl residue and the 5-and 6-positions of the /?-D-glucopyranosyl residue (which become the 3-and 4-positions of the D-erythritol residue). This modified Smith degradation ** P.-E.Jansson, L. Kenne, B. Lindberg, H. Ljunggren, J. Lonngren, U. Ruden, and S. Svensson, J. Am. Chem. SOC.,1977,99, 3812. Lindberg therefore gives further information and demonstrates that the p-D-galactopyrano- syl residue is linked to the branching p-D-glucopyranosyl residue., as in (24). In the conventional Smith degradation, acetal migration with the formation of cyclic acetals often becomes a side reaction,21 but this complication is avoided in the modified procedure discussed above. The Smith degradation is especially suitable for polysaccharides containing 2-acetamido-2-deoxyhexopyranosylresidues, as such residues, when substituted in either the 3- or the 4-position, are not oxidized by periodate.In studies of the capsular polysaccharide from Streptococcus pneumoniae type 1 2F,7 which is composed of hexasaccharide repeating units (26), the side chains were eliminated --+ 4)a-~-FucpNAc-( 1-+3)-/3-~-GalpNAc-( 1+4)-P-~-ManpNAcA-( 13 i 31 1 a-D-Galp a-D-G ICp-( I+~)-u-D-G ICP by Smith degradation, and a linear polymer was obtained. Analysis of this product demonstrated that the 3-positions of the 2-acetamido-2-deoxy-a-~-fucopyranosyl and 2-acetamido-2-deoxy-~-~-mannopyranosyluronicacid resi- dues had become exposed. The side chains were consequently linked to these positions. The hydroxyls at C-3 and C-4 in the a-D-galactopyranosyl group in (26) are the only cis-oriented vicinal hydroxyls in this polysaccharide.The a-D-galactosyl group could therefore be selectively oxidized by periodate, and on Smith degradation of this material, the 3-position of the 2-acetamido-2-deoxy- 'a-D-fucopyranosyl residue became exposed, demonstrating that the a-D-galactopyranosyl group was linked to this position. The possibility of performing similar periodate oxidations increases the usefulness of the Smith degradation. C./%Elimination Reactions.-(i) Uronic Acid Degradation. Several polysaccharides contain uronic acid residues. During the Hakomori methylation of such poly- saccharides, the carboxy-groups are simultaneously esterified. On treatment of the methylated product [e.g.(27)]with base, p-elimination occurs, and we have used this reaction in structural studies of several acidic polysaccharides.18 It was first believed that the primary product (28), which contains a vinyl ether linkage, should be sensitive to acid and that the aglycon should be released during hydrolysis with acid under mild conditions. Upon further reaction of the derived 4-deoxy-5-ulosuronide, with formation of a furan derivative, the sub- stituents at C-2 and C-3 should also be released. The unsaturated uronide (28) is, however, remarkably stable to a~id.~3 The substituents may nevertheless be split off, but during the alkaline treatment, with the formation of a 4-pyrone derivati~e.~~This is in agreement with the results of Aspinall and Rose11,25 23 K.Shimizu, Curbohydr. Res., 1981, 92, 219. 24 P. KOV~C,J. Hirsch, and V. Kovricik, Curbohydr. Res., 1977, 58, 327. z5 G. 0. Aspinall and K.G. Rosell, Carboh-vdr.Rm., 1977, 57, C23. Structural Studies of Polysaccharides COzMe CO2Me R%cL OR1 Base , &\OR1+R40H ~30 OR2 ~30 OR2 (28) (27) J who demonstrated that the glycosidic linkage may be cleaved during the treat- ment with base. Solutions of xanthan gum, a polysaccharide elaborated by Xanthomonas campestris, have unique physical properties and the polysaccharide is produced industrially.26 It is composed of pentasaccharide repeating units (30). In about half of these units, pyruvic acid is acetalically linked to the 4-and 6-positions of the terminal /3-D-mannopyranosyl group.In the structural studies of this polysaccharide,27 uronic acid degradation (Scheme 3) was an essential tool. The terminal and next-to-terminal sugars, D-mannose and D-glucuronic acid, were eliminated when the fully methylated polysaccharide was treated with base. In the polymeric residue, the hydroxy-group at C-2 in the second D-mannosyl residue had become exposed, as demonstrated by trideuteriomethylation and analysis of a hydrolysate. This result, in conjunction with the methylation analysis of the original polysaccharide, gave the structure of the trisaccharide side-chain in (30). 28 P. A. Sandford, Adv. Carbohydr. Chem., 1979, 36, 265. P.-E. Jansson, L. Kenne, and B. Lindberg, Carbohydr.Res., 1975, 45, 275. 424 Lindberg ‘OMe OH Scheme 3 The course of the uronic acid degradation becomes more complicated when the uronic acid residue is part of the main chain. For such examples, however, it may also give increased structural information.18 (ii) Oxidation-/%Elimination. There are different methods for preparing poly- saccharides in which all hydroxy-groups, except a limited number in defined positions, are methylated. One such method is the uronic acid degradation Structural Studies of Polysaccharides discussed above. These hydroxyls can be oxidized to carbonyl groups and the product subjected to /%elimination by treatment with base. The unsaturated sugars formed are sensitive to acid hydrolysis. This type of degradation was used in structural studies of xanthan gum (Scheme 4).27 The partially methylated polysaccharide obtained on uronic acid degradation (Scheme 3) was oxidized, CH2OMe Me0 Me0 OMe MeOH2C Me0 OMe CHzOMe AcidI Man\---70’ OMe CHzOMe Scheme 4 Lindberg using chlorine-dimethyl sulphoxide-triethylamine,28 treated with base and subsequently with acid under mild conditions.Analysis of the polymeric product showed that it consisted of methylated cellulose, in which every second glucosyl residue contained a free hydroxy-group in the 3-position. The trisaccharide side-chain in xanthan gum is consequently linked to this position. In structural studies of the capsular polysaccharide from Rhizobium meliloti, the sugar residues in the tetrasaccharide side-chains (24) were eliminated one after the other by this method, thereby establishing the sequence.22 D.Damination.-Several polysaccharides contain amino-sugars, which offer starting points for specific degradations. The amino-groups are most often acetylated, and have first to be deacetylated. This is generally done by treatment with base, e.g. aqueous barium hydroxide or hydrazine. We showed that sodium hydroxide in aqueous dimethylsulphoxide, containing thiophenol, is an efficient N-deacetylating agent.29 A different method is treatment with trifluoroacetic acid and trifluoroacetic anhydride, and hydrolysis of the resulting trifluoro- acetamides, which can be done under mild conditi~ns.~O Because of the inductive effect of the introduced 0-trifluoroacetyl groups, the glycosidic linkages are stable during the reaction conditions.Some polysaccharides are, however, degraded during attempted N-deacetylation by either method, and there is a need for milder, but still efficient methods for N-deacetylation. The deamination of amino-sugars and their glycosides is accompanied by rearrangement^.^^ Thus, deamination of a 2-amino-2-deoxy-~-glucopyranoside, via thediazonium ion (31), was known togive 2,5-anhydro-~-mannose(32,R2=H), with release of the aglycon. We showed that part of the reaction ( N 20 "/d) takes another course, with formation of a 2-deoxy-2-C-formyl-~-ribo-hexoside(33).32 A substituent at C-3 is eliminated during this reaction, which explained some unexpected results in the literature.The 0-specific polysaccharide from Shigella jlexneri, variant Y, is composed of tetrasaccharide repeating units (34) and deamination was used in the structural studies of this polysaccharide. 33 The N-deacetylated polysaccharide was deami- nated by treatment with nitrous acid, yielding tetrasaccharide (35) as the main product. Methylation analysis of the reduced tetrasaccharide showed that one of the 2-substituted a-L-rhamnopyranosyl residues in the original polysaccharide had become the terminal a-L-rhamnopyranosyl group in the tetrasaccharide. The 2-acetamido-2-deoxy-~-glucoseis consequently linked to 0-2 of L-rhamnose. On treatment of tetrasaccharide (35) with base, the trisaccharide linked to 0-3 of the 2,5-anhydromannose residue was eliminated.Methylation analysis of the trisaccharide alditol(36) gave the sequence of the three a-L-rhamnopyranosyl residues in (34). 28 E. J. Corey and C. U. Kim, Tetrahedron Lett., 1973, 919. as C. Erbing, K. Granath, L. Kenne, and B. Lindberg, Carbohydr. Res., 1976, 47, C5. 30 B. Nilsson and S. Svensson, Curbohydr. Res., 1978, 62, 377. 31 J. M. Williams, Adv. Curbohydr. Chem., 1975, 31, 9. 33 C. Erbing,. B. Lindberg, and S. Svensson, Actu Chem. Scund., 1973, 27, 3699. 33 L. Kenne, B. Lindberg, K. Peterson, and E. Romanowska, Curbohydr. Re&.,1977,56, 363. 427 Structural Studies of Polysuccharides HOHzC + RlOH HOCHzOH / (32) HO CHO (33) +2)-a-L-Rhap-(1+2) -a-~-Rhap-(I+ 3)-a-~-Rhap-(1+ 3)- p-~-GlcpNAc-( 1--+ (34) a-~-Rhap-(1+2)-a-~-Rhap-( 1+3)-a-~-Rhap- ;> (35) ~-L-R to1hap-(1+2)-a-~-Rhap-(1+3)-~-rhamni (36) Deamination reactions have also been applied to polysaccharides containing other amino-sugars.N-Deacylation of a polysaccharide from Vibrio cholerae yielded a polymer composed of (1 -2)-linked 4-amino-4,6-dideoxy-a-~-mannopyranosyl residues (37).* The main products of deamination, formed vig the epoxonium ion (38), are the 6-deoxy-a-~-mannopyranosyl (39) and 6-deoxy-P-L-allofuranosyl(40)residues, as confirmed by sugar and methylation analyses of the product. This established the nature of the amino-sugar and its mode of linkage. Lindberg Me In two polysaccharides containing 2-acetamido-4-amino-2,4,6-trideoxy-u-galactopyranosyl residues (41) this sugar was degraded during acid hydrolysis H2N 0 /-o*NHAc and could therefore not be isolated, but was identified through its deamination prod~cts.~**~sAs the free amino-group is axial, the deamination follows a dif- ferent course from those discussed above. A direct attack by water upon the diazonium ion (42)gives a 2-acetamido-2,6-dideoxy-~-glucopyranosylresidue (43).Alternatively, hydride shifts of H-3 or H-5 gives 2-acetamido-2,4,6- trideoxyhexos-3- or 5-ulose residues [(44)and (45)],with simultaneous cleavage of the linkage at 0-3 or 0-1, respectively. Sugar analysis of the deaminated and 34 B. Lindberg, B. Lindqvist, J. Lonngren, and D.A. Powell, Carbohydr. Res., 1980, 78, 111. 35 L. Kenne, B. Lindberg, K. Peterson, E. Katzenellenbogen, and E. Romanowska, Carbohydr. Res., 1980, 78, 119. 429 Structural Studies of Polysaccharides Me +N2Me t /-o NHAc /-+ NHAc 1 Me 0 reduced product gave 2-acetamido-2,6-dideoxy-~-glucoseand a mixture of 2-acetamido-2,4,6-trideoxyhexoses,which established the structure of the new arn ino-s ugar . 5 Anomeric Configuration Investigation of oligosaccharides obtained by graded acid hydrolysis, using either enzymic methods or determination of their optical rotations, was until recently the most important method for determining the anomeric configurations of the sugar residues in a polysaccharide. Now lH and 13C n.m.r.of the poly-saccharide and its degradation products has become the method of choice.36 It is often sufficient to determine chemical shifts and JH-1.H-2. For pyranosides 36 D. R. Bundle and R. U. Lemieux, A4erhod.s Carbohydr. Chem., 1976, 7,79. Lindberg JC-1,H-l also has considerable diagnostic value, being -160 Hz when H-1 is axial and N 170 Hz when it is eq~atorial.~~ Angyal and James38 showed that a fully acetylated glycopyranoside with an equatorial aglycon, e.g. (46), is oxidized by chromium trioxide in acetic acid, yielding the ester of a glyc-5-ulosonic acid (47). The corresponding glycopyrano- side with an axial aglycon (48) is not reactive. We have adopted this method CH20Ac CHzOAcAcosoR-C~O,-ACOH A C O a ,(OR AcO OAc AcO AcO 0 CHzOAc CrOB-AcOH AcO \ 7 NO REACTION for oligo- and polysac~harides.~~ Oxidation of the fully acetylated product, using myo-inositol hexa-acetate as an internal standard, and sugar analysis of the product, reveals the anomeric configurations of the different sugar residues. It is essential that the acetylation is exhaustive and also that there is considerable difference in energy between the two alternative chair forms of the sugar residues involved.The method cannot be used for furanosides. Chromium trioxide oxidation may also, in favourable cases, be used for sequence analysis. The capsular polysaccharide from Klebsiella type 37 is composed of tetrasaccharide repeating units (49),40 in which A is the lactic acid +4)-P-D-Glcp-( 1+3)-/3-~-Galp-( l+ 4 t 1 /3-Ap-( 1+6)-a-~-Glcp 37 K.Bock and C. Pedersen, J. Chem. SOC.,Perkin Trans. 2, 1974, 293. 38 S. J. Angyal and K. James, Curbohydr. Res., 1970, 12, 147. 39 J. Hocman, B. Lindberg, and S. Svensson, Acta Chem. Scand., 1972, 26, 661. roB. Lindberg. B. Lindqvist, J. Lonngren, and W. Nimmich, Curbohydr. Res., 1977, 58, 443. 431 Structural Studies of Polysaccharides ether of D-glucuronic acid (7) previously discussed. At the final stage of the structural studies, the only unknown feature was which of the two D-glucopyrano- syl residues was a-linked. In order to determine this, the acetylated polysaccharide was allowed to react with chromium trioxide in acetic acid. On treatment of the product (50) with sodium borodeuteride the carbonyl and the aldonate ester groups were reduced.The deacetylated product contained a mixture of two disaccharide alditols (51), of which the D-galactitol derivative predominated. I C2HzOH CHzOH I HCOH AcQ*Q AcO I _II_+ CHzOAc Ho I I HOCH 0-CH I C2H,0HI CHzOH 0 The structure, determined by g.1.c.-m.s. of the permethylated material and confirmed by methylation analysis, demonstrated that the a-D-glucopyranosyl residue is actually linked to 0-4of the /h-gaIactopyranosyl residue, as in (49). 6 Conformation Analysis In studies aiming at a better understanding of the biological and physical properties of a polysaccharide, determination of its structure is only the first step.The next step should be the determination of its conformation in solution. This has been rather difficult and has only been done for a limited number of polysaccharides; the pioneers in this field being E. D. T. Atkins and D. A. Rees. Recently, however, Lemieux et al.41 have used simple, hard-sphere calculations, with correction for the exoanomeric effect, for the determination of oligo- saccharide conformations. They have also demonstrated, using high-field 1H n.m.r. studies, that there is a good agreement between the calculated structures and those actually present in solution. Using their program, we have determined 41 R. U. Lemieux, K. Bock, L. T. J. Delbaere, S. Koto, and V. S. Rao, Can. J. Chem., 1980, 58, 631. 432 Lindberg Figure 3 Conformation of the capsular polysaccharide from Haemophilus influenzae type e the conformation of some bacterial polysaccharides.One of these is the capsular polysaccharide from Haemophilus infhenzae type e, composed of disaccharide repeating units (52),42 which has the conformation depicted in Figure 3.43 It is my firm belief that, in the near future, determination of conformation will be included in all studies of biologically important polysaccharides and oligosaccharidesa 7 Conclusion There has been considerable development in structural polysaccharide chemistry and even rather complicated structures can now be determined with moderate effort. Before 1970 only about 10 complete and wellfounded structures of oligo-saccharide repeating units in bacterial polysaccharides had been published.As a result of the improved methods, this number has now increased to about 200. There are also good methods for the determination of polysaccharide conforma- tions. As less time has to be devoted to the structural studies one may predict that polysaccharide chemists will pass on to other problems. The most fascinating of these is probably the specific interaction between polysaccharides 42 P. Branefors-Helander, L. Kenne, B. Lindberg, K. Petersson, and P. Unger, Curboh>&. Res., 1981, 88, 77. 43 P.-E. Jansson and L. Kenne, Personal communic.ation. Structural Studies of Polysaccharides (or oligosaccharide chains in glycoconjugates) and proteins, such as antibodies, lectines, and enzymes.It is a great honour to present the Haworth Memorial Lecture and I am both glad and grateful for this privilege. I am, however, also well aware that an organic chemist does not stand alone but depends heavily upon his co-workers. I have been especially fortunate and have had a number of skilled and enthusiastic co-workers. Several of these, from Great Britain, Canada, and the U.S.A. are, scientifically, grandchildren or great grandchildren of Sir Norman Haworth and have brought with them something of the spirit of the Haworth carbohydrate school to our laboratory.

ISSN:0306-0012    

DOI:10.1039/CS9811000409

出版商:RSC    

年代:1981

数据来源: RSC

摘要:

Methyl Group Removal in Steroid Biosynthesis By H. L. Holland DEPARTMENT OF CHEMISTRY, BROCK UNIVERSITY, ST. CATHARINES, ONTARIO, L2S 3A1, CANADA 1 Introduction A. Steroid Biosynthesis.-The biosynthesis of the physiologically active steroid hormones in mammals can be conveniently divided into two processes, as sum- marized in Scheme 1. Firstly, an anabolic (synthetic) conversion of acetate via mevalonic acid and squalene into the c30 molecule lanosterol (l), followed by a catabolic (degradative) conversion of lanosterol via cholesterol [&, (2)] into the steroid hormones such as cortisol [GI,(3)], testosterone [Clg, (4)], and oestradiol [ClS, (91.The route by which acetate is converted into lanosterol was extensively investi- gated and described some time ago,1v2 and many of the mechanistic features of this conversion have now been el~cidated.~ The transformation of lanosterol into the c27, C21, C19, and CISsteroids involves a complex series of enzymic reactions, many of which are imperfectly understood at the present time.The changes brought about are: (i) the saturation of C=C bonds and introduction of new sites of unsaturation; (ii) progressive cleavage of the C-17 side-chain; (iii) the removal of the methyl groups at C-4 (C-30 and C-31) and C-14 (C-32), and for the CISsteroids at C-10 ((2-19); (iv) the introduction of hydroxy-groups at specific sites in the steroid nucleus; and (v) keto-alcohol interconversions. The biosynthetic interconversions which are possible among the C21, C19, and CIS steroids are manif~ld,~ and the above list is not intended to imply a specific sequence of events.However, it is known that the gross skeletal changes occur in the following order: lanosterol + cholesterol -+ pregnanes (GI)-+ androstanes (C19) -+ oestranes (CIS).These changes are brought about by both side-chain cleavage (c27 -, C21+ C19) and by loss of methyl groups (c30 + c27 and C19 -+ CIS). This review is concerned with the removal of the C-30-32 and C-19 methyl carbons, and will therefore be dealing with reactions that occur at both early (loss of C-30-C-32) and late (loss of C-19) stages of the overall pathway. The actual sequence of loss of C-30-C-32 is considered in more detail in Section 4 (below). R. B. Clayton, Q.Rev. Chem. Soc., 1965, 19, 168, 201. 1. D. Franz and G. J Schroepfer, Annu. Rev. Biochem., 1967, 36, 656. T. W. Goodwin, ‘Rodd’s Chemistry of Carbon Compounds’, 2nd Edn., ed. M. F. Ansell, Elsevier, Amster- dam, vol. 2, 1974, p. 237. D. E. Cane, Tetrahedron, 1980, 36, 1109. R. I. Dorfman and F. Ungar, ‘Metabolism of Steroid Hormones,’ Academic Press, New York, 1965. Methyl Group Removal in Steroid Biosynthesis CH3COz-+ (I) Lanosterol (3) Cortisol (2) Cholesterol Scheme 1 Biosynthesis of steroid hormones B. Mono-oxygenase Enzymes.-The cleavage of the C-17 side-chain and the removal of methyl groups during steroid biosynthesis are oxidative processes, catalysed by mono-o~ygenases.~ This group of enzymes catalyses the introduction of oxygen into an organic substrate according to Equation 1.R-H + NADPH + 02 +RdH + NADP+ + OH-(1) The key features of this conversion are: (i) the introduction of one atom of molecular oxygen into R-H, the other being incorporated into a molecule of water; (ii) the requirement of one equivalent of an NADPH co-factor; (iii) the apparent absence of any required activation of the C-H bond of the substrate; and (iv) the involvement (not shown in Equation 1) of a cytochrome co-factor. M.J. Coon and R. E. White, in ‘Metal Ion Activation of Dioxygen,’ ed. T. G. Spiro,Wiley, New York, 1980, p. 73. Holland 2 Removal of Methyl Groups from C-4 of Lanosterol Of the methyl-group removals in steroid biosynthesis, those from C-4 are perhaps the best understood. Pioneering investigation by Bloch and co-workers6 in the 1950's established that rat liver homogenates were able efficiently to convert lanosterol (1) into cholesterol (2), that the reaction required molecular oxygen and NADPH, and that the methyl carbons were released as carbon dioxide (but vide infra, Section 3).It stands as a tribute to the insight of this group that the pathway for the lanosterol-cholesterol conversion proposed by them in 1956 contained many of the features of the currently accepted route for C-4 demethyla- tion (Scheme 2). The latter was elucidated almost a decade ago, largely by the efforts of Gaylor, Sharpless, and Clayton and their co-~orkers.~-~l The route shown in Scheme 2 was deduced by the classical methods of isolation of intermediates, and incubation of these and other postulated intermediates with an enzymic system capable of performing the overall transformation, The isolated intermediates include 3~-hydroxy-4/3-methyl-4~-carboxylicacids (8),11-13 4-a-methyl-3-keto-steroids(1 1),14J5 a 3/3-hydroxy-4cu-methy1-steroid( 12),9 and a 3/3-hydroxy-4a-carboxylicacid (1 9.16 Efficient transformation into 4-demethyl- steroids has been observed with a 3/3-hydroxy-4~-methyl-4cu-carboxylicacid (8),15 a 4a-methyl-3-keto-steroid (1 1),17 3/3-hydroxy-4a-methyl-steroids (12),11914*16J7 a 3~-hydroxy-4a-hydroxymethyl-steroid( 13),8 a 319-hydroxy-4cu-formyl-steroid (14),18 and a 3/3-hydroxy-4a-carboxylicacid (1 9.15 Crucial evidence for the sequence of events in this pathway was provided first by Sharpless and co-workers,S who demonstrated that a 4fl-methyl-4a-hydroxy- methyl-steroid (6) was converted efficiently into cholestanol by a rat liver homo- genate, whereas the isomeric 4a-methyl-4~-hydroxymethyl-steroid(1 8) was not metabolized.This firmly established the initial site of oxidation as the C-4a methyl group (C-31). The next identified intermediate in the pathway, the carboxylic acid (€9,was obtained from incubations in which the enzyme co-factor NAD+ was absent.12 The significance of the 3-keto-intermediates (9) and (16) was deduced from a requirement of the overall conversion for the alcohol dehydro- genase co-factor NAD+,ll necessary for oxidation of the C-3p alcohol gro~p.~~?~~ J.A. Olsen, M. Lindberg, and K. Bloch, J. Bioi. Chem., 1957, 226, 941. 'I J. L. Gaylor and C. V. Delwiche, Steroids, 1964, 4, 207.* K. B. Sharpless, T. E. Synder, T. A. Spencer, K. K. Maheshwari, G. Guhn, and R. B. Clayton, J. Am. Chem. Soc., 1968,90,6874. R. Rahman, K. B. Sharpless, T. A. Spencer, and R. B. Clayton, J. Bid. Chem., 1970, 245, 2667. lo E. L. Ghisalberti, N. J. DeSouza, H. H. Rees, L. J. Goad, and T. W. Goodwin, Chem. Comm., 1969, 1403. 11 W. L. Miller and J. L. Gaylor, J. Biol. Chem., 1970, 245, 5375. l2 G. M. Hornby and G. S Boyd, Biochem. Biophys. Res. Commun., 1970, 40, 1452. 13 D. P. Bloxham and M. Akhtar, Biochem. J., 1971, 123, 275. l4 A. C. Swindell and J. L. Gaylor, J. Biol. Chem., 1968, 243, 5546.l5 A. D. Rahimtula and J. L. Gaylor, J. Biol. Chem., 1972, 247, 9. l6 W. L. Miller and J. L. Gaylor, J. Biol. Chem., 1970, 245, 5369. l7 K. B. Sharpless, T. E. Synder, T. A. Spencer, K. K. Maheshwari, J. A. Nelson, and R. B. Clayton, J. Am. Chem. SOC.,1969, 91, 3394. la D. R. Brady, R. D. Crowder, and W. Ja Hayes, J. Biol. Chem., 1980, 255, 10624. l9 N. J. Moir, W. L. Miller, and J. L. Gaylor, Biochem. Biophys. Res. Commun., 1968, 33, 916. 437 Methyl Group Removal in Steroid Biosynthesis H3C kHzOH HC kHO 30 31 (7) (1) (6) 0,\1NADPH H CH3 CH3 CHzOH (13) 0, NADPH1 V (17) Scheme 2 Removal of the methyl groups from C-4 of lanosterol Holland HO HOH2 CH3 The presence of a carbonyl group at C-3 serves not only to facilitate the loss of carbon dioxide from the resulting P-keto-acids (9) and (16),20 but also to effect, via enolization, the epimerization of the C-4P methyl group of (10) to the cu-p0sition.~5 It has been demonstrated that oxidative attack at the C-4 methyl groups occurs only from the a-side :a C-4P monomethyl-steroid such as ( 19)is not transformed by the enzymes of this path~ay.1~ A requirement for exogenous NADPH, and the observation14 that, in its absence, C-3 keto-steroids such as (1 1) are not transformed, led to the conclusion that the P-alcohol functionality at C-3 must be regenerated by an NADPH dependent reductase before oxidation of C-4 monomethyl-steroids can proceed.The absence of NAD+ leads to the accumulation of the carboxylic acid (1 9,in addition to (8),15 implicating a further oxidation of the C-3 alcohol to carbonyl before decarboxylation occurs to produce (17).The final step in the sequence, the reduction of the C-3 carbonyl of (17) to alcohol, also requires NADPH.l5 Several of the enzymes which catalyse reactions of Scheme 2 have been partially purified and characterized by Gaylor and co-workers, and data thus obtained have been of decisive value in the elucidation of the sequence of reactions portrayed in that Scheme. In spite of a requirement for NADPH and molecular oxygen, the oxidative enzymes in rat liver capable of demethylating 4,4-dimethyl- cholest-7-ene-3P-ol at C-4 do not require cytochrome P-450 or cytochrome b5,21 both of which are common mono-oxygenase co-factors:s the identities of the electron transfer components of the C-4 methyl-sterol oxidases are unknown at the present time.The alcohol dehydrogenase which catalyses oxidation at C-3 during demethylation has been partially purified and characterized.15 Indirect evidence suggests that this oxidation is rate limiting in the decarboxylation of (8) 4 (lo), and that the latter may in fact be a non-enzymic process at the pH (9.0) optimal for the activity of the dehydr~genase.l~-~~ 3 Removal of the C-14a Methyl Group In spite of having been subjected to the same intense effort as that expended upon demethylation at C-4, the C-14a demethylation reaction remains incompletely understood, It was originally proposed6 that the methyl carbon was liberated as carbon dioxide, but later work22 demonstrated that, in contrast to the losses from *O M.Lindberg, F. Gautschi, and K. Bloch, J. Bid. Chem., 1963, 238, 1661. 21 J. L. Gaylor and H. S. Mason, J. Biol. Chem., 1968, 243, 4966. 22 K. Alexander, M. Akhtar, R. B. Boar, J. F. McGhie, and D. H. R. Barton,J. Chrm. Soc., Chem. Commiin., 1972, 383. 439 Methyl Group RemoVal in Steroid Biosynthesis C-4, the loss of C-32 from the C-14a position occurs at the aldehyde oxidation level, resulting in the release of formic acid. This was subsequently oxidized to carbon dioxide by other enzymes present in the liver homogenates used.23 Also in contrast to C-4 demethylation, the removal of the C-14a methyl requires a cytochrome P-450 ~o-factor~~ in addition to NADPH and molecular oxygen, although it appears that the cytochrome P-450 may be necessary only for the initial oxidative step (-CH3 -+ --CHZOH).~~ It was suggested some time ago that loss of methyl from C-14 was accompanied by the lossof hydrogen from C-15/3,26 but it was later demonstrated unequivocally that the loss occurs from the C-15a position,27 the original assignments of stereo- chemistry of label having been in error. No loss of hydrogen from the C-16 position occurs during the conversion of lanosterol into cholesterol by rat liver homogenate.2B The possible role of a da(g)or d7double bond in C-14a demethylation has received much attention. The presence of unsaturation in a position P,y to the carbon that is lost led to the postulate29 that the activation so achieved may be instrumental in promoting the loss of C-32 as formic acid, as shown in Scheme 3.However, the observation^^^ that the steroid (21) is only poorly converted into cholesterol, and that conversion of the C-14a aldehyde (20) into cholesterol requires NADPH and oxygen, together with the observed loss of the C-15a hydrogen discussed above, have been cited30 as evidence against this proposal. An observation of significance with respect to the loss of hydrogen from C-15 is that the initial products of enzymic C-14a demethylation of c30steroids with a 47 (22) 0rd8(~) (1) double bonds are the correspondingd7J4- and dR@)J4-dienes, (23) and (24), re~pectively.~~ This led to the proposal of the mechanisms shown in Scheme 4,30 for which the following evidence was cited.Firstly, C-14a hydroxy- methyl intermediates of type (25) are dealkylated efficiently by rat liver prepara- tions, liberating C-32 as formic a~id30-3~ and the corresponding aldehyde (20) is similarly transformed.30J2 The liberation of formic acid from (25) and the production of C-14a demethyl-steroids was reported to be dependent upon the presence of NADPH and oxygen, no significant transformation occurring when this combination was replaced by NADP+, NAD+, or NADPH under nitrogen.30 a3 S. Trowbridge, Y. C. Lu, R. Shaw, J. Chan, and T. Spike, Fed. Proc., Fed. Am. SOC.Exp. Biol., 1975, 34, 560. ap G. F. Gibbons and K. A. Mitropoulos, Eur. J. Biochem., 1973, 40, 267.85 G. F. Gibbons, C. R. Pullinger, and K. A. Mitropoulos, Biochem. J., 1979, 183, 309. I6 L. Canonica, A. Fiecchi, M. Galli Kienle, A. Scala, G. Galli, E. Grossi Paoletti, and R. Paoletti, J. Am. Chem. SOC.,1968, 90, 3597. *' G. F. Gibbons, L. J. Goad, and T. W. Goodwin, Chem. Commun., 1968, 1458. ** M. Akhtar, A. D. Rahimtula, I. A. Watkinson, D. C. Wilton, and K. A. Munday, Eur. J. Biochem., 1969, 9, 107. L. Richards and .I.B. Hendrickson, 'The Biosynthesis of Sterols, Terpenes, and Aceto- genins,' Benjamin, New York, 1964. 30 M. Akhtar, K. Alexander, R. B. Boar, J. F. McGhie, and D. H. R. Barton, Biochem. J., 1978,169,449. 31 M. Akhtar, C. W. Freeman, D. C. Wilton, R. B. Boar, and D. B. Copsey, Bioorg. Chem., 1977, 6, 473.38 J. Fried, A. Dudowitz, and J. W. Brown, Biochom. Biophys. Res. Commun., 1968, 32, 568. Holfund \k NADPH 0, NADPH\1 d/ *' H L + HCOzH \I/ \@L Scheme 3 Loss of C-32 from lanosterol involving a AR@)or A' bond 441 Methyl Group Removal in Steroid Biosynthesis CHO (29) I I (22) (23) Scheme 4 Formation of dienes on removal of C-32 from C,, sterols* *Illustrated in detail only for a substrate. A similar mechanistic proposal applies to A' substrates, yielding dienes. Akhtar and Barton and their co-w~rkers~~ also established that the conversion of (25) into (20) required NADPH (and, by extension, oxygen). This conversion has been stated to occur in the presence of only NAD+ under nitrogen or helium,33 but the absence of endogenous NADPH and absorbed oxygen, which could support the reaction, was not established in this case.The work of Akhtar et al.30 which demonstrates the accumulation of the aldehyde (20) under con- ditions of limiting NADPH concentration suggests that NADPH (and, by in- ference, oxygen) may be required for further conversion of (20). The liberation of 33 A. E. Dudowitz and J. Fried, Fed. Proc., Fed. Am. Soc. Exp. Bid., 1969, 28, 665. Holland formic acid from the aldehyde (20) also appears to be dependent upon NADPH and ~xygen.~~~~~ However, a recent reporP4 states that the 4,4-demethyl-l4a- hydroxymethyl-d7-steroid (26) is converted into the d8(14)-ene (27) by liver H microsomes under anaerobic conditions and with only NAD+ as a co-factor.On the basis of this and other evidence, the latter w0rkers3~ have indicated support for the mechanism of C-32 removal outlined in Scheme 3, but offer no explanation for the observed loss of hydrogen from C-15a of lanosterol. This loss, however, has not been demonstrated for 4,4-demethyl c28 substrates. In addition, the product obtained in this study contained only an isolated double bond; the formation of conjugated dienes such as (23) and (24), obtained by Akhtar ef a1.,30 was not observed. These results, obtained with similar enzyme preparations, are obviously at variance; the differences may be attributable to the use of CZS substrates, devoid of the C-4 methyl groups, in the more recent study,34 but the reasons for them are not clear.Both 4,4-demethyl-d8!14)-enes[e.g. (27)]35 and d8(9),14-dienes [e.g. (24)]36 are further metabolized to cholesterol by rat liver preparations. However, since c27 mono-enes [e.g. (27)J have only been obtained as a product when the substrate [e.g. (26)] is devoid of methyl groups at C-4, and are only efficiently converted into cholesterol in the absence of these sub- stit~ents,~~.~~and the co-factor requirements for these conversions may be different, the influence of the C-4 methyl groups on the process of C-14a demethy- lation may be significant. The ultimate interpretation of the available mechanistic data on the role and nature of unsaturation at carbons 7, 8, 9, 14, and 15 during C-14a demethylation may therefore depend upon the rigid establishment of the sequence of demethylation of C-4 and C-14a of lanosterol (see Section 4).The later stages of Scheme 4 [path (a)] included an intermediate oxygenated at C-15 (28). The possibility that C-15 oxygenated steroids may be intermediates in the C-14a demethylation reaction was suggested by the loss of the C-15a hydro- gen from lanosterol during its conversion into cholesterol. The apparent require- ment for NADPH and oxygen of the dealkylation of (20)30$33 is consistent with 34 R. A. Pascal, P. Chang, and G. J. Schroepfer, J. Am. Chem. SOC., 1980, 102, 6599. 35 G. J. Schroepfer, B. N. Lutsky, J. A. Martin, S. Huntoon, B. Fourcans, W.-H. Lee, and J. Vermilion, Proc. R. SOC. London, Ser. B, 1972, 180, 125.38 A. Fiecchi, L. Canonica, A. Scala, F. Cattabeni, E. Grossi Paoletti, and R. Paoletti, Life Sciences, 1969, 8, 629. Methyl Group Removal in Steroid Biosynthesis the involvement in this step of a mono-oxygenase enzyme; enzymes of this type are known to hydroxylate steroids with retention of stereochemistry.37 However, although the 15a-hydroxy-4,4-dimethyl-steroid(30) gives rise to c27 steroids on incubation with rat liver homogenate, so does its C-lSP epimer (31), and the CH3 CH3 (30) CH3 CH3 (31) conversion efficiencies are low, both being extensively metabolized by other routes38 The conversion of 14a-methyl-d7-cholestene-3~,15P-diol (32) into H cholesterol by a rat liver homogenate preparation has been rep0rted,39?~0 but the intermediacy of a C-lSg alcohol is inconsistent with the observed loss (from c30 substrates) of the C-15ahydrogen occurring by a mono-oxygenase catalysed oxidation with retention of configuration.The possibility that this hydroxylation may proceed with inversion of configuration has been ~uggested,~" but there is lack of adequate precedent for this. Both the 15a-and 15~-hydroxycholestenes (33) and (34),41 and the corresponding ketone (35)42are converted efficiently into C-15 deoxygenated c27 steroids, including cholesterol (2), by rat liver homo- s' L. L. Smith in 'Terpenoids and Steroids,' A Specialist Periodical Report, The Chemical Society, London, vol. 4, 1974, p. 394. 38 G. F. Gibbons, K. A. Mitropouious, and C. R. Pullinger, Biochem. Biophys.Res. Commun., 1976, 69, 781. 39 J. A. Martin, S. Huntoon, and G.J. Schroepfer, Biochem. Biophys. Res. Commun., 1970,39, 1170. 40 T. E. Spike, A. H.-J. Wang, I. C. Paul, and G.J. Schroepfer,J. Chem. SOC.,Chem. Commun., 1974,477. 41 S. Huntoon, B. Fourcans, B. N. Lutsky, E. J. Parish, H. Emery, F. F. Knapp, and G. J. Schroepfer, J. Biol.' Chem., 1978, 253, 775. 42 D. J. Monger, E. J. Parish, and G. J. Schroepfer, J. Biol. Chem., 1980, 255, 11122. Holland (35) genates, but the relevance of these findings to the possible involvement of C-15 oxygenated intermediates in C-14a demethylation is not clear. An alternative pathway to account for the loss of the C-15a hydrogen, path (b) (Scheme 4j, involves stereospecific loss of this hydrogen from the C-14a formate (29), produced by an enzymic ‘Baeyer-Villiger’ reaction on the aldehyde (20).Similar oxidative reactions are to be involved in the cleavage of the steroid side-chain between C-20 and C-17, and may also be involved in the loss of the C-19 carbon in oestrogen biosynthesis (see Section 5). In the absence of any definitive data on the proposed intermediates (28) and (29), however, their involvement in the C-14a demethylation of lanosterol is still speculative. 4 Sequence of the Demethylations at C-4 and C-14a As discussed in the preceding section, the presence or absence of methyl groups at C-4 in substrates which have been employed in the study of C-14a demethylation can lead to differing conclusions regarding the mechanism of the latter trans- formation.Jt is therefore of some concern that the sequence of demethylation of c30 precursors be firmly established. Many of the substrates used in the study of C-4 demethylation (Section 2) were 4-methyl-or 4,4-dimethyl-cholesterol derivatives, devoid of the C-14a methyl. This has been justified by the common assumption that demethylation at C-14a premeds that at C-4, an assumption based on the isolation of C-14a monodemethyl-steroids from both mam-malian43-45 and micro-~rganism~~ sources capable of synthesizing cholesterol and ergosterol, respectively. However, one of these reports44 also established the F. Gautschi and K. Bloch, J. Am. Chem. Soc., 1957, 79, 684. 44 J.-A. Gustafsson and P.Eneroth, Proc.R. Soc. London, Ser. B, 1972, 180, 179. 46 K. A. Mitropoulos, G. F. Gibbons, and 9. E. A. Reeves, Steroids, 1976, 27, 821. Methyl Group Removul in Steroid Biosynthesis presence in meconium from newborn infants of the 4/3-monodemethyl-steroid (36) [cf.part structure (1 2), Scheme 21, a result which has been quoted34 in support of a pathway for cholesterol biosynthesis in which demethylation commences at C-4, and subsequent loss of the C-14 methyl group occurs as outlined in Schenie 3. Other reports of the isolation of 4-demethyl-14a-methyl-steroidsconcern ~lant~6-48or yeast mutant49 sources, or enzyme systems operating in the presence of specific inhibitors;50-52 the relevance of these findings to the normal pathway for cholesterol biosynthesis in mammals remains to be established.The available evidence therefore favours C-14a as the first site of demethylation of lanosterol, but in the absence of definitive data available from purified enzyme systems capable of performing this reaction, this conclusion must be regarded as tentative. 5 Removal of C-19 and the Mechanism of Oestrogen Biosynthesis The conversion of C19 steroids such as testosterone (4)or 4-androstene-3,17- dione (37) into the corresponding cl8hormones oestradiol (5) and oestrone (38) (37) (38) involves both removal of the C-19 methyl group from C-10 and the aromatization of ring A. Both (4) and (37) have been used as substrates in the study of this transformation, which is normally carried out using a niicrosomal preparation O6 C.Djerassi, J. C. Knight, and D. I. Wilkinson, J. Am. Chem. Sor., 1963, 85, 835.''L. J. Goad, B. L. Williams, and T. W. Goodwin, Eur. J. Biochem., 1967, 3, 232. 48 A. M. Atallah and H. J. Nicholas, S/eroids, 1981, 17, 611. P. J. Trocha, S. J. Jasne, and D. 9. Sprinson, Biochemistry, 1977, 16, 4721. 50 P. J. Doyle, G. W. Patterson, S. R. Dutky, and C. F. Cohen, Phytochemisrr,v, 1971,10,2093. 61 G. F. Gibbons and K. A. Mitropoulous, Biochem. J., 1973, 132, 439. 53 N. N. Ragsdale, Biochim. Biophys. Acta, 1975, 380, 81. Holland from human placenta ;the available evidence indicates that both are transformed at the same enzyme site by the same mechani~m,~~,~~ and may be interconverted during the transformation by reversible oxidation-reduction at C-17.55 Although the initial stages of methyl group removal have been thoroughly investigated, later reactions of the aromatization sequence are not so well understood. The overall transformation of (4) or (37) to the corresponding oestrogenic steroid requires three equivalents of NADPH and molecular oxygen,s6 indicating the involvement of a mono-oxygenase enzyme.As is the case for C-4 demethylation, a requirement for a cytochrome P-450 co-factor has not been rigidly established for the aromatization reaction. The latter conversion is insensitive to carbon monoxide, an effective inhibitor of cytochrome P-450- dependent oxidations, suggesting the involvement of either a different electron carrier or a CO-insensitive form of cytochrome P-45O.s7 Two of the required oxidizing equivalents are consumed in the conversion of C-19 from the methyl into the aldehyde oxidation levels, as outlined in Scheme 5.The intermediacy of the C-19 alcohol (39) and the aldehyde (41), the latter formed by collapse of the gem-diol (40), has been confirmed by their isolati~n,~~~~~ their ability to act as substrates for the aromatase enzyme ~y~tem,~~~6~~~~ and their effectiveness as competitive inhibitors of the normal substrate (4) or (37).6l The loss of C-19 has been demonstrated to occur as formic acid;62 the C-19 carboxylic acid (42) is not aromatized by placental micro some^.^^ The stereochemical fate of the hydrogens involved was determined to be that shown in Scheme 5,64965 and the fate of the oxygen atoms involved in the oxidation at C-19 was deduced by Akhtar and co-workers from experiments with 180-labelled materials.66f67 The outstanding problems in oestrogen biosynthesis concern the function of the third oxidizing equivalent and the pathway by which the C-19 aldehyde (41) is aromatized.Available evidence from the use of labelled substrates has con- 53 W. Gibb and J.-C. Lavoie, Steroids, 1980, 36, 507. 54 K. C. Reed and S. Ohno, J. Biol. Chem., 1976, 251, 1625. 55 W. E. Braselton, L. L. Engel, and J. C. Orr, Eur. J. Biochem., 1974, 48, 35. 56 E. A. Thompson and P. K. Siiteri, J. Biol. Chem., 1974, 249, 5364. 67 R. A. Meigs and K. J. Ryan, Biochim. Biophys. Acfa, 1968, 165, 476. 58 A. S.Meyer, Experientia, 1955, 11, 99. 59 M. Akhtar and S. J. M. Skinner, Biochem. J., 1968, 109, 318. A. S. Meyer, Biochim. Biophys. Acta, 1955, 17, 441. 01 N. Hollander, Endocrinology, 1962, 71, 723; W. G. Kelly, D. Judd, and A. Stolee, Bio-chemistry, 1977, 16, 140. 62 L. R. Axelrod, C. Matthijssen, P. N. Rao, and J. W. Goldzieher, Acta Endocrinol., 1965,48, 383. 63 S. Dell’Acqua, S. Mancuso, N. Wiquist, J. L. Ruse, S. Soloman, and E. Diczfalusy, Acta Endocrinol., 1967, 55, 389. 64 D. Arigoni, R. Battaglia, M. Akhtar, and T. Smith, J. Chem. Soc., Chem. Commun., 1975, 185. 65 E. Caspi, E. Santaniello, K. Patel, T. Arunachalam, and C. Eck, J. Am. Chem. Soc., 1978, 100, 5223. 66 M. Akhtar, M. R. Calder, D. L. Corina, and J. N. Wright, J. Chem. Soc., Chem.Commun., 1981, 129. 67 M. Akhtar, D. Corina, J. Pratt, and T. Smith, J. Chem. Soc., Chem. Commun., 1976, 854. 447 Methyl Group Removal in Steroid Biosynthesis 0 NADPH 0s -0B NADPH 0% 0*11+ HC-OH Scheme 5 Oxidation at the C-10 methyl group in the aromatization of CISsteroids CO2H Holland firmed the retention of oxygen at C-3,68eliminating the involvement of Schiff base intermediates, and the retention of hydrogens at C-l~t,~~ C-4, C-~CU,C-201,~~ and C-6p71(Scheme 6). Pathways and intermediates that have been proposed in > Scheme 6 Retention of ring A and B substituents (a)during enzymic aromatization of CIQsteroids. recent years for the aromatization of (41) are presented in Scheme 7. Pathway (a)72 involves a 4,5p-epoxy-intermediate (43), of which the conversion into oestradiol has been reported.73 However, the recent observation66 that the oxygen atom introduced on oxidation of (41) is incorporated into the formic acid so liberated, and not into the aqueous medium (see Scheme 9,militates against the intermediacy of (43) and its derived alcohol (44),the C-4 oxygen of which must be lost to the aqueous environment.The observed fate of the oxygen introduced during the third oxidation step would seem to support the intermediacy of the C-log formate ester (49,formed by an enzymic Baeyer-Villiger oxidation (6:Section 3). However, (45) is not aromatized by placental microsomes.66 Nevertheless, the existence of pathway (b) and the intermediacy of a structure such as (49,perhaps as an enzyme-bound species, cannot be totally disregarded on this evidence alone (vide injia).An alternative pathway (c) involves a Ip-hydroxy-steroid (46). This has been proposed on the basis of the observation that human placental microsomes are capable of C-lp hydroxylation of the 19-nor-steroid (53).74 However, the same transformation of C19 substrates has yet to be reported. If pathway (c) is opera-tive, then the results of Akhtar and co-workers66 demonstrating incorporation of the third oxidizing equivalent of oxygen into formic acid eliminate from consideration a simple dehydration of (46) via (47) [pathway (c, i)], and reqiiire the formation of the internal hemi-acetal(48) and the C-lp formate (49) [pathway (c, ii)].The formation of (48) is stereochemically improbable, and in the absence 68 H. L. Holland and G. J. Taylor, Can. J. Chem., 1980, 58, 2326. 6s J. Fishman and M. S. Raju, J. Bid. Chem., 1981, 256, 4472. 70 J. Fishman, H. Guzik, and D. Dixon, Biochemistry, 1969, 8, 4304. 71 H. L. Holland and G. J. Taylor, Can.J. Chem., 1981, 59, 2809. 72 P. Morand, M. Kalapurackal, L. Lompa-Krzymien, and A. Van Tongerloo, J. Theoret. Biol., 1976, 56, 503. 73 P. Morand, D. G. Williamson, D. S. Layne, L. Lompa-Krzymien, and J. Salvador, Biochemistry, 1975, 14, 635. 74 M. Ganguly, K. L. Cheo, and H. J. Brodie, Biochim. Bioph~s.Actu, 1976, 431, 326. 449 Methyl Group Removal in Steroid Biosynthesis CHO HO CHO CHO 0BB 0mH:m03 (43) IJ 0P CHO HO HO Scheme 7 Proposed routes for the aromatization of (41) Holland of further supportive data, pathway (c) must be regarded as an unattractive option at the present time.In pathway (d), Scheme 7, aromatization occurs via the C-2P-hydroxy-deriva- tive (50). In order to be consistent with the data from the oxygen labelling experiments discussed above,66 pathway (d, i) must now be eliminated from consideration, and discussion centred on pathway (d, ii) involving the hemi- acetal (51) and the 19-nor-2P-formate (52). Human placental microsomes are capable of hydroxylation of (53) at C-@,75 and indeed the Zp-hydroxy-inter- mediate (50) has been trapped (in very low yield) from incubations of labelled (37) with placental mi~rosomes.~~ The C-Zg alcohol (50) rapidly aromatizes non-enzymically at neutral or basic pH, but the C-201alcohol (54)does not;77 CHO 0 in the presence of placental microsomes, the conversion occurs with stereo- specific loss of the C-lp hydrogen.69 The alcohol (50) forms the internal henii- acetal (51) very readil~;~7 this reaction has not been invoked in explaining the facile non-enzymic aromatization of (50), but may be involved in the enzymic process, The data that are currently available therefore do not allow for a clear dis- tinction between pathway (d, ii), and pathway (b), Scheme 7.The latter route, if operative, must involve the intermediacy of the formate (45) or its equivalent as a tightly bound intermediate, which is not in equilibium with the corresponding exogenous material (vide supra).Akhtar and co-workers66 have proposed that this occurs in the form of the enzymic peroxy-acetal(S), which decomposes as shown with concerted loss of the C-lg hydrogen. An alternative route is proposed in Scheme 8.This involves binding of the substrate to the enzyme as the d2*4-dienol 75 H. J. Brodie, A. K. Pillai, and C. E. Hay, Biochim. Biophys. Acta, 1969, 187, 275. 76 J. Goto and H. Fishman, Science, 1977, 195, 80. ” H. Hosoda and J. Fishman, J. Am. Chem. Soc., 1974, 96, 7325. 45 1 Methyl Group Removal in Steroid Biosynthesis 0 (56): a process which would result in the observed stereospecific loss of the C-2P (axial) hydrogen.7s The C-19 alcohol and aldehyde intermediates, (57) and (58), respectively, are known to be present as tightly enzyme-bound species,61 not in rapid equilibium with corresponding exogenous material.A similar phenomenon for (59) may prevent it from acting as an effective substrate for aromatization when added exogenously to the enzyme system. CH3 CHzOH 1 (56) I (57)Enzyme Enzyme CHO I CHO 0& 0m I (58)Enzyme 0I fl Enzyme Scheme 8 Proposed route for oestrogen biosynthesis via a C-10/3formate ester 78 E. J. Corey and-R. A. Sneen, J. Am. Cham. SOC.,1956, 78, 6269. Hollund 6 Summary and Comparison of Demethyiation Reactions in Steroid Biosynthesis From the data presented in Sections 2, 3, and 5 it is apparent that the loss of the C-4 methyl groups may be distinguished mechanistically from other methyl losses.The enzymic strategy of using the C-3 carbonyl group as an electron acceptor in a ,8-keto-acid decarboxylation necessitates the loss of carbon from C-4 as carbon dioxide. Losses from C-14 and C-10 occur as formate, in what may be formally regarded as a retro-Claisen reaction, the electron acceptors being a proton and the C-3 carbonyl of a d*-3-keto-system9 respectively; or these losses may occur by parallel pathways involving oxidation of the methyl group to the aldehyde level followed by Baeyer-Villiger oxidation to produce a formateester, which then loses the elements of formic acid in a 1,2-eIimination. In this event, the loss of carbon from C-10 and C-14 would not require activation by the presence of an adjacent functional group.Although all three of the methyl group removals discussed herein require molecular oxygen and NADPH, an absolute requirement for a cytochrome P-450electron carrier co-factor has been demonstrated only in the case of C-14a demethylation. The involvement of cytochrome P-450 in the removal of C-19 has been proposed,79 but remains to be rigidly established, whereas its presence as a co-factor in the C-4 demethylating system has been excluded. I wish to thank sincerely my former co-worker, G. J. Taylor, whose interest and enthusiasm in oestrogen biosynthesis was instrumental in the conception of this review. '* E.A. Thompson an P.K. Siiteri, Ann. N. Y.Acad. Sci., 1973, 212, 378.

ISSN:0306-0012    

DOI:10.1039/CS9811000435

出版商:RSC    

年代:1981

数据来源: RSC

摘要:

CENTENARY LECTURE* Metal Clusters in Biology: Quest for a Synthetic Representation of the Catalytic Site of Nitrogenase By R. H. Holm DEPARTMENT OF CHEMISTRY, HARVARD UNIVERSITY, CAMBRIDGE, MASSACHUSETTS 021 38, U.S.A. 1 Introduction It is now well established that there occur, in certain proteins and enzymes, aggregates of two or more metal atoms that are bridged by oxygen or sulphur atoms and terminally co-ordinated mainly or entirely by functional side-groups of amino-acid residues comprising polypeptide chains.1 In those instances where the collective properties of an individual metal site depart significantly from those of an isolated co-ordination unit of comparable constitution and geometry, the aggregate -by the usual definition of the inorganic chemist -may be considered a cluster.Such is the case for the 2-Fe and 4-Fe prosthetic groups (1) and (2) of cys-s,7, Fe-I‘STFe ,S-CYS cys-s\ ..-S-..Fe,S-cys cys-sHF%sr ‘s-cys S-1\ -Fe,(\‘S-cys /Fe-S (1) s-cys (2) iron-sulphur electron-transfer proteins, the ferredoxins (Fd). These entities, which are the most thoroughly investigated biological clusters,2-6 contain tetrahedrally co-ordinated iron atoms which are antiferromagnetically spin- coupled in all oxidation levels in vitro. Accurate synthetic representations (3) and *This is an expanded and updated version of the Centenary Lecture presented by Professor R. H. Holm in London in April, 1980. J. A. lbers and R. H. Holm, Science, 1980, 209, 223. ‘Iron-Sulfur Proteins’, ed.W. Lovenberg, Academic Press, New York; (a) 1973, Vols. 1 and 2; (b) 1977, Vol. 3. G. Palmer, in ‘The Enzymes’, (3rd edn.), Vol. XII, Part B, ed. P. D. Boyer, Academic Press, New York, 1975, pp. 1-56. R. H. Sands and W. R. Dunham, Q. Rev. Bioph,vs., 1975, 7, 443. B. A. Averill and W. H. Orme-Johnson, Met. Ions Biol. S-vst., 1978, 7, 127. W. V. Sweeney and J. C. Rabinowitz, Annir. RPV.Biochcm., 1980, 49, 139. Metal Clusters iti Biology (3) (4) (4) of these centres can be readily assembled in simple preparative systems (see below), and their structural and electronic properties and reaction chemistry have been elucidated in considerable detai1.197-13 In these species, Fe-Fe distances of -2.7 8, allow direct orbital overlap, which, in addition to inter- actions through intermediary sulphur atoms, leads to electronic coupling between or amongst Fe sites.The spectroscopic and magnetic properties of clusters (3) and (4) closely approach those of protein groups (1) and (2), respectively, in equivalent oxidation levels, and thus serve as synthetic analogues of the latter. Among the noteworthy features of (1)-(4) is the electronically delocalized nature of the cubane-type clusters (2) and (4) in those oxidation levels that require formally inequivalent oxidation states of iron, a matter most clearly revealed by Mossbauer spectroscopy of Fd proteins14 and their redox-group analogue~.~?l~Both the Mossbauer spectral and bulk magnetic properties15 of these clusters are accountable in terms of a description involving antiferro- magnetic spin-coupling of iron atoms.Over the past decade, a new type of biological cluster, as yet undefined in detail, and not anticipated by serendipitous inorganic cluster chemistry, has emerged. This is the Fe-Mo-S entity that is present in the native FeMo protein of the nitrogenase enzyme complex, and which is removable from it in the form of a cofactor (FeMo-co).16 The present extensive body of enzymological and physical evidence, while not decisive, is suggestive in respect of FeMo-co (in a reduced oxidation level) being the catalytic site for reduction of dinitrogen to ammonia. Stimulated by this possibility, and abetted by increasing physico- chemical definition of the biological cluster and our experience in Fe-S R.H. Holm and J. A. Ibers, in ref. 26,Ch. 7. R. H. Holm, Acc. Chem. Res., 1977, 10,427. a E. J. Laskowski, R. B. Frankel, W. 0. Gillum, G. C. Papaefthymiou, J. Rena ud, J. A. Ibers, and R. H. Holm, J. Am. Chem. SOC.,1978,100,5322. 10 E. J. Laskowski, J. G. Reynolds, R. B. Frankel, S. Foner, G. C. Papaefthymiou, and R. H. Holm, J. Am. Chem. SOC.,1979,101,6562. J. G. Reynolds, C. L. Coyle, and R. H. Holm, J. Am. Chem. SOC., 1980,102, 4350. l2 P. K. Mascharak, G. C. Papaefthymiou, R. B. Frankel, and R. H. Holm, J. Am. Chem. SOC.1981,103,61 10. l3 J. M. Berg and R. H. Holm, in ‘Iron-Sulfur Biochemistry’, ed. T. G. Spiro, Wiley, New York, in the press. l4 P. Middleton, D. P. E. Dickson, C.E. Johnson, and J. D. Rush, Eur. J. Biochem., 1978,88, 135; ibid., 1980, 104,289. l5 G. C.Papaefthymiou, E. J. Laskowski, S. Frota-Pessoa, R. B. Frankel, and R. H. Holm, Znorg. Chem., in the press. 1E V. K. Shah and W. J. Brill, Proc. Natl. Acad. Sci. USA, 1977,74,3249. Holm analogue chemistry, we have initiated research directed toward the synthesis of this cluster. Unlike the cases of prosthetic groups of Fd proteins, for which synthetic analogues have been prepared, 7-10~13917 the target cluster enjoys only a modicum of direct structural chara~terization.~8J~ Consequently, our endeavour is, in Hill’s description,20 a venture in speculative rather than corroborative modelling of a prosthetic group. The leading results of the endeavour form the substance of this report. For summaries or reviews of the biochemistry of nitro- of FeMo-c0,2~-~~ and of gena~e,~l-2~ of relevant Fe-Mo-S chemi~try,~~?~~,~O chemical approaches to the complexation and reduction of dinitro-gen,21~22~253 other sources should be consulted.27 ~29~1~32 2 Metal Clusters of Nitrogenase A.The Native Enzyme.-The nitrogenase enzyme complex consists of two types of proteins: the Fe protein, which transfers electrons that are supplied by an external reductant, with concomitant hydrolysis of ATP; and the FeMo protein, which is reduced by the Fe protein, contains the catalytic site(s) for reduction of substrate, and is the source of FeMo-co. The sequence shown in Scheme 1 is an abbreviated representation of the current view of coupled reactions in a reconstituted enzyme system, resulting in the reduction of physio- logical (N2, H+) and a number of non-physiological substrates, including acetylene. The Fe protein from all organisms has four iron and ‘inorganic’ sulphur (S*) atoms in an a2 subunit structure of molecular weight -57OOO.The structural l7 D. Coucouvanis, D. Swenson, N. C. Baenziger, C. Murphy, D. G. Holah, N. Sfarnas, A. Simopoulos, and A. Kostikas, J. Am. Chem. SOC.,1981, 103, 3350. l8 S. P. Cramer, K. 8.Hodgson, W. 0. Gillum, and L. E. Mortenson, J. Am. Chem. SOC., 1978, 100, 3398. l@S. P. Cramer, W. 0.Gillum, K. 0.Hodgson, L. E. Mortenson, E. I. Stiefel, J. R. Chisnell, W. J. Brill, and V. K. Shah, J. Am. Chem. SOC.,1978,100, 3814.H. A. 0. Hill, Chem. Br., 1976, 12, 119. .dl‘Recent Developments in Nitrogen Fixation’, ed. W. Newton, J. R. Postgate, and C. Rodriguez-Barrueco, Academic Press, New York, 1977. az ‘A Treatise on Dinitrogen Fixation, Sections I and 11: Inorganic and Physical Chemistry and Biochemistry’, ed. R. W. F. Hardy, F. Bottomley, and R. C. Burns, Wiley, New York, 1977. 23 W. H. Orme-Johnson and L. C. Davis, in ref. 2b, Ch. 2. ar L. E. Mortenson and R. N. F. Thorneley, Annu. Rev. Biochem., 1979,48, 387. 25 ‘Nitrogen Fixation’, ed. W. E. Newton and W. H. Orme-Johnson, University Park Press, Baltimore, 1980, Vols. I and 11. aa ‘Molybdenum and Molybdenum-Containing Enzymes’, ed. M. P. Coughlan, Pergamon Press, New York, 1980. 27 ‘Current Perspectives in Nitrogen Fixation’, ed.A. H. Gibson and W. E. Newton, Australian Academy of Science, Canberra, 198 1. B. K. Burgess and W. E. Newton, in ‘Nitrogen Fixation : Chemical/Biochemical/GeneticsInterface’, ed. A. Muller and W. E. Newton, Plenum Press, New York, in the press. 2@ ‘Molybdenum Chemistry of Biological Significance’, ed. W. E. Newton and S. Otsuka, Plenum Press, New York, 1980. 30 D. Coucouvanis, Acc. Chem. Res., 1981, 14, 201. 31 J. Chatt, J. R. Dilworth, and R. L. Richards, Chem. Rev., 1978, 6, 589. 32 W. E. Newton, Adv. Chem. Ser., 1980, 191, 351. 457 Metal Clusters in Biology MgATP [FeMo proteinIred [FeMo proteinlo, MgADP Scheme 1 organization of the Fe-S content of this protein has been examined by the core extrusion method, which has been described at length elsewhere.13J3-37 The method is based on the reversible ligand-substitution reaction (1) (where n = 2 or 4 and x = 1-4), first established with the analogue dianion complexes (3)38 and (4).39 Subsequently, a demonstration of the extrusion reaction [react ion (2)] [FenSn(SR)J2-+ xR’SH + [FenSn(SR)4-z(SR’)z]z-+ xRSH (I) unfoldingholoprotein + RSH +[FenSn(SR)4I2-+ apoprotein (2)solvent was provided.33~36J~+~* Here the treatment of a small Fd protein, unfolded in an aqueous/organic solvent that contains a large excess of the thiol which acts as an extrusion reagent, results in the quantitative removal of [FenSnI2+cores of (I) and (2) in the form of their analogue complexes [FenSn(SR)4I2- (n = 2 or 4).The latter, which can be independently synthesized, are assayed by an appropriate spectroscopic technique (u.v.-visible spectra, 19F n.m.r., or e.p.r. of reduced clusters). Application of the method to a clostridial Fe protein has shown the presence of one Fed34 cluster (2),23933 possibly bridging the two subunits. The FeMo proteins have an ~$2 subunit structure (the range of molecular weights is 220000-240000) with two molybdenum atoms and about equal amounts of iron and inorganic sulphur. More recent preparations contain -30-32 iron atoms. Our approach to the investigation of the structural 33 W. 0.Gillum, L. E. Mortenson, J.-S. Chen, and R. H. Holm,J. Am. ChLJm.Soc., 1977,99, 584. 34 R. H. Holm, in ‘Biological Aspects of Inorganic Chemistry’, ed.A. W. Addison, W. R. Cullen, and D. Dolphin, Wiley, New York, 1977, pp. 71-1 11. 35 W. H. Orme-Johnson and R. H. Holm, Mrthods Enzymo!., 1978, 53, 268. 36 G. B. Wong, D. M. Kurtz, Jr., R. H. Holm, L. E. Mortenson, and R. G. Upcliurch,J. Am. Chem. Soc., 1979, 101, 3078. 37 B. A. Averill, J. R. Bale, and W. H. Orme-Johnson, J. Am. Chcm. Soc.., 1978, 100, 3034. 38 L. Que, Jr., M. A. Bobrik, J. A. Ibers, and R. H. Holm,./. Am. Chcm. Sac,., 1974, 96, 4168. 39 J. J. Mayerle, S. E. Denmark, B. V. DePamphilis, J. A. Ibers, and R. H. Holm, J. Am. Chem. SOC.,1975, 97,1032. 40 L. Que, Jr., R. H. Holm, and L. E. Mortenson, J. An?. Chrm. Soc.., 1975. 97, 463. Holm organization of this formidable metal content has utilized core extrusion.Initial experiments36 were based on reaction (3) in Scheme 2. Here, semi-reduced (5) n = 2 (6) n = 4 [reaction (4)] [reaction (S)] (Fed34 [S(SH)-o-~yll4}~-(7)(9) (RFSH = P-CF~CGH~SH) Scheme 2 (as isolated) proteins were placed in the unfolding medium 4: 1 v/v hexamethyl-phosphoramide (HMPA) :aqueous buffer containing p-CF3CsH4SH. Extrusion products, assayed by I9Fn.m.r. spectroscopy, consisted of small amounts of the Fed32 complex (5)and much larger quantities of the Fe4S4 complex (6).However, 525% of the total iron content was removed in these reactions, suggestive of incomplete extrusion, In subsequent e~periments,~l using the same protein samples, reaction conditions, and method of assay of the product, o-xylyl-a,a'- dithiol (7) was introduced as the extrusion reagent in reaction (4).The species that were'liberated were subjected to the ligand-substitution reaction of reaction (9,which is a specific example of reaction (1). Only cluster (6)was detected, indicating removal of protein Fe/S* in the form of (8) rather than (9). Of the predetermined iron content of the protein, 49-56 % was liberated. When normalized to 30-32 iron atoms, the results indicate about four Fed34 clusters per a& unit. Under the same conditions, separate samples of FeMo-co afforded no evidence of formation of (5) and (6), consistent with an earlier report that benzenethiol does not disrupt FeMo-co.42 These findings lead to the conclusion that only non-cofactor iron is extruded, and that the large majority of this iron content is organized in the form of Fe& clusters.The first of these conclusions is supported by chemical analyses of FeMo-co, dl D. M. Kurtz, Jr., R.S. McMillan, B. K. Burgess, L. E. Mortenson, and R. H. Holm, Proc. Natl. Acad. Sci. USA, 1979, 16, 4986. 42 J. Rawlings, V. K. Shah, J. R. Chisnell, W. J. Brill, R. Zimmermann, E. Munck, and W. H. Orme-Johnson, J. Biol. Chrm., 1978, 253, 1001. Metal Clusters in Biology which show that about half of the iron content of an FeMo protein (7-8 iron atoms pel molybden~m~~*~3-~5) is associated with the cofactor. Extrusion results gained from the sequential application of reactions (4) and (5) are encouragingly consistent with the deduction from Mossbauer spectros- copy that FeMo proteins contain four Fe4S4 ('P') cluster^.^^^^^ These may form the electron-transfer conduit that couples the Fe protein to the catalytic site(s).However, the nature of these clusters differs from that of conventional Fd-type and m.~.d.~* groups (2). MOs~bauer~69~~ spectral features in particular are unusual. Further, except for transient signals, these clusters are e.p.r.-silent in isolated, oxidized, and re-reduced protein sample~,~~~*~ despite the fact that sufficient electrons have been subtracted or added to change the oxidation level of P-clusters by one electron. A minority population of iron atoms, accounting for -6% of the total, and of unknown structure, has also been detected in the Mossbauer spectra of FeMo protein^.^^^^^ Although the properties of P-clusters might be subject to some clarification by application of the synthetic analogue approach, the main concern here is with synthetic clusters that are possibly related to FeMo-co, whose properties are considered next.B. FeMo-co.-If, as seems likely, the essential catalytic machinery (excluding electron-transfer apparatus) for the reduction of substrates of nitrogenase is contained in FeMo-co, its isolation by Shah and Brill16 in 1977 has opened the way for the eventual elucidation of the structure of the catalytic site and its mechanism at an atomic level of resolution. In the original procedure,16 FeMo-co was extracted with N-rnethylformamide (NMF) from a pellet of FeMo protein that had previously been denatured with citric acid.Modified and improved procedures are now a~ailable.*~-~~ In NMF, FeMo-co forms a brown, oxygen- sensitive solution. It is not as yet a fully characterized entity; selected properties, at the current stage of definition, are collected in Table 1.50-S7 In particular, neither the ratios of constituent atoms nor the molecular weight45 are precisely 43 B. K. Burgess, D. B. Jacobs, and E. I. Stiefel, Biochim. Biophys. Acra, 1980, 614, 196. 44 S.-S. Yang, W.-H. Pan, G. D. Friesen, B. K. Burgess, J. L. Corbin, E. 1. Stiefel, and W. E. Newton, submitted for publication. 45 B. E. Smith, in ref. 29, pp. 179-190. 46 R. Zimmermann, E. Munck, W. J. Brill, V. K. Shah, M. T. Henzl, J. Rawlings, and W.H. Orme-Johnson, Biochim. Biophys. Acta, 1978, 537, 185. 47 B. H. Huynh, M. T. Henzl, J. A. Christner, R. Zimmermann, W. H. Orme-Johnson, and E. Munck, Biochim. Biophys. Actu, 1980, 623, 124. 48P.J. Stephens, C. E. McKenna, M. C. McKenna, H. T. Nguyen, and F. Devlin, Bio-chemistry, 198 1, 20, 2857. 49 G. D. Watt, A. Burns, S. Lough, and D. L. Tennent, Biochemistry, 1980, 21,4926. 50 B. H. Hunyh, E. Munck, and W. H. Orme-Johnson, Biochim. Biophys. Am, 1979,527,192. 51 W. E. Newton, B. K. Burgess, and E. I. Stiefel, in ref. 29, pp. 191-202. 5B E. Munck, H. Rhodes, W. H. Orme-Johnson7L. C. Davis, W. J. Brill, and V. K. Shah, Biochim. Biophvs. Acta, 1975, 400, 32. 63 B. K. Burgess, S.4. Yang, C.-B. You, J.-G. Li, G. D. Friesen, W.-H.Pan, E. I. Stiefel, W. E. Newton, S. D. Conradson, and K. 0.Hodgson, in ref. 27, pp. 71-74. 54 G. D. Watt, in ref. 29, pp. 3-21. 55 B. E. Smith, D. J. Lowe, and R. C. Bray, Biochem. J., 1979, 135, 331. B. K. Burgess, E. I. Stiefel, and W. E. Newton, J. Bid. Chem., 1980, 255, 353. 57 V. K. Shah, in ref. 25, Vol. I, pp. 237-247. 460 Holm Table 1 Selected properties of FeMo-co Property References Atom ratio: (6--8)Fe: (4-6)S* :Mo 16,4245, 50 No amino-acids or identified endogenous organic 44,45 components Anionic (possible ligands : citrate, phosphate, 28, 51 C1-, S2042-, sCh2-,H20, OH-, or NMF) E.p.r. + Mossbauer spectral properties :a iron atoms 42, 50, 52 are spin-coupled; S = 4 per Mo; g x 4.6, 3.3, <2.0 I & +>, 5.8-5.9 (weak) I k2 > ; zero-field splitting x + 6 cm-l; e.p.r.hyperfine broadening with 5’Fe but not 95Mo enrichment Molybdenum XAS: 3 or 4 S at 2.36 A, 2 or 3 Fe at 19, 53 2.68 A, 2 or 3 N,O at 2.10 A (EXAFS) Redox? 7[FeMo-coIred ---_.rm m I[FeMo-coj --mmv LreMo-ca)]ox 42, 46, 49, 51s>1 t -s = f -, (e.p.r.-silent) 54-56(e.p.r.-silent)-+-Activity: -250-300 nmole of C2H4 are formed per 43-45, 51, 53, minute per nano (gram atom) of molybdenum. 57 Reconstitutes cofactor-deficient FeMo protein from a 16 mutant organism (Azotobacter vinelandii UW45) to full activity (a) These results were obtained in part from studies of native FeMo proteins. known. However, the near-identity of spectroscopic properties and reconstitution activities of samples of FeMo-co from five different organisms points to an identical cofactor from all sources.FeMo-co is distinct from a molybdenum cofactor (Mo-co) that is common to all other molybdo-en~ymes.5~ While some nitrogen-fixing organisms produce both cofactors, FeMo-co and Mo-co are not genetically related,28 and their activation of protein extracts from mutant organ- isms is mutually exclusive.59 Very recently, a second species, designated ‘FeMo-cluster’, and having the atom ratio (6.0 k 0.5) iron per molybdenum atom, has been isolated.60 It is obtained by extraction of HCI-treated FeMo-protein with butan-2-one, in which solvent it displays an e.p.r. spectrum quite different from that of FeMo-co. After transfer to NMF, the distinctive S = 9 e.p.r.spectrum, although weak, appears. In NMF, FeMo-cluster does not activate Azotobacter vinelandii UW45, but, as s* J. L. Johnson, in ref. 26, Ch. 10. P. T. Pienkos, V. K. Shah, and W. J. Brill, Proc. Natl. Acad. Sci. USA, 1977, 74, 5468. 6o V. K. Shah and W. J. Brill, Proc. Nurl. Acad. Sri. USA, 1981, 78, 3438. 461 Metal Clusters in Biology FeMo-co,61 it reduces acetylene to ethylene in the presence of sodium boro- hydride. Other evidence60 indicates that FeMo-cluster is derived from FeMo-co. Thus it appears that a content of >6 iron atoms per molybdenum atom is required for activity, but not for the e.p.r. spectrum; this spectrum is unique in biology. FeMo-cluster may be the centre ‘MEPR’, containing the spin system of S = 3 and which was earlier deduced to contain, most probably, 6Fe per Mo atom from a detailed Mossbauer and e.p.r.spectral analysis of native proteins46*47*52and FeMo-c0.~~-50 The only direct structural information for the Fe-Mo-S cluster of nitrogenase follows from X-ray absorption spectra (XAS) of molybdenum and associated extended X-ray absorption fine structure (EXAFS) of several FeMo proteinsls and the cofact0r.~~*53 Analysis of the EXAFS by the Stanford group has led to the conclusions in Table 1 and the proposal of structures (10)and (1 1) as models for the co-ordination unit of molybdenum in the proteins. Inasmuch as the technique62 does not reliably sense atoms that are 23.5 A from the atom whose X-ray spectrum is excited, the presence of the sulphur atom that is diagonally opposite the molybdenum atom in (10) is conjectural.The similarity of the cubane-type MoFe3S4 core unit of (10) to the Fed34 core in (2) and (4) is obvious. Ligands of the molybdenum atom that are external to the core framework are less well defined, but may consist in part of one or two sulphur atoms at about 2.5 A. 0x0 ligands (as in Mo-10) are absent. No Mo -. -Mo interactions of 5 3.5 A have been detected, suggesting (together with spectros~opic~~~~~ and molecular-weight45 evidence) that two cofactors, each containing one molyb- denum atom, are present rather than one cofactor, containing two molybdenum atoms. Upon removal from the protein, FeMo-co retains the 2 or 3 iron and 3 or 4 sulphur atoms around the molybdenum atom.A recent EXAFS analysis53 suggests the presence of 2 or 3 oxygen or nitrogen atoms, which presumably arise from exogenous ligands that are introduced in the isolation procedure. Likely candidates are given in Table 1. A schematic representation of the current view of the clusters that are present in FeMo proteins is given in Figure 1. Based on the ratios of atoms and results from EXAFS, including a somewhat different interpretation of the latte1-,~3 various structural models for the Mo site in 61 V. K. Shah, J. R. Chisnell, and W. J. Brill. Biochrm. Biophys. Res. Commrrn., 1978, 81, 232. 62 S. P. Cramer and K. 0. Hodgson, Prog. Inorg. Chcm., 1979, 25, I. 63 R.-K. Teo and B. A. Averill, Biochcm.Bioph.vs. RPS.Commun., 1979. 88, 1454. Holm \ \ Fe-S Fy--S\ /1‘s 4e’ I S-Fe %l-k w+l. Fe -S Fe --S / / Figure 1 Clusters in the FeMo protein of nitrogenase, according to current evidence nitrogenase have been propo~ed~~-~~ subsequent to the suggestions of (10) and (11). While some are of arresting conception, none of these models has been synthesized. It has proven possible to prepare clusters that contain the core unit in (10).The developing chemistry of these species is described in the follow- ing sections. Because they are the first mixed-metal MnM’4-nS4 cubane-type clusters to be isolated, MoFesS4 species are of interest in the context of abio- logical cluster chemistry. In addition, sufficient information has been accumu- lated to suggest the appropriateness of these clusters as preliminary models of the co-ordination unit of molybdenum in the enzyme.3 The Assembly of Fe& and MoFe3S4 Clusters Initial syntheses of the vast majority of clusters that are now known were not deliberate, in the sense that the reactants and conditions employed could not necessarily have afforded molecules of uniquely predictable composition and structure. This was the case in our original synthesis of [Fed%(SR)4I2-64 Lu Jiaxi, in ref. 25, Vol. I, pp. 343-371. 65 K. R. Tsai, in ref. 25, Vol. I, pp. 373-387. W. E. Newton, J. W. McDonald, G. D. Friesen, B. K. Burgess, S. D. Conradson, and K. 0. Hodgson, in ref. 27, pp. 30-39. 463 Metal Clusters in Biology clusters,67~6*where structure (4) was only one of the conceivable possibilities.It is now abundantly clear that these clusters, derived from substitutionally labile iron(i1, 111) reactants, form as a consequence of being the thermodynamically most stable, soluble reaction products. The term ‘spontaneous self-assembly’ has been introducedl as a reminder of the thermodynamic origin of cluster synthesis. Before proceeding to the preparation of MoFe3S4 species, an examina- tion of the course of assembly reactions that afford [Fe4S4(SR)4I2- will prove-instructive. A. Fe4S4 Clusters.-The [Fe4S4(SR)4I2- clusters are assembled in a series of reactions, dependent upon mole ratios of reactants. In recent work,69 component reactions have been identified by the use of spectrophotometric and 1H n.m.r.spectral observations, and products of these reactions have been isolated, The original assembly reaction, which is applicable to virtually all thiolates, and which affords yields of -80 % in situ, has the limiting stoicheiometry of reaction (6). However, this reaction proved too rapid for convenient study. Attention was 4FeC1, + 4HS-[Fe,S,(SR),I2-+ RSSR + + 6RS-+ 40Me-12C1-++ 4MeOH (6) turned to the system in reaction (7),70 utilizing R = Ph and elemental sulphur as the source of cluster sulphide. With the mole ratio RS--: Fe = 3.5: I, in methanol or acetonitrile as solvent, the rapid reaction (8) ensues, affording, the complex [Fe4(SR)1ol2-(12) in high yield. Subsequent introduction of sulphur affords fFe4S4(SR)4l2-.The overall reaction is ((8) + (9)) = (7). When the mole ratio RS-: Fe >, 5: 1 is employed, in acetonitrile, the initial identifiable product is the 4FeCI, + 4s + 14RS-+ [Fe,S,(SR),l2-+ SRSSR + 12CI-(7) 4FeC1, + 14RS-+ [Fe4(SR)lo]2-+ 2RSSR + 12C1-(8) [Fe,(SR),,J2-+ 4s [Fe4S4(SR)J2-+ 3RSSR (9)--f mononuclear tetrahedral complex TFe(SR)4I2- (13) that is produced in reaction (10). Addition of sulphur results in reaction (I I), by which the well-characterized binucleav clusters [Fe2Sz(SR)4l2- (3) are formed. No further reaction occurs in acetonitrile, but addition of methanol results in the slow formation of the final cluster product by means of reaction (12). The sum of reactions {[4 x (lo)] + [2 x (]I)] + (12)) is reaction (7).The course of reactions in the two assembly FeCl, + 5RS-[Fe(SR),I2-+ gRSSR + 3CI-(10)--f 2[Fe(SR),I2-+ 2s + [Fe,S2(SR),I2-+ RSSR + 2RS-(1 1) 2[Fe2S2(SR),l2--+ [Fe4S,(SR),*l2-+ RSSR + 2RS-(1 2) 67 T. Herskovitz, B. A. Averill, R. H Holm, J. A. Ibers, W. D. Phillips, and J. F. Weiher, Pror. Natl. Acad. Sci. USA, 1972, 69, 2437. 6B B. A. Averill, T. Herskovitz, R. H. Holm, and J. A. Ibers, J. Am. Chem. SOC.,1973, 95, 3523. e9 K. S. Hagen, J. G. Reynolds, and R. H. Holm, J. Am. Chem. Soc., 1981, 103, 4054. ’O G. Christou and C. D. Garner, J. Chem. Sor., Dalton Trans., 1979, 1093. Holm systems is summarized in Scheme 3. These reaction schemes appear to apply just as well to those alkylthiolate systems in which insoluble polymers are not formed prior to the introduction of sulphur.In addition, iron(r1) salts may be used as reactants, resulting in obvious changes in the stoicheiometries of reactions (7), (8), and (10). FeCh 3.5 RS-25 RS-J\ [reaction (8)] [reaction (1011 SR 2-I [reaction (1 I)] 4s [reaction (9)]sl I 2-(3) J Scheme 3 The reaction sequences depicted in Scheme 3 are notable in several respects. When conducted in methanol solutions, reactions ((8) + (9)) and ((10) + (11)+ (12) 1afford the cluster (4) in quantitative (> 99 %) yields in siru. In the former sequence, the adamantane-like structure (12) has been demonstrated crystallo- graphically for [Fe4(SPh)l0]2-,~~ and it is very similar to that of [Co4(SPh)1ol2-, 71 K.S. Hagen, J. M.Berg, and R. H.Holm, Inorg. Chim. Acra, 1980, 45, L17. Metal Clusters in Biology prepared ea~-lier.~2 The cluster [Fe4(SPh)10l2- reacts with elemental sulphur in an ‘all-or-nothing’ fashion, forming (4) (and no other product) in proportion to the amount of sulphur present. As seen from the stoicheiometry of reaction (9), the reducing equivalents that are required to form the requisite amount of sulphide are contained within the molecule. The quantitative nature of this reaction is impressive upon the observation that all twelve skeletal bonds of (12) must be ruptured in the course of formation of the product cluster. The other reaction sequence is of significance because it provides the demonstration that tetra-nuclear clusters can be elaborated by a series of spontaneous irreversible reactions, commencing with trivial reagents and passing through successive mononuclear and binuclear intermediates.In this sequence, the structures of the intermediates [Fe(SPh)4I2- (1 3)’7 and [Fe2S2(SPh)4I2- (3)39 have been established. Several implications concerning the biosynthesis of Fed& protein groups (2) may be drawn from these results. Chief among these are that no protein is required to guide the assembly of a cluster from simple reactants and, given the occurrence of reaction (1) and the reverse of reaction (2) when protein is re-folded, the insertion of a cluster into a peptide environment may be a non-enzymatic process. B. MoFe3S4 Clusters.-Identification of reaction sequences that terminate in the formation of [Fe4S4(SR)4I2- has provided a reasonably satisfactory (albeit non-mechanistic) picture of the modes of self-assembly of these clusters.Similar orderly chemistry may ultimately be shown to yield MoFesS4 clusters. However, our initial synthetic approaches have relied on the assumption that such clusters might spontaneously assemble from appropriate reactants, as has been shown for the clusters (4). Purposeful synthetic pursuit then reduces to elementary considerations of the choice of reactants and reaction stoicheiometry. Because it is the simplest soluble source of molybdenum and sulphide, and in view of its demonstrated prowess as a ligand,73 MoS42- was employed, together with FeC13 and thiolate, in the successful reaction system that is shown in Scheme 4.Yields of the three cluster assembly products, i.e. [MozFesS~(pz-S) (/-L.z-SR)~(SR)~]~-(14), [M02Fe6S8(~2-SR)3(SR)6I3-(1 5), and [M02Fe&(p2-sR)6- (SR)C]~-~~-(16), can be optimized by the adjustment of reaction conditions.74-77 Clusters are readily isolated in the form of quaternary ammonium salts. The indicated bridged ‘double-cubane’ structures have been demonstrated by X-ray diffra~tion.~~*78The analogous tungsten clusters can be prepared from WS42 -. 72 1. G. Dance, J. Am. Chem. SOC.,1979, 101, 6264. 73 E. Diemann and A. Muller, Coord. Chem. Rev., 1973, 10, 79.’* T. E. Wolff, J. M. Berg, C. Warrick, K. 0. Hodgson, R. H. Holm, and R. B. Frankel, J. Am. Chem. SOC.,1978, 100, 4630.75 T. E. Wolff, J. M. Berg, K. 0. Hodgson, R. B. Frankel, and R. H. Holm, J. Am. Chem. SOC.,1979, 101, 4140. T. E. Wolff, J. M. Berg, P. P. Power, K. 0. Hodgson, R. H. Holm, and R. B. Frankel, J. Am. Chem. SOC.,1979, 101, 5454. 77 T. E. Wolff, P. P. Power, R. B. Frankel, and R. H. Holm, J. Am. Chem. Soc., 1980, 102, 4694. 78T.E. Wolff, .I.M. Berg, P. P. Power, K. 0. Hodgson, and R. H. Holm, Inorg. Chem., 1980, 19, 430. Holm I M I I I m d Y 'v)N fit! d + L v 0 zgE I + d' 467 Metal Clijsters in Biology Simultaneous with our initial report of a Fe-Mo-S cluster, i.e. [Mo2Fe6Sg-(SEf)gl3- (14;R = Et), in 1978,74 the structure and several properties of [M02Fe6S8(SPh)9]~- (15;R = Ph) were described by Christou, Garner, and co-workers.79980 Thereafter this group, using essentially the same reaction system as that shown in Scheme 4, has reported the synthesis, structures, and electronic properties of a number of clusters (15) and their tungsten ana- logues.81-85 Clusters (15) and (16) have been the most thoroughly investigated.Their formation is accountable in terms of the limiting stoicheiometries of reactions (1 3) and (14). Purified yields of cluster salts that exceed 50 % from both reactions, ~MoS,~-+ 6FeCI, + 17RS-+ [Mo,F~,S,(SR),]~-+ 4RSSR + 18CI-(13) --f2MoSa2-+ 7FeCI, + 20RS-[Mo,F~,S,(SR),,]~-+ 4RSSR + 21CI-(14) are usual in the author's laboratory. A different type of bridged double-cubane, [MO~F~~S~(~FL~-OM~)~(SR)~]~-(17), is isolated when reaction (13) (if R = Ph) is RS 3-\ Me 0 RS SR conducted in the presence of excess methoxide The tungsten analogue is formed in methanol in the absence of methoxide.84986 The use of Buts- in reaction (13), with no methoxide ion, affords the corresponding cluster (17; R = In this case the Mo(,uz-SBut)3Mo bridge unit is presumably destabi- lized by steric interactions. The reaction system given in Scheme 4 has been shown to lead to the assembly of a new family of clusters, the double-cubanes (14)-(17).Each of these contains 79 G. Christou, C. D. Garner, and F. E. Mabbs. Inorg. Chim. Acra, 1978, 28, L189. 8o G. Christou, C. D. Garner, F. E. Mabbs. and T. J. King, J. Chem. Sot-., Chem. Commun., 1978, 740.S. R. Acott, G. Christou, C. D. Garner, T. J. King, F. E. Mabbs, and R. M. Miller, Inorg. Chim. Acra, 1979, 35, L337. 82 G. Christou, C. D. Garner, and R. M. Miller, J. Inorg. Biochem., 1979, 11, 349. 83 G. Christou, C. D. Garner, F. E. Mabbs, and M. G. B. Drew, J. Chem. SOC.,Chem. Commun., 1979, 91. 84 G. Christou and C. D. Garner, J. Chem. Soc., Dalron Trans., 1980, 2354. 85 G. Christou, C. D. Garner, R. M. Miller, C. E. Johnson. and J. D. Rush, J. Chem. SOC., Dulron Trans., 1980, 2363. G. Christou, C. D. Garner, T. J. King, C. E. Johnson, and J. D. Rush, J. Chem. SOC., Chem. Commim., 1979, 503. G. Christou, P. K. Mascharak, W. H. Armstrong, G. C. Papaefthymiou, R. B. Frankel, and R. H. Holm, J. Am. Chem. SOC..1982, 104, in the press.Holm the desired MoFe3S4 unit, incorporated in the structural proposal (10) for the Mo site in nitrogenase. As yet, no individual MoFesS4 cluster has been identified among the products of this system, whose initial composition does not evidently preclude the formation of species such as [MOF~~S~(SR)~]~-~~-. The reaction steps in the assembly system shown in Scheme 4 are currently under investigation. In reaction (13) the first identifiable species formed is (18;R = Ph), which has been prepared ~eparately.~~?~~ This is one member of an expanding group of recently synthesized, smaller Fe-Mo-S complexes, also including [C12FeMoS4I2- (19),89-91, [(PhS)2Fe2MoS6I3-(20),92 [Cl4Fe2MoS4l2-(21),90p93[FeM02s8]~-(22),9*$95[(C2H4S2)2FeM02S6]~- (23),96 and [(NO)2FeMoS4l2 -.97 Certain of these complexes may represent fragments of the Mo site in the enzyme and FeMo-co. For example, there is a resemblance between (21) and the structural J (18) L = RS (19) L = CI D.Coucouvanis, E. D. Simhon, D. Swenson, and N. C. Baenziger, J. Chem. SOC.,Chem. Commun., 1979, 361. R:H. Tieckelmann, H. C. Silvis, T. A. Kent, B. H. Huynh, J. V. Waszczak, B.-K. Teo, and B. A. Averill, J. Am. Chem. SOC.,1980, 102, 5550. so D. Coucouvanis, N. C. Baenziger, E. D. Simhon, P. Stremple, D. Swenson, A. Simopoulos,A. Kostikas, V. Petrouleas, and V. Papaefthymiou, J. Am. Chem. SOC.,1980, 102, 1732. 91 A. Miiller, H.-G. Tolle, and H. Bogge, Z. Anorg. Allg. Chem., 1980, 471. 115. gz R. H. Tieckelmann and B.A. Averill, Inorg. Chim. Acta, 1980, 46, L35. 93 A. Miiller, S.Sarkar, A,-M. Dommrose, and R. Filgueira, Z. Nautrforsch., Teil.B, 1980, 35, 1592. s4 J. W. McDonald, G. D. Friesen, and W. E. Newton, Inorg. Chim. Acta, 1980, 46, L79. 95 D. Coucouvanis, E. D. Simhon, and N. C. Baenziger, J. Am. Chem. SOC.,1980, 102,6644. 96 P. L. Dahlstrom, S. Kumar, and J. Zubieta, J. Chem. SOC.,Chem. Commun., 1981, 411. D7 D. Coucouvanis, E. D. Simhon, P. Stremple, and N. C. Baenziger, Inorg. Chim. Acta, 1981, 53, L135. Metal Clusters in Biology proposal (1 l), and (22) exhibits an S = $-type e.p.r. spectr~rn.~~*g~ The synthesis and properties of a number of these complexes and their tungsten analogues have been reviewed by Couco~vanis.~~ 4 Properties of MoFesS4 Clusters A.Structures.-Single-crystal X-ray determinations have proven indispensable in the recognition of these clusters. Thus far, structures of [MOzFesSg(SEt)s]3-(14;R = Et),75 [Mo2Fe~&(sEt)g]~- (15; R = Et),75981 [M02Fe&8(SPh)g]~- (15;R = Ph),8o [M02Fe7Ss(SEt)12]~-(16;R = Et),78and [MozFe7Ss(SCH2Ph)l2I4- (16;R = CHZP~)~~have been solved. In addition, the structure of a cluster that is described as [MO~F~&~(SCH~CH~OH)~]~-has been reported.83 Our results indicate that this species is the mixed-ligand cluster [Mo2Fe&s(p-sEt)s- (SCHzCHzOH)6]3-. Structures of the tungsten analogues, i.e. [WzFesSs-[%“7%‘ ScTs’12y-[Mo,Fc, %(SCp,),r-(4’) Ma MO 6638 Ma Fe(41 3 319 Ma Fe 2 7x) Fe Fe 2 712 Ma Fc 2 72312) Fc Fe 2687(3) Figure 2 Structures of [Mo,Fe6S,(SEt),l3-(l4; R = Et), [Mo,Fe6S,(SEt),l3- (15;R= Et), [Mo,Fe,S,(SEt),,13- (16;R = Et) and [Mo,F~,S,(SCH,P~),,]~-(16;R = CH,Ph), excluding carbon atom and protons (Reproduced by permission from J.Am. Chem. Soc., 1979, 101, 4140 and Inorg.Chem., 1980, 19,430) Holm and [W~F~~SS(OM~)~(SP~)&-,*~(SEt)g]3-,82 [WzFe&(ScHzPh)12]~-,~~ have also been determined. The latter provides a characterized example of the M(w2-OMe)3M bridge unit, assuring its presence in the molybdenum clusters (17), which are of corresponding composition and closely related physicochemical properties. The replacement of molybdenum for tungsten effects only small changes in the dimensions of the cluster, as expected from the difference of -0.01 8, in the six-co-ordinate radii of these elements.98 Four detailed structures of specific examples of clusters (14)-(16) are set out in Figure 2, and a stereo-view of a cluster of type (1 5) is shown in Figure 3.Each Figure 3 A stereo-view of the structitre of [Mo,Fe,S,(SEt),13-(1 5; R = Et),excluding ethyl groups (Reproduced by permission from J. Am, Chem. Soc., 1979,101, 4140) structure consists of two MoFe3S4 sub-clusters, related by a symmetry centre or plane and connected by different types of bridging units to afford the overall double-cubane arrangements. The sub-clusters, which resemble the cubane-like Fe4S4 clusters (4), consist of interpenetrating, nearly concentric, and imperfect MoFe3 and S4 tetrahedra that afford MoFe3S4 cores closely approaching idealized CsV symmetry. Each iron atom is terminally co-ordinated by one thiolate ligand.Although all clusters are necessarily mixed-valence in iron, no structure shows any evidence for localized iron(i1,iri) sites, a property in common with the clusters (4).13 Six-co-ordination at the molybdenum sites in (14)-( 17) is completed by ligation to three bridging atoms. Because of disorder the bridge structure of cluster (14) has not been unambiguously established, but there is no doubt as to the presence of three bridging atoms. Chemical anal~ses~~9~7 are consistent with the S:Fe:Mo atom ratio in the formulation (14), which is employed in the following sections. This type of bridge is readily distinguished from that in (15), which is -0.4 8, longer and not disordered.The clusters (16) are bridged by a central iron atom, with trigonally distorted octahedral co-ordination furnished by the two sub-clusters acting as terdentate ligands. The Fe-S bond distances in the bridge78 and the S7Fe isomer shifts77 clearly show the presence of iron(iir) and iron(ii) in the cluster trianion and tetra- anion, respectively. 98 R.D. Shannon, Artu Cr,vstullogr., &ct. A, 1976, 32, 751. 471 Metal Clusters in Biology Whereas MoFe3S4 and WFe& clusters and the bridging arrangement in (16) are new structural components that have thus far been formed only in the reaction system shown in Scheme 4, the bridge units in (14), (15), and (17) are found in other compounds.Recent structural work has demonstrated the presence of M(p2-SR)3M in [(q7-C7H7)Mo2(SR)3(CO)zL],where L is Cog9 or P(OMe)3,100 [(q5-C5H5)2M02(SMe)3C13],101 [W2(SEtj3Ch(SMe2)2] (24).lo2 Similarly,and Wr(p2-S)(p-SEt)2Woccurs in [W2S(SE~)~C~~(C~HBS)~](25)lo3and M(p2-OMe)3M in [(q7-C7H7)(q3-C7H7)Mo2(OMe)3(C0)2]104and in [(q3-C7H7)(q4-C7H~)W2-(OMe)3(C0)4].104 With the exception of the last two compounds, M Ma separations are substantially shorter than those in (14), (15), and (17) (M -. -M 2 3.2 A), where direct metal-metal interactions between sub-clusters contribute no appreciable stability to the double-cubane structures. The W-W distances in (24) [2.505(1) A] and (25) [2.524(1) A] are very short, and nearly the same, in Et Et /S\Cl\ /s, ,c1,c1Me2S-W-S-W-SMe2 -Me2S-,w,-~CI / y/\C]CI’ Et contrast to the corresponding distances in the molybdenum clusters (15) and (14).Strong metal-metal bonding occurs in the two tungsten complexes, which contain unexceptional terminal ligands and whose oxidation states of tungsten atoms are the same or one unit higher than those that are considered most probable for molybdenum atoms in (14) and (15) (see below). Hence it appears that the incorporation of molybdenum (or tungsten) atoms into MFe3S4 clusters markedly attenuates the metal-metal interactions that are intrinsic to the MIv(p2-S)(p2-SR)2MIv and M111(p2-SR)3MIVbridge units in classical complexes. The double-cubanes (15)-( 17) contain sub-clusters with core [MoFe3S4]3 oxidation levels.In (14) the sub-clusters are formally inequivalent, having [MOF~~S~]~+>~+oxidation levels, but are crystallographically indistinguishable in the particular salt in~estigated.~~The newer clusters [MoFe4Sd(SEt)3-(cat)3I3-(26)l05 and [Mo~Fe6S&~2-sEt)2(SEt)4(3,6-Pr~2cat)2]~-(27)106 (cat = catecholate), whose syntheses and properties are described in a later section, also contain cores with a net charge of 3 +.Their detailed structures are presented in 9s K. Weidenhammer and M. L. Ziegler, Z. Anorg. Allg. Chem., 1979, 455, 29. looI. B. Benson, S. A. R. Knox, P. J. Naish, and A. J. Welch, J. Chem. Soc., Chem. Conimun., 1978, 878. lol C. Couldwell, B. Meunier, and K. Prout, Acta Crystallogr., Sect.B, 1979, 35, 603. lo2 P. M. Boorman, V. D. Patel, K. A. Kerr, P. W. Codding, and P. Van Roey, Inorg. Chem., 1980, 19, 3508. Io3 P. M. Boorman, K. A. Kerr, and V. D. Patel, J. Chcm. Suc., Dalron Trans., 1981, 506. lo4 K. Weidenhammer and M. L. Ziegler, Z. Anorg. Allg. Chem., 1979, 455, 43. T. E. Wolff, J. M. Berg, and R. H. Holm, /norg. Chcm., 1981, 20, 174. lo6 W. H. Armstrong and R. H. Holm, J. Am. Chem. Suc., 1981, 103, 6246; and research in progress. Holm -3 4- EtS \ El Fe-S 0 0 EtS (26) L Figure 4.Consistent with the identity or near-identity of oxidation levels, (sub) cluster Mo-S and Mo-Fe distances occur in the narrow intervals 2.34-2.36 and 2.69-2.73 A, respectively. These values are in good agreement with those deduced from EXAFS analysis of native FeMo proteinsls and of FeMo-co (see Table 1).Further, the cubane-type MoFe3S4 configuration provides shells of three sulphur and three iron atoms around the molybdenum atom, a con- figuration that is compatible with the ranges of EXAFS occupancy numbers. Complex (26), with a mean Mo-0 distance of 2.15(2) W,l05 affords an EXAFS spectrum qualitatively resembling that of FeMo-co, in which the molybdenum atom presumably experiences O/N ligand-atom interactions in NMF solution.53 Other conceivable representations of the Mo site in the native enzyme and FeMo- co have not yet been adequately examined, including an EXAFS test of proposal (11) with a complex such as (21). In the absence of other Mo-Fe-S cluster species of demonstrably more faithful structural features, those containing MoEe3S4 units have been pursued as preliminary models on the basis that they contain some structural elements in common with Mo sites in the native enzyme and cofactor.B. Ligand-substitution Reactions.-The chemistry of the Fe-S clusters (3) and (4) has been significantly expanded by the occurrence of reactions resulting in substitution of thiolate ligands. Effective reactants include electrophiles such as thiols [reaction (l)], acid halide~,10~~1~~ and, more recently, phenol, which affords an isolable salt of [Fe4S4(0Ph)4I2- .log With benzoyl chloride, the fully substituted clusters [FenSnC14]2- (n = 2 or 4)have been isolated and structurally The reactions of [Mo~FesSs(SEt)g]~-chara~terized.l~~~~~O (15; R = Et) and [M2Fe7Ss(SEt)12l3- [M = Mo, i.e.(16;R= Et), or W] with thiols and acetyl chloride at ambient temperature have been examined in some detail in the systems shown in Scheme 5 [reactions (15)-(l8)].l1l Of the two types of sulphur atoms that are potentially subject to attack by electrophiles, only those of terminal thiolates proved reactive under conditions in which (sub) cluster lo’ G. B. Wong, M. A. Bobrik, and R. H. Holm, Innrg. Chem., 1978, 17, 578. lo* R. W. Johnson and R. H. Holm, J. Am. Chem. Soc., 1978, 100, 5338. loSW. E. Cleland and B. A. Averill, Inorg. Chim. Acra, 1981, 56, L9. 110 M. A. Bobrik, K. 0. Hodgson, and R. H. Holm, Inorg. Chem., 1977, 16, 1851 ll1 R.E. Palermo, P. P. Power, and R. H. Holm, Inorg. Chrm., 1981, 20, in the press. Metal Clusters in Biology (7) Mo Mo Fe(4)3092 Fe 2725 Fe Fe 2709 [MoZFe6s~'s~~'6(C,~H~60~)~]e S(6) S Mo Mo 3 El3 Mo Fe 2 760 Fe...h 2 697 --Figure 4 Stritctures of [MoFe,S,(SE t)3(~at)3]3(26), [MolFe,S,( SEt),(3 ,6-Prn,cat)(27; R = Et), and [Mo,Fe,S,(SPh),15-. Carbon atoms of thiolatr ligands and a portron of the catecholate (cat) rings are omitted (Reproduced by permission from Inorg, Chem., 1981, 20, 174 and J. Am. Chem. Soc., 1981, 103, 6246) Holm structures were left intact. Formation of the products [Mo2Fe&(p~-SR)3- (SR')613-(28), [MO~F~~SE(~L~-SR)~C~~]~- (30),(29), [M~F~~S~(~~-SR)G(SR')~]~-and [M2Fe7S~(p2-SR)sCls]~- (31) was monitored by spectroscopic and electro- chemical methods. Examples of each type of mixed-ligand cluster were isolated.With one exception, our findings are in agreement with results described by other investigator^.^^^^^^^^^ Treatment of [MozFe6S~(SEt)g]~-with excess 2-hydroxyethanethiol affords (28;R = Et, R' = CHZCH~OH),and not the fully substituted cluster (15;R= CH2CH20H). Identity of the reaction products obtained in different laboratoriess3y is assured by the essentially indistinguish- able cell constants of their Et4N+ salts, which differ from those of authentic (E~~N)~[Mo~F~~SE(SCH~CH~OH)~]prepared by reaction (1 3).l11 Resolution of this matter presents a consistent picture of the reactivity of the clusters (15) and (1 6) with thiols and acetyl chloride.Evidently the adjacent metal centres reduce the nucleophilicity of bridging sulphur atoms such that they are not protonated by thiol acids as strong as benzenethiol (pKa z 6.5) nor attacked by the strong electrophile acetyl chloride. Kinetic studies of reaction (1) indicate that pro- tonation of bound thiolate is the rate-determining step in s~ibstitution,~~~and detection of thioester products in the reaction of (3) or (4) with acid halide~~O~~~~* demonstrates direct attack of electrophile on thiolate. These processes are likely to occur when (15) or (16) react with these reagents. The reaction systems shown in Scheme 5 do not offer a route to individual MoFesS4 clusters nor to single- or double-cubane species carrying a labile substituent at the molybdenum site, which is a possible requisite to the binding and activation of substrates of nitrogenase (excluding H+;see below) by this series of compounds.Other experi- ments have shown that the iron(I1)-bridged form of (16) is more reactive than its iron(Ii1) counterpart, and that the bridge structure of the latter may be disrupted with catechols. Some results from the second approach are described subse- quently. C. Electron-transfer Reactions.-Electrochemical studies have established the electron-transfer series shown in equilibria (19)-(21), based on the trianionic clusters (14)-(17).779859879111 Evidence for these series, in the form of cyclic voltammograms of clusters in which R is ethyl, is presented in Figure 5.In each, the steps with potentials E1 and E2 correspond to one-electron reactions of sub- clusters, resulting in changes in their oxidation levels and in the internal El E2 (15), (17): [(2u)l3-+ [(a+ /3)14-+ [(2P)15-(20) El (16): [(Fe3+ + 2a)I3-+ [(Fe*+ + 2a)l' + [(Fe2++ a + ,@)I5-ES+ [(Fez++ 2/3)6-(21) a = [M03+Fe2.67+S2-, y = [Mo~+F~*.G'+.S*-34]I+/3 = [M03+Fe2.33+S2-]L+ 6 = [M04+Fe2.33+S2-]3+34 9 34 'la G. Christou and C. D. Garner, J. Chem. Soc., Chem. Conmiin., 1980, 613. 113 G. R.Dukes and R. H. Holm, J. Am. Chem. Snr., 1975, 97, 528. 475 Metal Clusters in Biology I d m m n00 s Ir, - 0,n d 476 Holm -1.60 V Figure 5 Cyclic voltammograms of [Mo,Fe,S,(SEt),13-(14;R = Et), [Mo,Fe,S,-(SEt),13-(15;R= Et), and [Mo2Fe,S,(SEt),,l3-(16;R = Et), recorded in DMFsolutions at 100mV s-l.Peak potentialsiv versus the saturated calomel electrode (s.c.e.) are indicated. A platinum working electrode was used distribution of electrons, as represented by formulations a-8. The series (21) also contains the Fe3+/Fe2+ step of the bridging iron atom, which occurs at potentials that are less negative than El and E2. In addition, (2-) and (1 -) 417 Metal Clusters in Biology clusters of type (1 5) are detectable by cyclic voltammetry, as is the (2 -) species of cluster (14). In this regard, the two one-electron oxidations of the cluster (17;R = But) are particularly well defined by both cyclic voltammetry and differential pulse polarography,s7 but, as yet, no oxidized species have been isolated.There is also electrochemical evidence for the (6-) and (7 -) forms of certain clusters (15).85 The very negative potentials (5-2 V) at which these species are produced render their isolation extremely difficult. Consequently, effective experimentation is limited to the reduced members within the series of equilibria (19)-(21). Inasmuch as all transformations of substrates of nitro-genase are reductive, these species are of principal concern in the context of models of the enzyme. The electron-transfer steps in the series of equilibria (19)--(21) do not meet all electrochemical criteria for reversible transfer of charge, but they are effectively reversible, chemically, in that anodic and cathodic peak currents for each are essentially equal.Chemical reversibility is also demonstrated by the spectroelectrochemical experiment depicted in Figure 6 for a typical cluster of type (15). Here the cycle (3-) -+ (4-) -(5-) -+ (4-) -(3-) is traversed over a period of about four hours, with 5-7% loss in intensity of the 394 nm band of the re-oxidized (3-) cluster, Reduction of [M02Fe&(sPh)g]~- with sodium acenaphthylenide affords [M~zFet&(SPh)g]~-, which, although very 355 nm 355 Figure 6 Absorption specfra of [ MoaFe6Ss(SEt)J-** , measured in art optically -15--transparent thin-layer electrochemical (OTTLE)cell in DMF solution Holm sensitive to oxidation, has been isolated as its crystalline Et4N+ salt.Its structure, shown in Figure 4, reveals that there is retention of the double-cubane arrange- ment, with a substantial lengthening (by 0.15 A) of the Mo --Mo distance as + compared to (3-) clusters. Oxidized and reduced clusters, in solution, im- mediately equilibrate by the comproportionation reaction (22), for which log Kcom = (El -E2)/0.059. For the redox series based on clusters (14), (15), and (17), (El -Ez)= 200 k 20 mV and KcomE lo4. The difference of potentials is much greater than the statistical value of 36 mV, indicating that there are interactions between sub-clusters when they are 3.2-3.8 A apart. In clusters of type (1 6),where the Mo * -Mo separation is 6.9 8,(see Figure 2) prior to reduc- tion, (El -E2) is much smaller, and usually is not resolved. This behaviour is evident in the cyclic voltammogram of [M02Fe&-(SEt)12]~- in Figure 5, where (El -Ez)< 100mV is a reasonable estimate.These separations of potentials provide a simple means of identifying products of synthesis that contain triply bridged double-cubanes, and they are frequently used in the author's laboratory for that purpose. One application has been the demon- stration of the retention of bridge structure in the ligand-substitution products [(28)-(3 l)].lll Proton n.m.r. spectra, which exhibit isotropically shifted resonances, owing to paramagnetism of the cluster, are also valuable in identifying double-cubane structural types and the positions in which ligands have sub- st it uted .779 l1849 D.Electron Distribution in Sub-clusters.-In addition to a longer Mo * -. Mo distance and attendant small increases in bridge Mo-S distances and Mo-S-Mo angles, the structure of [Mo2Fe&@Ph)9I5- (Figure 4) reveals other differences when compared to those of several [Mo2Fe&(SR)9l3- structure^.^^ The Mo-S, Mo . . Fe, and Fe-S distances in'sub-clusters are marginally longer (by 0.01-0.02 A), resulting in a mean increase in the volume of MoFesS4 of 2.6%. The mean terminal Fe-SPh distance is significantly longer (by -0.07 A), Similar behaviour is found in structural comparisons of the (2-) and (3-) clusters (4). In the pairs [Fe4S4(SPh)4I2-l3- (1.9%, 0.032 &9p38 and [Fe4S4(SCH2Ph)4I2-f3- (2.6%, 0.046 A),689114the increases in the volume of the Fe& core and of the Fe-SR bond length assume the stated values.Unlike these clusters, in which structural changes of the core are essentially localized in particular bonds, leading to a change from an idealized geometry to another, changes in bond distances in the sub-clusters in [MO~F~&(SP~)~]~-are nearly uniformly dis- tributed. The structural results indicate small alterations of the Fe sites in clusters of type (I 5) upon reduction of each sub-cluster by one electron. In order to pursue the relationship between these changes and the distribution of electrons in the sub-cluster in different oxidation levels of the cluster, a procedure based on 57Fe isomer shifts (s),from Mossbauer spectra, has been e~plored.~~?~~ For lI4 J.M. Berg, K. 0. Hodgson, and R. H. Holm, J. Am. ChcJm.Soc., 1979, 101, 4586. 479 Metal Clusters in Biology various Fe-S complexes whose compositions define the (mean) oxidation states s of iron (ranging from 2+ to 3+), values of 6 are well fitted by the linear equation (23). This equation refers to values of 6 for tetrahedral FeS4 sites that S/mm s-l = 1.44 -0.43 s (23) are measured at 4.2 K and for which the reference is iron metal at this tempera- ture. As examples, the dianionic and trianionic clusters (4) provide cases of Fe2'5+ and Fe2.25+, respectively. Mossbauer spectra of the trianionic clusters (14)-( 17),759779s59S7and of the tetra-anionic (FeII-bridged) form of (16)77 have been described. Spectra of sub-clusters have been interpreted in terms of two or three incompletely resolved quadrupole doublets, with very similar values of 6.In none of these clusters (or in any considered below) do isomer shifts correspond to localized iron(I1,III) sites; this is a behaviour that has previously been noted for the clusters (4). clusters haveMossbauer spectra of the [MO~F~~SS(SP~)~]~-I*-.~-been examined in frozen solutions at 4.2-80K;87 the (4-) cluster, which has not been isolated, was generated by reaction (22). The spectra were analysed as two quadrupole doublets whose isomer shifts yield, from equation (23), the oxidation-state formulations a and p as indicated in the series of equilibria (20). In these and other formulations the superscripted oxidation state of iron is the mean value of the states of the three iron atoms.Spectra of the (4-) cluster are interpretable by a model involving localized (a + p) sub-clusters, indicating that the rate-constant for intramolecular exchange of electrons, k, is 5 107 s-1 at T G 80 K. Similar experiments with the [M02FesSg(SEt)s]~-~~- clusters afford the sub-cluster formulations in the series of equilibria (19). The descriptions of the (4-) cluster, whose spectrum was not determined, have been inferred from the results for the (3-) and (5-) clusters. In all cases, the oxidation states of molybdenum were obtained by difference. Reduced members of the series of equilibria (20), based on the clusters (17), and of the series (21) have not been examined by Mossbauer spectroscopy.The descriptions that are shown are based on the indistinguishability of values of 6 of the oxidized clusters and of [Mo~F~~S~(SR)~]~-and by analogy with the series of equilibria (20) derived from the latter cluster. The formulations a--6 for the sub-clusters are not intended as literal de-scriptions but only as the best current estimates of distribution of charge over the metal sites in these electronically complex molecules. The more specific de- scription Fe2.C7+ is preferred to the value +2.5 ( i-O.l), which has been proposed by other investigator^,^^ for clusters of the types (15) and (17). The latter mean oxidation state requires electronically inequivalent sub-clusters in double-cubane trianions of these types; this is a feature for which there is no clear spectroscopic or structural evidence.The presence of localized sub-clusters in [MOzFesSs- (SR)g]4- argues against equilibration of inequivalent electron distributions in sub-clusters by internal transfer of electrons at a rate that is fast when compared to the time-scale of Mossbauer spectra. This may not be the case for the members of the series of equilibria (19), which possibly possess an electronically delocal- 480 Holm ized Mo-S-Mo bridge. In this event, the description M03*5+ is more appropriate than ones with integral oxidation states. The interpretations of charge distribution in the series of equilibria (19)-(21) lead to the proposal that changes in electronic structure that are consequent to electron-transfer reactions of double-cu banes are largely confined to sub-cluster Fe3 portions and associated sulphur atoms.The oxidation state of the molybdenum atom in a given series is considered to remain nearly constant. Several other observations are supportive of this proposal. The increase in Fe-SPh bond distances upon reduction of a cluster of type (1 5) to the (5 -) form must be associated with the larger radius of tetrahedral iron(1r) vs. that of iron(r1r) (0.14) Redox potentials of the couples [Mo2Fe6S8(SR)9I3-J- [series (20)] and [Fe4S4(SR)4I2-J- vary linearly [according to the relation (24)] as the sub- stituent R is varied, and at parity of R they are very nearly equal.87 These results indicate that, in the first couple, the orbitals in the sub-cluster that are involved in the redox process are appreciably Fe-S in character.These and other points of evidence concerning changes in electronic structure in the redox series, as well as a fuller description of comparative [M02Fe6Ss(SR)9]3-*5- structures, are presented elsewhere. 759 779 87 [E,(Fe-Mo-S)/V] = [0.85 E (Fe-S)/V] -0.16 (in DMF, 1’s the s.c.e.) (24) E. The Reduction of Substrates.-A reduced double-cubane cluster, such as [MOzFe6Ss(SR)9]5-, presents the uncommon property of being an electron carrier that is capable of delivering two electrons at strongly reducing potentials that are separated by only -200 mV. This property raises the possibility of exploiting reduced clusters in the reduction of substrates that are generally considered to be transformed in one or more ‘concerted’ two-electron steps, with accompanying protonation.Substrates of nitrogenase, such as those shown in Scheme 1, are of this type. The clusters could act as electron donors to other molecules that contain the site of reaction, or they themselves might function both as carriers and reactants in stoicheiometric or catalytic processes. An example of the first type of behaviour has been found. A water-soluble version of cluster (15) has been shown to be capable of replacing Fd in an illuminated chloroplast/hydrogenase system that evolves dihydrogen.ll5 The oxidation level(s) of the cluster under turnover conditions is (are) not known.The presence of hydrogenase is obligatory to activity inasmuch as the cluster does not evolve dihydrogen in the presence of reduced Fd. To examine the second possibility, research on reductive transformations of substrates in systems that contain reduced double-cubanes has been initiated. Using the isolated salt of [M02Fe6S8(SPh)g]~-, and benzenethiol as a protic substrate, the formation of dihydrogen in solutions in NN-dimethylacetamide, at 25 “C,has been demonstrated.116 In the presence of a sufficient excess of thiol (-500 equivalents), a quantitative yield of dihydrogen, based on the overall 115 M. W. W. Adams, K. K. Rao, D. 0. Hall, G. Christou, and C. D. Garner, Biockim. Biophys, Acta, 1980, 589, 1. 116 G. Christou, R. V. Hageman, and R.H. Holm, J. Am. CIiowi. Soc., 1980, 102, 7600. 481 Metal Clusters in Biology reaction (25), is obtained. With smaller excesses of thiol, the yields of dihydrogen + 2PhSH -5 [MO~F~&(SP~)~]~-[MO~F~,S~(SP~)~]~-+ 2PhS-+ H, (25) are reduced, and the (4-) cluster accumulates, at least partly as a consequence of reaction (22). Kinetic studies of the evolution of dihydrogen in systems that contain the (4-) cluster [generated by reaction (22)] are in progress.llT The sequence of reactions (26)-(29), corresponding to the overall reaction (30), is provisionally offered as a description of the course of events leading to the formation of dihydrogen. Protonation occurs at a bridging or terminal thiolate sulphur atom. The two-electron-transfer property of the double-cu bane occurs in reaction (28), in which a transient hydride, presumably stabilized to an extent by Mo-H-or Fe-H- interactions, is proposed.This species is then protonated in a slow step (29) to afford dihydrogen and the oxidized cluster. The reaction sum ((22) + (30)) = (25). (4-)a + PhSH + (4-)/HT + PhS-(26) (4-)/H+ + (4-) + (5-)/H+ + (3-) (27) (5-)/H+ + (3-)/H-(28) (3-)/H-+ PhSH -+ (3-) + PhS-+ HZ (29) 2 (4-) + 2PhSH -+ 2 (3-) + 2PhS-+ H, (30) a(4-) refers to the cluster [Mo2Fe,S8(SR)g]4-; (3-) and (5-) refer to similar clusters. In similar reaction systems, based on [MozFe1&(SPh)9]~- and oxygen acids, or other thiols, the evolution of dihydrogen is found, but yields are less than quantitative when 500 equivalents are used.With only 2 equivalents of Et3NH as the protic substrate, N 80%of the theoretical amount of dihydrogen (compared to -30% with 2 equivalents of benzenethiol) is ev01ved.l'~ Kinetic studies on the latter systems are under way. These results are not to be construed as requiring a single two-electron-donor cluster for the formation of dihydrogen. Under the same conditions, a yield of N 30% of dihydrogen, based on reaction (31), is obtained with N 500 equivalents of thio1.1167117 This reaction is the more 2[Fe4S4(SPh)J3-+ 2PhSH -+ 2[Fe4S,(SPh),12-+ 2PhS-+ H, (31) appropriate as a model system for hydrogenase inasmuch as these enzymes118 contain no molybdenum, and there is some evidence for the presence of Fe& ~lusters.3~9~~~A reaction sequence analogous to reactions (26)-(29), in which one reduced cluster acts as a reductant and another as both a reductant and a reaction site, is readily conceived.Incorporation of a reaction site and a capacity to transfer two electrons into the same molecule may provide kinetic and thermodynamic advantages, however. At present we view a reaction system such as reaction (25), together with its final mechanistic description, as being potentially useful in interpreting the formation of dihydrogen in enzymes that contain several clusters that are juxtaposed, so as to promote the efficient internal transfer of electons to the substrate. No substrate-reducing ability has yet been detected for the oxidized clusters [MO~F~GS~(SR)~]~- and [Fe4S4(SR)4I2-.11' T. Yamamura, G. Christou, and R. H. Holm, unpublished results. 118 M. W. W. Adams, L. E. Mortenson, and J.-S. Chen, Biochim. Biopfrys. Acm, 1981, 594, 105. Holm 5 Structural Alterations of the Molybdenum Site in Clusters At present there is no unequivocal evidence that binding and activation of substrates of nitrogenase takes place at a molybdenum-containing site in the native enzyme or in FeMo-co. The frequently espoused view that such a site is directly involved in enzymatic catalysis derives a measure of support from observations of abiological systems. Stable molybdenum-dinitrogen complexes have been isolated and structurally characterized,31 patterns of the protonation of dinitrogen that is co-ordinated at electron-rich Mo atom centres have been establi~hed,~~~-~~~and appreciable quantities of ammonia have been detected among reaction products from Mo/N2 systems that had been treated with protic or hydridic reagents.119-121*123-128Detailed consideration of these results, many of which have been reviewed,31*S2 is beyond the purview of this report.While the systems employed are decidedly non-physiological in terms of the ligation of the molybdenum atom and/or its oxidation state, they have provided the stimulating result that dinitrogen can be bound to and activated by molybdenum, and converted into ammonia under ambient conditions. Further, the establishment of modes of protonation of co-ordinated dinitrogen provides a valuable mechanistic baseline for any such systems with more physiologically relevant molybdenum complexes that may be shown to be capable of reducing dini trogen.In the present context, it should be remembered that a comparable effort has not been expended by the inorganic chemists to devise dinitrogen-fixing systems that are based on iron complexes. The occurrence of reaction (31) is one indica- tion that Fe-S clusters can effect reductive transformations. Another is reaction (32),which gives 560% yields of eth~1ene.l~~ When a deuterio-acid is present, the predominant sterlochemical product is cis-1,2-CsHzDz, as is found in the reduction of acetylene by nitrogenase. 2[Fe,S3(SPh)J3-+ 2HOAc + C2H2-+ ~[F~,S,(SP~I)~]~-+ 20Ac-+ C2H4(32) While mindful of these results, we are inclined toward the position that any IlQJ.Chatt, A. J. Pearman, and R. L. Richards, J. Chem. SOC.,Dalton Trans., 1977, 1852, 2139; ibid., 1978, 1766. 120 R. N. F. Thorneley, J. Chatt, R. R. Eady, D. J. Lowe, M. J. O'Donnell, J. R. Postgate, R. I-. kichards, and B. E. Smith, in ref. 25, Vol. I, pp. 171-193. lal T. Takahashi, Y. Mizobe, M. Sato, Y. Uchida, and M. Hidai, J. Am. Chem. SOC.,1980, 102, 7461; ibid., 1979, 101, 3405. lP2R. A. Henderson, J. Orgonomet. Chem., 1981, 208, C51. 123 G. E. Bossard, D. C. Busby, M. Chang, T. A. George, and S. D. Iske, Jr., J. Am. Chem. Soc., 1980, 102, 1001. 124 J. A. Baumann and T. A. George, J. Am. Chem. Sac., 1980, 102, 6153. lZ5B. J. Weathers, J. H. Grate, N. A. Strampach, and G.N. Schrauzer, J. Am. Chem. Soc., 1979, 101, 925. Iz6 E. L. Moorehead, P. R. Robinson, T. M. Vickrey, and G. N. Schrauzer, J. Am. Chem. SOC.,1976, 98: 6555. 12' G. N. Schrauzer, P. R. Robinson, E. L. Moorehead, and T. M. Vickrey,J. Am. Chem. SOC., 1976, 98, 2815. 12* C. R. BrDlet and E. E. van Tamelen, J. Am. Chem. Soc., 1975, 97, 91 1. lZQR. S. McMillan, J. Renaud, J. G. Reynolds, and R. H. Holm, J. Znnrg. Biorhem., 1979, 11, 213. Metal Clusters in Biology eventually productive reaction chemistry of MoFe3S4 clusters with dinitrogen is likely to involve the molybdenum site. Although a reaction scheme such as reactions (26)-(29), involving protonation of a ligand followed by bond-breaking and internal transfer of an electron, leading to a transitory metal hydride, can be entertained with protic molecules, clusters of the types (14)-(17) and their reduced forms are likely to prove ineffective in the activation of other substrates of nitrogenase.Co-ordinative saturation of the molybdenum atom is evident. Indeed, the clusters [Mo~Fe&s(SPh)g]3-9~- {as well as [Fe4S4(SPh)4]2-y3- ) do not react with dinitrogen at ambient temperature. Current research is directed at the introduction of more labile and chemically manipulable ligand environ- ments external to the MoFe3S4 cluster structure. A related goal is the isolation of single MoFe3S4 clusters in order that their electronic features, unperturbed by a neighbouring cluster [e.g.(14), (15), or (17)] or paramagnetic ion [e.g.(16)], can be examined.A. Bridge-cleavage Reactions and Products.-Inasmuch as substitution or cleavage of the bridge of clusters (15) and (16) does not result from their treat- ment with thiols or acetyl chloride under mild conditions, the reactivity of the iron(i1i)-bridged cluster (16) with other reagents has been explored. Because of the high affinity of catechols for iron(111),~~0 reaction (33) was carried 0~t.lO5 The unanticipated cluster (26) was isolated as its Et4N+ salt in 550% yield, 6 catH, [MO~F~,S~(SE~)~~]~--[MoF~,S,(SE~),(C~~),]~-(33) 12Et,N (26) based on molybdenum. Other molybdenum-containing products have not been purified sufficiently for identification. The structure of (26) (see Figure 4) consists of a single MoFe3S4 cluster with the a electronic formulation, and a [Fe(cat)3J3- subunit is triply bridged to the molybdenum atom.lO5 This arrange- ment leads to a decrease of the trigonal twist angle of the subunit by -12' compared to free [Fe(cat)3]3-,l31 and an overall symmetry of the cluster that closely approches C3.Cluster (26), while of interest in its own right, does not provide a magnetically unperturbed single-cubane species. Means of removing the [Fe(cat)3I3- subunit from the molybdenum site are under investigation. However, this cluster has provided a simple clue leading to entry into a new type of cluster structure. Inspection of molecular scale-models indicates that there are unfavourable steric interactions between ethanethiolate ligands and the 3-substituent on the catecholate ligand when the latter is a methyl or a larger group.This feature suggests that a different reaction product might be obtained from cluster (16) and an appropriate 3,6-disubstituted catechol. Reaction (34) (where R is C3H5 or Prn) proceeds smoothly at room temperature to afford the previously unknown clusters (27), obtained as their Et4N+ salts in lroA. Avdeef, S. R. Sofen, T. L. Bregante, and K. N. Raymond, J. Am. Chenz. Soc., 1978,100, 5362. 13' K. N. Raymond, S. S. Isied, L,. D. Brown, F. R. Fronczek, and J. N. Nibert, J. Am. Chem. SOC.,1976, 98, 1767. Holm [Mo,Fe,S,(SEt),,13-+ 5(3,6-R’,catH2)+ 4 Et,N -+ (34)[Mo,Fe,S,(SEt),(3,6-R’,cat),14-(27;R = Et)+ [Fe(3,6-R’,cat),I3-+ 6EtSH + 4Et,NH+ -50% yield after purification.lo6 Tungsten clusters can be prepared in an analogous manner.The structure of [MOzFesS~(SEt)s(3,6-Pr“2Cat)2]~-,shown in Figure 4, is that of a centrosymmetric double-cubane, having sub-clusters of the 01 electronic formulation.106 Each molybdenum atom is chelated by a catecho- late, and six-co-ordination is completed by thiolate ligation in the form of a Mo(p2-SEt)Fe bridge unit. The bridges, which are of a type that has not pre- viously been encountered, present a particularly interesting point of reactivity. The Fe-S bond is 0.04 A longer than the average Fe-SEt terminal bonds and the Mo-S bond is -0.12 A longer than Mo-SR distances in the bridges of clusters of the types (14)-(16). The possible substitutional lability of the bridge that is implied by these results has been investigated. In contrast to the outcome of reactions (15) and (17), treatment of the clusters (27) with a small excess of arylthiol, as shown in reaction (39, results in the [Mo,Fe6S,(SEt),(3,6-R’,cat),]*-[Mo,Fe,S,(SAr),(3,6-R’,~at),]~-+ + + (35) 6 ArSH 6EtSH substitution of both terminal and bridging ligands.The structure of one reaction product, [MOzFesSs(SPh)s {3,6-(C3H&cat }214-, as its Et4N+ salt, has been deter- mined, and it was found to have the doubly-bridged double-cubane arrange- ment (27).lo6 When dissolved in polar aprotic solvents such as dimethyl sulphoxide (DMSO), at ambient temperatures, the clusters (27) do not appear to retain the bridged structures that are present in the crystalline state.Isotro- pically shifted lH n.m.r. spectra demonstrate the presence of co-ordinated catecholate, and are consistent with a structure having all thiolates bound as terminal Fe-SR ligands. The situation is clearest for clusters with Ar = Ph or p-tolyl. Here the integration of the sharp meta-and para-proton or the methyl resonances vs the signals of the catecholate ring and the cation demonstrates that at least -90 % of the signal intensities of the aryl group occur in the form of resonances of terminal ligands. In no cases were signals that possibly originated from thiolate ligands in the bridge observed in spectral scans of k400 p.p.m. (from Me& as the reference peak). While the exact structures of the solute species are not known, the presently available results support disruption of the bridge and the attendant formation of single or bridged cubanes, involving the binding of solvent (L) at the molybdenum site.Possible formulations include [MoFe&(SR)3( 3,6-R’2cat)LI2-and [MozFesSs(SR)6(p2-L)(3,6-R’2cat)2I4-.Thus the solute clusters present the potentiality of molybdenum sites that have ligands that are sufficiently labile that they can be displaced by substrates of nitrogenase when the clusters are reduced. Further, clusters in which R is aryl are reducible at about the same potential (--1.1 V vs the s.c.e., in aprotic solvents) as that required for the formation of [MO~F~~SS(SP~)~]~-.~~ which 485 Metal Clusters in Biology can readily be manipulated under anaerobic conditions.Ongoing experimenta- tion is aimed toward the isolation and determination of the structure of these reduced clusters and the elucidation of their reactivities. B. Electronic Features of Bridge-cleavage Products.-The cluster [MozFesSs- (SEt)9]3-, of type (15), exhibits a magnetic susceptibility behaviour in the solid state, at 4.2-150 K, that is consistent with that of a Curie paramagnet having two sub-clusters, each with S = +.*7 This is a property that is of considerable interest in view of the presence of clusters in nitrogenase and FeMo-co with a spin system of S = 3 per molybdenum atom. In the solid and solution states, at cryogenic temperatures, this cluster displays rather complicated e.p.r.spectra which may arise from interactions between the sub-clusters and intermolecular interactions that are not well revealed in the magnetic susceptibility behaviour. Certainly these spectra bear little resemblance to an isolated S = 3 spin system of the type that is found in the native enzyme and in FeMo-~o.~~~*~~~~In this regard, the e.p.r. spectra of solutions obtained from salts of [Mo2Fe&(SAr)6- (3,6-R’zcat)2l4- are more encouraging.lo6 A case in point is the spectrum of a frozen DMSO solution, prepared from (E t4N)4[M02Fe&s(SPh)6 {3,6-(C3H5) 2-cat)z] and presented in Figure 7. This spectrum corresponds to an effectively axial S = 3 species with apparent g values of 4.38 and 1.99, associated with the Ho -3.06 I.99 Figure 7 The X-bund e.p.r. spectrum of a -10 mmol 1-’ solution in DMSO, preparedfrom (Et4N)o[M02Fe6S8(SPh),{3,6-(C,H,),cat) 2] and recorded at 8 K; selected g-values are indicutcci Holm I k9 > Kramers’ doublet.Using theoretical expressions for g values,52 the assign- ments g, r 4.38, gs > 3.0, and gz r 1.99 are reasonable for this doublet. If the crossing point on the baseline is taken as gx z 3.6, the spectrum corresponds to the spin Hamiltonian parameter ratio, IE/D I, of -0.06, This estimate signifies a slightly rhombic g-tensor ; purely axial electronic symmetry would afford a spectrum with g, = 4 and g,, = 2. The large linewidth of the gyfeature obscures the small rhombic splitting. In all systems thus far examined, including those prepared from analogous tungsten clusters, the gx and g, components have not been resolved.Detailed studies of magnetic susceptibility at low temperatures have not been carried out. Magnetic moments of 4 PB per molybdenum atom that were obtained in DMSO solutions at ambient temperature, however, are consistent with a spin-quartet ground state. Several other features of the e.p.r. spectra that are typified by that in Figure 7 deserve mentim. A weak, incompletely resolved, feature at g N“ 5.9 is consistently present, and it possibly corresponds to the gz component of the I+$> Kramers’ doublet. The cluster [MozFesSs(SEt)6(3,6-Pr*~cat)2]~-has been prepared in two isotopically enriched forms, containing >99 atom percent of 95Mo (I = 3) or 57Fe (I = 4.).These species were subjected to the ligand- substitution reaction (35)with benzenethiol and the e.p.r. spectra of the resulting clusters were examined in situ. The 95Mo- and 57Fe-enriched clusters and the natural-abundance cluster gave essentially identical spectra. There .is no clear evidence for hyperfine broadening of the g x 2 signal upon 57Fe enrichment. Line broadening of the g 5 2 signal has been reported for one enriched FeMo protein.52 6 Current Status and Prospects The information presented here is intended as a conspectus of our approach to a synthetic representation of the Mo-Fe-S cluster catalytic site of nitrogenase. The research is predicated on the viability of MoFe&-type clusters as at least pre- liminary models.The results described are those obtained through August, 1981. For more detailed accounts of the synthesis, structures, and many of the pro- perties of these clusters outlined here, reference is made to the original publica- tions from the author’s laboratory.74-7~~87~1~~~~~6~1~~~~~GIn pursuing the objective, our efforts have benefited from a continually improving state of physicochemical definition of the biological cluster. With due recognition that FeMo-co has not yet been shown to reduce dinitrogen to ammonia6’ and is not the functional equivalent of the native enzyme in the reduction of at least one other ~ubstrate,l3~ it remains as the most immediate object of synthesis. As a means of assessing the current status of the synthetic approach, the selected properties (1)-(7) of FeMo-co in Table 1 are compared with those of synthetic clusters.(1) The ratios of Fe, S*, and Mo atoms attained in synthetic species are 3:4.5:1 for (14), 3:4:1 for (15), (17), and (27), 3.5:4:1 for (16), and 4:4:1 for (26). The smaller complexes (1 8)-(23) have less favourable Fe: Mo ratios. No 13* C. E. McKenna, J. B. Jones. H. Eran, and C. W. Huang, Nuture (London), 1979, 280. 61 I. 487 Metal Clusters irt Biology discrete cluster has yet been prepared that has atom ratios falling within the indicated ranges for cofactor. (2),(3) All clusters are anionic and all contain organic ligands. Since cysteine is absent in FeMo-co, ligation of other than ‘inorganic’ sulphur presum- ably involves one or another of the indicated possible ligands, whose presence is likely to depend on the particular procedure used to prepare the cofactor.Ligation of cysteinate in the native enzymes cannot be discounted, MoFe3S4 clusters are stable when terminal Fe-SR ligands are replaced by chloride, e.g. (29) and (3 l), and Mo-0 interactions are found in clusters (17), (26), and (27). Terminal Fe-0 binding appears to be possible, at least with anionic ligands, judging from the isolation of [Fe4S4(0Ph)4I2-109 and [(PhO)2FeMoS4I2-133 and the generation in solution of [Fe&(SR),-(OAC)~-~]~-(n = 0-3).loS Another conceivable function of oxygen ligands, viz. the bridging of metal sites within a cluster by oxide or hydroxide, remains an open question.No such clusters containing also molybdenum and iron and inorganic sulphur have been prepared. (4) All MoFeaS4 clusters contain iron atoms that are spin-coupled within sub-clusters; one model for spin-exchange interactions in the form of anti- ferromagnetic coupling has been proposed.134 The clusters (27; R = Ph) and (27; R =p-tolyl), in solution, give S = 3 e.p.r. spectra; g2and g, resonances are not resolved. The representative spectrum in Figure 7 has g values com- parable to those of the native enzyme (e.g. 4.32, 3.66, and 1.9852) and of FeMo-co. Spectral linewidths for the enzyme are narrower than those of FeMo-co, and the extent of rhornbicity ( /E/DI = 0.055) is very close to that estimated for the synthetic cluster.The sign and value of the zero-field split- ting of the Kramers’ doublets in the latter species have not been determined. Linewidths for clusters are larger than those of cofactor, whose rhombicity is well resolved and which exceeds that of the native enzyme and those of synthetic clusters. Line broadening was not observed in 95Mo- and S7Fe- enriched clusters. (5) Dimensions of MoFe3S4 sub-clusters in (14)-(17), (26), and (27) (Figures 2, 3, and 4) are essentially invariant. Values of Mo-S* and Mo . Fe distances and the presence of three S* and three Fe atoms at these distances are in good accord with conclusions from molybdenum EXAFS analysis of FeMo- co and native enzymes.ls Clusters (17) and (26) provide possible models of the Mo site in the cofactor, for which recent EXAFS results implicate 0-or N-~o-ordination.~~ (6) Redox activity of synthetic clusters is demonstrated by the existence of the series of electron-transfer equilibria (19)-(21).Sub-clusters with electronic formulation aare those with S = 4. Reduction to the p formulation, as in [Mo2Fe&(SR)9l4-*5-, results in species whose magnetic moments at ambient 133 H. G. Silvis and B. A. Averill, Inorg. Chim. Acta, 1981, 54, L57. 134 G. Christou, D. Collison, C. D. Garner, F. E. Mabbs, and V. Petrouleas, Inorg. Nucl. Chem. Lett., 1981, 17, 137. 488 Holm temperature are consistent with S = 2 for their sub-cl~sters.8~ A change from S = 3 to an even spin (deduced from the lack of an e.p.r. signal) occurs upon reduction of the biological cluster in the native enzyme and in cofactor.(7) No synthetic cluster has yet been shown to reconstitute the FeMo protein of Azotobacter vinelandii UW45, but not all types have been tested. The reported lack of activation of this protein by FeMo-cluster,GO which appears to be derived from (and to possess a lower ratio of Fe:Mo atoms than) FeMo-co, suggests that reconstitution activity may be the single most stringent criterion of the faithfulness of a synthetic cluster to the cofactor. That certain properties (e.g. the MoFe2,&,4 structural arrangement and the S = 4 system) are common to enzyme and cofactor indicates that the basic structure of the cluster core is probably the same in both environments. (Ligands that are external to the core may differ, however.) If these structures are in fact the same, the expressed criterion is appropriate to the assessment of synthetic ciusters as models of the Mo-Fe-S cluster of nitrogenase.It is evident that a model that is consistent with the key properties of FeMo-co has not yet been achieved. Nonetheless we are persuaded by the collective properties of MoFe&-type clusters that certain elements of these species may be correct. Important individual properties, such as the structural relation of (21) to the site proposal (ll), and the e.p.r.66.94 and magneticl35 properties of (22), which are indicative of a S = 3 system, are found in other Fe-Mo-S species, as noted earlier. Whatever are their deficits, the clusters described here at least offer the merit of approaching properties (4)-(6) of the biological cluster. In a structural sense, we regard the MoFesSs fragment of a MoFesS4 cluster to be the most likely element in common with the biological cluster, This fragment, of course, does not carry the full complement of iron (and possibly inorganic sulphur) atoms that are indicated by chemical anaiysis of FeMo-co. One conception for incorporating this fragment into a larger cluster, consistent with the analytical ranges for the cofactor, has arisen from recent experiments by G.Christou and K. S. Hagen in the author's laboratory. In this work the new cluster [coSSS(s ph)8l4 -has been structurally characterized. It contains the [coS(~4-s)6]*+core, composed of a cube of cobalt atoms with a sulphur atom above each face, forming an octahedron.The structure approaches idealized 0tL symmetry. Each cobalt atom occupies a tetrahedral site and has one terminal thiolate ligand. In terms of population of metal- and sulphur-atom sites it is the inverse of [Fe6S8(PEts)6]2+,136 symmetry)which has a [Fes(p3-S)8I2+core (of 01, and iron(~rr) atoms in five-co-ordinate sites. From the structure of the cobalt cluster, the species (MOF~~S~(SR)~L~]~-, which has structure (32) and is of trigonal symmetry, has been devised by Christou. Iron atoms occupy tetrahedral sites, with terminal thiolate ligands specified for the sake of definiteness. The MoFe& fragment, with a geometry similar to that in MoFesS4 clusters, is apparent ; six-co-ordination at the molybdenum atom is completed by three 135 A.Simopoulos, V. Papaefthymiou, A. Kostikas, V. Petrouleas, D. Coucouvanis, E. D. Simhon, and P. Stremple, Chem. Phys. Lett., 1981, 81, 261. 136 F. Cecconi, C. A. Ghilardi, and S. Midollini, J. Chem. Soc., Chem. Commrm., 1981, 640. Metal Clusters in Biology SR R RS ligands L. The strilcture has the appealing feature of being built largely of Fe2S2 rhombs such as are present in clusters (3) and (4). In Fe-S cluster chemistry the p4-S bridging atoms find precedent in [F~~S~(SBU~)~]~-.~~~Jn terms of its topoIogy, the ‘n-type string bag cluster’ of LuG4 is one-half of the core polyhedron of (32). This structural proposal of the nitrogenase cluster is offered in the spirit of speculative modelling. As with model structures proposed by 0thers,~3-66 the object of the proposal remains to be synthesized. Acknowledgments. Research in the author’s laboratory has been supported by grants from the National Institutes of Health and the National Science Foundation. Thanks are due to Drs. W. H. Armstrong, G. Christou, and P. K. Mascharak for preparation of Figures, and to these and the remainder of my enthusiastic research collaborators whose names are found in the references. 13’ G. Christou, R.H. Holm, M. Sabat, and J. A. lbers, J. Am. Clwm. Soc.. 1981, 103, 6269.

ISSN:0306-0012    

DOI:10.1039/CS9811000455

出版商:RSC    

年代:1981

数据来源: RSC

摘要:

Physical Chemistry of Biological Morphogenesis By L. G. Harrison DEPARTMENT OF CHEMISTRY, UNIVERSITY OF BRITISH COLUMBIA, VANCOUVER, B.C., CANADA V6T 1Y6 1 Introduction Morphogenesis is the creation of a complicated shape out of a simpler one by chemical processes in living organisms. To the physical scientist, the essence of it is symmetry-breaking. Yet the precept that ‘asymmetry begets asymmetry’ is not necessarily violated. Natural disturbances, which are continually present every- where, contain adequate asymmetry to serve as antecedent for any shape, however complex. But how do living organisms go about making a rather precise selection of what parts of the available asymmetry to amplify? We know that the specification for the development of an organism is written in the genetic code.For some thirty years, the most rapidly advancing divisions of biological science have been those dealing with DNA, RNA, and proteins. The powerful theories here have been those of molecular geometry, statically perceived in terms of building block or jigsaw puzzle fitting together of parts. Genetic information does not, however, specify how much of a protein is to be produced at any time, nor where it is to go. No-one today believes that the nucleus contains a reduced spatial map from which the organism is built, as a building is constructed from an architect’s plans, or as a human sperm was once believed from vague microscopical resemblance to contain a ‘homunculus’. There is a wide gap in understanding between macromolecular geometry and the large- scale shape of the whole organism, and this gap is not being closed very quickly.This review is intended, therefore, to show physical chemists a field in which experiment and theory are rather far apart, and in which they might be able to make sof-ne contribution towards bringing them closer together. All physical chemistry is divided into three parts: structure, equilibrium, and kinetics. In approaching a set of phenomena for which there are no definitely proved or generally accepted explanations, the physical chemist must ask himself in which of these fields the answers are likely to lie. Two accounts with almost identical titles have no overlap whatever in outlook. These are a paper written in 1952 by Turing,’ entitled ‘The Chemical Basis of Morphogenesis’, and a chapter of Lehninger’s ‘Biochemistry’,2 entitled ‘The Molecular Basis of Morphogenesis’. The former expounds a model, in terms of rate equations for reaction and diffusion, showing how spatially inhomogeneous arrangements of material might be established and maintained kinetically in a system in which the equilibrium state is a homogeneous uniform distribution.This type of theory has later been A. M. Turing, Philos. Trans. R. SOC.London, Ser. B, 1952, 237,37. A. L. Lelminger, ‘Biochemistry’, (2nd Edn.), Worth Publishers, 1975, chap. 36. Physical Chemistry of Biological Morphogenesis extensively elaborated in the theory of ‘dissipative structures’ of Prigogine and his coliab~rators,~-~ and more specifically tied to biological examples in the work of Gierer and Meinhardt.‘-+B The structural approach2 is based on precise evidence for the geometry of assemblies of quite large numbers of protein molecules in viruses,299 micro- tubules,lO and microfilaments.ll Its successes stop short of the cellular level.Studies of microstructure with the aid of optical and electron microscopes are, however, providing increasing evidence that microtubules and microfilaments are often present at places and times of morphogenetic significance, especially at the onset of cell division in both plants (‘pre-prophase band’ of microtubulesl2) and animals (contractile ring of microfilamentsl3). Many biologists expect that the ‘cytoskeleton’ of these structural proteins will prove to be the geometrical link between the molecular and the macroscopic scale.Living organisms are never at equilibrium, and are probably farthest away from it when undergoing morphogenesis. Nevertheless, there may be cases in which the appearance of disparate parts is to be accounted for principally in terms of a drive towards minimum free energy, rather than in terms of geo- metrical fitting, or imbalances in rates of reaction and transport processes. There is then some correspondence between the process concerned and phase transitions. In respect of some particular kinds of interaction, the ‘equilibrium’ state is heterogeneous. Classical theories of phase nucleation, critical supersaturation, critical micelle concentration, and spinodal decomposition have been used in discussion of pattern formation.14,15 The behaviour of mixtures of cells sometimes mimics molecular mixtures in ways that suggest a concept of cell-as-molecule.Such mixtures frequently ‘sort out’ into two separate aggregates, each of a single type of cell. SteinberglG showed that this phenomenon could be explained in some detail by differential adhesions between cells. This leads to an analogue of surface tension, measurable by the sessile drop method,17 for an assembly of I. Prigogine, in ‘Fast Reactions and Primary Processes in Chemical Kinetics’, 5th Nobel Symposium, ed. S. Claesson, Interscience, New York, 1967, p. 371. P. Glansdorff and I. Prigogine, ‘Thermodynamics of Structure, Stability, and Fluctuations’, Wiley-Interscience, New York, 1971.G. Nicolis and I. Prigogine, ‘Self-Organization in Non-equilibrium Systems’, Wiley, New York, 1977. A. Gierer and H. Meinhardt, Kybernetik, 1972, 12, 30. H. Meinhardt, J. Cell Sci., 1977, 23, 117. * A. Gierer, Prog. Biophys. Mol. Biol., 1981, 37, 1. sA. Klug, Fed. Proc., Fed. Am. SOC.Exp. Bioi., 1972, 31, 40. lo ‘Microtubules’, ed. K. Roberts and J. S. Hyams, Academic Press, 1979. l1 ‘Cell Motility: Molecules and Organization’, ed. S. Hatano, H. Ishikawa, and H. Sato, University Park Press, Baltimore, 1979. l2 J. D. Pickett-Heaps and D. H. Northcote, J. Cell Sci., 1966, 1, 109. l3 R. Rappaport, Znt. Rev. Cytol., 1971, 31, 169; T. Schroeder, Z. Zellforsch., 1970, 109,431, J.Cell Biol., 1972, 53, 419, Proc. Natl. Acad. Sci. USA, 1973,70, 1688; R. E. Kane, ref. 1 I, p. 639. l4 T. C. Lacalli and L. G. Harrison, J. Theor. Biol., 1978, 74, 109. l5 J. W. Cahn, Acta Metallurg., 1961, 9, 795; J. Chem. Phys., 1965, 42, 93. la M. S. Steinberg, J. Exp. Zool., 1970, 173, 395. l7 H. M. Phillips and M. S. Steinberg, Proc. Natl. Acad. Sci. USA, 1969, 64, 121. Harrison cells, and is in general closely analogous to classical concepts of solution thermo- dynamics and immiscibility. The term ‘self-assembly’ has commonly specified the structural approach, but is nowadays being extended to include sorting-out by differential adhesion. The dangerously similar term ‘self-organization’ is used by Prigogines for the kinetic approach.It is perhaps least confusing to avoid these terms, unless one uses them to cover the whole topic, regardless of mechanism. In the 1930’s, a term ‘morphogenetic substance’ was current, and it later became clear18 that what was then envisaged is now known as messenger RNA, and that it does not solve the problems of morphogenesis. From the 1950’s onwards, the term ‘morphogen’ has been used, sometimes rather vaguely. Its most specific meaning is to specify either of two substances, an ‘activator’ and an ‘inhibitor’, commonly designated X and Y, which appear in Turing’s modell and most later versions of reaction-diffusion. Candidates for the style and title of morphogen range from ammonia via cyclic adenosine monophosphate (CAMP) to some rather large proteins and glycoproteins. In non-living chemical systems, cerium-catalysed oxidation of malonate by bromate (Belousov-Zhabotinski reaction) shows time and space periodicities, for which the analogues of three morphogens ‘X, Y, and Z’ are probably BrOz-, Ce4+, and Br-.5J9-21 No substance is yet definitely established as a morphogen in a living system.2 The Kinetic Approach :Reaction-diffusion A. Symmetry-breaking:Optical Resolution as an Example.-Reaction diffusion mechanisms for morphogenesis, when expressed as rate equations, commonly contain simple autocatalytic terms [equation (l)] where X represents a displace-axiat = kx (1) ment from equilibrium and may therefore assume both positive and negative values.Hypothetical chemical mechanisms, such as Prigogine’s ‘Brusselator’3-5 commonly show a bimolecular autocatalytic step [equation (2)], assumed to 2x + Y = 3x (2) have orders corresponding to molecularities. The essence of this concept of auto- catalysis was first mentioned, and is most simply illustrated, in regard to a diffeient but closely related problem, the origin of optical activity in nature, e.g. why living material contains, in general, only L-amino-acids and D-sugars. Mills,22 in a presidential address on inorganic stereochemistry to a regional meeting of the British Association in 1932, pointed out that, if a reaction in which a prochiral reactant A yields a chiral product D or L is autocatalysed bimole- cularly and stereospecifically [equation (3)], then the racemic state is unstable.J. Brachet, preface to S. Puiseux-Dao, ‘Acetabularia and Cell Biology’, trans]. P. Malpoix-Higgins, Springer-Verlag, New York Inc., 1970. A. M. Zhabotinski, Biojzika, 1964, 9, 306. 2o A. T. Winfree, Science, 1972, 175, 634; Sci. Am., 1974, 230, 82. 21 R. J. Field and R. M. Noyes, Furaday Symp. Chem. SOC.,1974,9,21;J. D. Murray,J. Theor. Biol., 1976,56, 329. %* W. H. Mills, Chem. Znd. (London), 1932, 750. Physical Chemistry of Biological Morphogenesis 2D 2L A-tDandA-tL, (3) If, for example, the D: L ratio is at any time 2:1, the rates of formation of D and L (with order as expected from molecularity) are in the ratio 4: 1 and the system is moving closer to resolution as D.Mills showed that fluctuations could be expected to be present in adequate amount to start a system on the road to resolution. Mills’ concept has been rediscovered or elaborated several times.23-27 At least two account~~~?~~ indicated that an intermediate stage should be an assemblage of separate territories, each occupied by pure D or pure L. This is a rudimentary morphology, but an unstable one, in that large areas will ultimately surround and destroy smaller ones. If we now use X to represent the measure of asymmetry [equation (4)], and not X=D-L (4) the concentration of a single substance [as one might do in relation to equation (2)], the rate of growth of asymmetry is: aX/at = kfAD2-kfAL2 = kiA(D -L)(D + L) = krA(D + L)X (5) There are many circumstances, especially when X is small, in which the total product (D + L) is likely to be varying much more slowly than the asymmetry X.Equation (5) is then, approximately, equivalent to equation (1); bimolecular auto- catalysis gives first-order exponential growth of asymmetry. The effective rate parameter is, however, a pseudo rate-constant, containing both the total reactant concentration A and the total product concentration (D + L), both assumed to be held roughly constant by external supply and removal. Figure 1 shows a more complete model of this kind. Catalysis takes place by adsorption of D and L on to sites on a catalytic surface. Arrangement of D and L is random, site activation is bimolecular and stereospecific, there are sufficient concentrations of D and L in solution to maintain adsorption saturation, and adsorption-desorption is rapid.Autocatalysis of D product ion then depends on D and L through the ratio D/(D + L). 9 is diffusivity along a co-ordinate s. aDpt = krAD2/@ + L)2 -krD3/(D + L)2 -kextD + 93a2D/3as2 (6) The equation is similar for aL/at, with D and L interchanged throughout. This model introduces the reverse reaction on the catalyst, an important feature neglected in Mills’ original suggestion. Since this is a cubic term, it is capable of preventing the asymmetrising effect of the squared term for the forward reaction. The symmetry-breaking is, indeed, made possible only because the non-stereo- specific removal, giving the linear term kext D, opposes the effect of the cubic term.I showed2’ that, in a well-stirred system with the diffusion term omitted from equation (6),symmetry-breaking will occur from the racemic steady state only if: *3 F. C. Frank, Biochim. Biophys. Am, 1953, 11, 459. F. F. Seelig, J. Theor. Biol., 1971, 31, 355; 1971, 32, 93. =E. P. Decker, Nature (London),New Biol., 1973, 241, 72. a6 L. G. Harrison, J. Theor. Biol., 1973,39, 333. 27 L. G. Harrison, J. Mol. Evol., 1974,4, 99. Harrison I R Figure I The simplest irreversible (dissipative) cycle for symmetry breaking by bimolecular aurocatalysis. When di8hsion is considered, the system is usu~lly envisaged as essentially one-dimensional, elongated along the direction s kext > kr/4 (7) If we put in the diffusion term, and examine what happens if a simple sinusoidal pattern of asymmetry is superimposed on the racemic steady state, then we find X = a(t)sin(2ns/A) (8) that for long wavelengths the amplitude u increases with time, while for short wavelengths it decreases.The threshold wavelength for growth is :28 A0 = 2~9+/(kext-kr/4)* (9) This simple example illustrates many of the important general features of kinetic mechanisms for the generation of spatial pattern : (a) A threshold rate of interference from the rest of the universe must be exceeded for symmetry-breaking to occur. The term kext is a measure of the rate of entropy increase, or dissipation of energy, in the surroundings, necessary to create and maintain a low-entropy ordered state in the sys- tem.Hence the Prigogine school use the term ‘dissipative structure’ for what I prefer to call ‘kinetically maintained structure’. (6) Reaction-diffusion mechanisms can set up quantitative scales of distance. This is seen in the dimensionality of (9/k)*, where k is a first-order L. G. Harrison, in ‘Origins of Optical Activity in Nature’, ed. D. C. Walker, Elsevier, 1979, chap. 10. 495 Physical Chemistry of Biological Morphogenesis constant. This form is exemplified, for a threshold spacing, in equation (9). More complex expressions for ‘chemical wavelengths’, e.g. the Turing wavelength given in equation (18) below, are variations on the same theme. This quantitative aspect is a particular strength of the reaction-diffusion mechanism that is not easily matched in structural or equilibrium models.(c) Reaction-diffusion tends to amplify long-range order. For example, the simplest possible autocatalysis-diffusion rate equation is : If we put into this the sinusoidal disturbance (S), we find that X grows according to a simple exponential growth law: where Figure 2(a) shows how kg increases with wavelength. If we use the more com- plicated model of equation (6),start from a small disturbance to the spatially uniform racemic steady state, and confine attention to the early stages of growth or decay of the disturbance, this is again exponential, and gives equation (13), which corresponds to Figure 2(a) with a different value for the threshold.This monotonic increase explains the eventual dominance of one product over the other, as in spontaneous optical resolution. Formation of a stable pattern requires a different dependence, e.g. as in Figure 2(b), in which some finite-wavelength disturbance grows fastest and hence is eventually selected by the system out of whatever nature happens to provide at all wavelengths. Turing’s model, des- cribed in Section 2B, is designed to produce this dependence of kgon wavelength. Spontaneous resolution has been discussed here as an analogy, which provides the quickest route to understanding some basic features of the kinetic approach to all kinds of symmetry breaking. Two questions more directly related to optical asymmetry immediately arise, however.First, since the kinetic dis- cussion shows the possibility, in a non-living chemical system, of spontaneous resolution in converting a prochiral substrate into a chiral product, without any preliminary doping of the catalyst with optically asymmetric material, has this ever been observed? In 1958, there was a claim29 that such spontaneous reso- lution had been found in the reduction of a-ketoglutaric acid and its oxime to a-hydroxyglutaric acid and glutamic acid by hydrogenation on Raney nickel [equation (14)]. BPT.Isoda, A. Ichikawa, and T. Shimamoto, Rikagaku Kenkyusho Hokoku (J. Inst. Phys. Chem. Res., Tokyo),1958,34,134 (In Japanese; translation by Y. Koga available from L. G. Harrison). Harrison x.-\ A0 TURING AUTOCATALYSIS Figure 2 Exponential growth rate constants for pattern (i.e.non-uniform concentration distribution) sinusoidal in position s: (a) for simple autocatafysis and difision of one substance, equation (1 0); more rapid amplification of longer wavelengths; (h) for Turing’s two-morphogen model, showing most rapid amplification at A,. For (a), A, =2n(/gk)B (Reproduced by permission from ref. 28) Ni, H,HO2CvO2H +HO2CWCO2H (14) NOH H NH2 I have tried to repeat this (unpublished; McGinnis, thesis30) and obtained equivocal data on resolution but definite indications of autocatalysis. This topic deserves further study on modern catalysts more sensitive to asymmetrising influences. Second, the question arises of just how close any symmetry-breaking in living systems may be to this model.It is well known that small chiral molecules are usually found in only one enantiomeric form in living organisms, and that this asymmetry carries through to their assembly into structures as large as proteins, including the assembly of several protein subunits into a polymer. I have pointed out, however, that on the large scale both enantiomers are present in structures as obvious as our right and left hands, and I have made a ‘wild surmise’ that the smallest spatial scale at which right- and left-handed structures appear might be an important scale to consider, and might be not far above the scale of organi- sation of a few protein subunits into a p0lymer.2~J~ B. Turing’s Equations.-Turing’s proposal1 was principally in the form of linear 30 M.J. McGinnis, M.Sc. thesis, University of British Columbia, 1977. 31 L. G, Harrison and T. C. Lacalli, Proc. R. SOC.London, Srr. B 1978, 202, 361. Physical Chemistry of Biological Morphogenesis differential rate equations involving two measures of displacement from equi- librium, X and Y. These are, for diffusion in one spatial dimension s: These equations, being linear, can be solved exactly in analytical form. The solutions are summations of real or complex exponentials in the time and distance variables. For them to represent morphogenetic behaviour, there are various restrictions on the values of the four rate constants and two diffusivities. The essence of the mode of operation of the Turing model has been described qualitatively by Ma~nard-Smith.~~ We suppose kl and k3 to be positive, /c2 to be negative, and ks to be zero; also 9s > 9~;i.e.X catalyses its own growth and that of Y, Y inhibits X, and the inhibitor diffuses faster than the activator (X). This implies that, from a localised centre of activity, Y will, at least in early stages of development, spread out further than X. The useful slogan ‘short-range activatian; long-range inhibition’ is therefore often used to characterise mech- anisms of the Turing type. But, in the full operation of a Turing model to give a precisely ordered pattern, both X and Y are periodically distributed over the whole region concerned. In Maynard-Smith’s illustration (Figure 3), we suppose that the system, an elongated one along one spatial dimension s, is initially at equilibrium through- a Figure 3 Qualitative picture of how the Turing model leads to X and Y waves in phase, redrawn following Maynard-Smith’s ill~stration~~ (schematic onfy; nor computed).See text for explanation J. Maynard-Smith, ‘Mathematical Ideas in Biology’, Cambridge University Press, 1968. Harrison out with respect to both X and Y. Somewhere, a small and fairly localised positive X-disturbance is introduced [Figure 3(a)]. It grows (kl catalysis) and spreads (9~diffusion), but a positive Y displacement grows within the X dis-placement and spreads faster (k3 and BYterms) [Figure 3(b)]. Where Y has spread beyond the effective boundaries of the X peak, its inhibitory effect (k2 term) produces X troughs [Figure 3(c)]. In these regions of negative X, the k3 term becomes an inhibition of Y, i.e.where X has been 'pushed down' it can 'pull Y down after it' [Figure 3(d)]. The implication is that X and Y are on the way to settling down into wavelike patterns in phase with each other.In this model, we do not find an activator at one end of the system and an inhibitor at the other; X and Y have their maxima and minima together. A convenient abbreviation of the mathematical treatment of Turing's equa- tion@ may be obtained by going directly to the apparent final state in Maynard- Smith's illustration, and taking initial X and Y disturbances to be sinusoidal in s, and exactly in phase or 180" out of phase, so that 8 = Y/X is everywhere the same and depends only on time, 8 = 8(t).Insertion of such disturbances into equations (15) confirms the possibility of this type of solution by yielding an ordinary differential equation for the evolution of 8 with time.Eventually, the X and Y waves will settle down to a constant ratio of amplitudes, and will grow or decay together in simple exponential fashion with a rate constant kg. How kB varies with wavelength depends on the values of the kinetic and diffusion parameters. A typical plot in the region of morphogenetic significance is shown in Figure 2(b). A maximum for kg occurs at a finite wavelength; given sufficient time, and sufficient concentration range available for exponential growth of both X and Y on both sides of equilibrium, a pattern of this wavelength will finally dominate the system.The growth rate constant (doubled, for convenience) is given by: 2kg = kl + k4 - (2n/A)'(9x+ 9~)+ (b' + 4kzkS)f (1 4) where b = k4 -k, - (2n/A)'(9~-Bx) (1 7) The maximum of kg is at: -Am = 249~-B~)~/[k4k, + (BY+ 9~)(-kaks/9~9~)']*(18) which is dimensionally of the form (9/k)* and in fact reduces for reasonable values of the parameters to 27r[gx9~/(-kzk~)]*. Since k2k3 is negative in the region of interest, k, may be complex, indicating time-oscillatory behaviour. When kg is real and positive, spatial pattern can be established without any time- oscillatory behaviour. For fairly wide ranges of the kinetic constants and diffusivities, kg varies with h as shown in Figure 2(b); kg is real at all finite positive wavelengths, and passes through a maximum.For the maximum to be sharp, with kg going negative at long wavelengths, kl and k4 must be chosen within a rather restricted range, and the latter must be negative (Y self-inhibitory). T. C. Lacalli and L. G. Harrison, J. Thror.Biol., 1978, 70, 273. Physical Chemistry of Biological Morphogenesis All the required conditions were given by Turing,l very briefly and without indication of derivation. Lacalli and 133934 gave simple derivations of these, and showed how the conditions could be plotted on a diagram of kl and kq. Turing’s model has the obvious defect of unrestricted exponential growth, which must always become unrealistic after some finite time, because concen- trations grow to exceed any possible value or, in the other direction, become negative.More realistic models give non-linear differential equations, often corresponding to practical limitations on concentrations, such as saturation effects. The strength of the Turing model is that, for the initial development of pattern out of uniformity, the mathematical process of linearisation casts most of these non-linear models back into the Turing form.34 The Turing model therefore shows how pattern should start to grow out of uniformity for most reaction- diffusion mechanisms; but it does not give a full account of how the pattern may change as it develops, because of non-linearities.Mathematical analysis of reaction-diffusion has so far been carried out mainly for fixed boundary conditions. Biological development is concerned largely with moving boundaries, because organisms grow. Experiments in this field often also involve changes in boundaries, e.g. when one chops an organism into pieces and watches what happens next. The term ‘regulation’ is used to describe the ability of an organism to maintain or re-establish pattern in the face of such natural or artificial changes in boundaries. Turing’s model has been criticised35736 for lack of regulatory capacity. A major objection has been that the Turing model requires a precise fit between some multiple or half-multiple of the ‘chemical wavelength’ corresponding to maximum kg and the size of the system.The former is fixed by the dynamic constants of the mechanism; the latter is continually changing. This type of objection is invalid, for two reasons. First, even if the fit of wavelength to system size is inexact, a pattern can develop, and one particular pattern may arise for quite a wide range of system size. Figure 4 shows a computation of the development of a half-wave of X and Y along an elongated system. The cal- culations were carried out for possible relevance to the morphogenesis of slime moulds (Plate 4). Computation of the effect of cutting the specimen into two unequal pieces showed re-establishment of the pattern in both pieces. Amplitudes are, however, shown normalised in the diagrams.In this, as in all calculations on the linear equations, there is a great increase in amplitude as time goes by, and it is a valid objection that regulation usually takes enough time that the system must have moved into a region of concentrations in which one should be using more realistic non-linear models. Second, as mentioned above in relation to equation (3,autocatalytic rate parameters may often turn out to be pseudo rate-constants, containing concealed concentrations that happen to be, in practice, constant, if one looks beyond the rate equations to the possible chemical mechanisms giving rise to them. In the 34 T. C. Lacalli and L. G. Harrison, J. Theor.Biol., 1979, 76,419. 35 J. Bard and I. Lauder, J. Theor.Biol., 1974, 45, 501.3e C. H. Waddington, ‘Principles of Embryology’, George Allen and Unwin, London, 1956, pp.422-423. 500 Harrison Figure 4 Computation of restoration of pattern in both parts of an elongated system cut into two unequal pieces, on the basis of the Turing model, for comparison with what happens in slime moulds (Plate 4). Amplitudes are shown normalized; there is a very large increase as time goes on case of equation (3,if the total reaction product is P = D + L, the auto- catalytic rate constant is kfAP; for the adsorption saturation model on a hetero- geneous catalyst [equation (6)],the corresponding expression is kfA/P.If this type of expression is used for the constant k in equations (10) and (12), the threshold wavelength for growth of a disturbance is: A, = 2n(gP/krA)+ (19) and is thus controlled by the reactant/product ratio A/P, which depends upon supply and removal and can certainly change as a system grows.It is, for example, quite easy to envisage circumstance^^^ in which reactant is supplied to the bulk of a system across its boundaries and used up in such a way that its steady-state concentration falls as the system grows. Equation (19) then shows ho increasing as the system grows. I have shown3l that the same kind of argument can be made for the ‘chemical wavelength’ Am, and that this can in fact grow in proportion to the size of the system, so that a pattern can be stable for indefinite increase in size. The important point here seems to be that the reaction scheme should have two sequences in parallel starting from the same reactant A, with the ‘morphogen variable’ X or Y being a measure of imbalance between the concentrations produced along the two branches, e.g.equation (20). 37 L. G. Harrison, in ‘Developmental Order: Its Origin and Regulation’, 40th Annual Sym- posium Society Developmental Biology, ed. S. Subtelny, Alan R. Liss Inc., 1981 (in press). Physical Chemistry of Biological Morphogenesis where the products PI and PZ and the parameters of rate processes leading to them need not be symmetrically related as closely as enantiomers. The model as so far described shows how non-uniform concentration dis- tributions in space could arise for two hypothetical substances X and Y,or for some greater number of substances PI, Pz, etc., with the symbols X and Y serving for quantitative measures of imbalance.The question remains of how X and Y are supposed to influence morphogenesis. In single-celled plants, which have rigid cell walls and are capable of converting differential reaction rates into a complicated shape (Plates 2 and 3), it is reasonable to envisage X or Y as a catalyst for growth processes at the cell surface. Embryology of multicellular animals is discussed very differently, in terms of a variety of levels of gene expression achieved by something analogous to the throwing of switches to turn on or off the activity of particular genes in certain individual cells or groups of cells. Ka~ffman~~-*O has suggested, first, that the notation 1 or 0 could be used to indicate on or off for each switch, leading to a ‘binary epigenetic code’ in which, for example, the notation 0100 on a region of an embryo would mean that of four genes A, B, C, D, only B is switched on in that region.Second, he suggested that the function of a chemical morphogen is to throw a switch, e.g. positive X turns A on while negative X turns A off. Third, he proposed that when a morphogen system goes through a succession of patterns as a system grows, each pattern can serve to switch on or off a new gene in some definite sequence, and that each switching is permanent. If we consider, for instance, two-dimensional Turing patterns on a growing ellipse, with the rate parameters truly constant so that increasingly complex patterns are favoured as the ellipse becomes larger, the expected succession of patterns has nodal lines and positive and negative regions as shown in Figure 5.Kauffman envisaged each pattern as switching one gene in 011 111 010 110@@@4 4 000 101001 100 Figure 5 Nodal lines of successive Turing waves on a growing ellbse (size shown nor- malized). At each stage,positive Xswitches aparticulargene on (1) and negative X switches ifoH(0). The resulting binary epigenetic code is shown for the first three stages. Nodes 1 and 2 may appear to have been established in the wrong order: for a full account, see Kauy- man,3e*40This model is hypothetical S. A. Kauffman, Science, 1973, 181, 310; ‘Cell Patterning’, CIBA Foundation Symposia, new series, no.29, Elsvier, Amsterdam, 1975, p. 201. 39 S. A. Kauffman, Am. Zool., 1977, 17, 631. loS. A. Kauffman, R. Shyrnko,and K. Trabert, Science, 1978, 199, 259. Harrison Plates 1-10 Some examples of morphogenesis Plate 1 Pores in the cellulosic cell wall of a single-celled alga, Cosmarium botrytis. The array of pores is not quite regular, but far from random. Nucleution-depletion model, section 3A and Figures 8 to 10 (Reproduced by permission from ref. 14) Physical Chemistry of Biological Morphogenesis Plate 2 Semicell morphogenesis in a single-celled alga, Micrasterias rotata. The cell has the shape of a biconvex lens, almost split in half (arrows) and with the semicircular margin of each half deeply indented.In vegetative reproduction, the cell splits at the isthmus between the two halves. A ‘bubble’ of cell wall grows out from each half, and develops a Harrison wavy outline, the lobes of which repeatedly square ofland bifurcate in a manner suggestingthe presence of non-linear concentration waves, sections 2B and C. Fully-grown, diameter-200pm.Numbers are hours ufter cell division Physical Chemistry of Biological Morphogenesis Plate 3 Growing t@ of a giant single-celled alga, Acetabularia mediterranea. The cell grows, by action mainly at rounded tip T, into a cylinder perhaps 4cm long 400pm in diameter. Every few days, the tip flattens and forms a ring of hair initials I from which a whorl W of hairs grows (W marks previous whorl; another is just forming at I).Reaction diffusion and Arrhenius-type temperature dependence of hair spacing at first appearance of initials, section 2D Harrison f 2 Plate 4 Multicellular morphogenesis without cell division or feeding: the cellular slime mould Dictyostelium discoideum. Thousands of independent amoebae, dividing and feeding on decaying vegetation, aggregate into an elongated (-1 mm) assembly (a to b to c) and differentiate into ‘pre-spore’ and ‘pre-stalk’ cells (c or e), which rearrange into the stalk and mass of spores of the fruiting stage d. Treatment of the differentiation by reaction-diflusion, section 2B. Restoration of pattern after cutting (e to f and g), computation shown in Figure 4 / Plate 5 Schematic representation of ‘engulfment’ and ‘sorting out’ experiments on two types of living cells from an animal embryo.Cells as analogues of immiscible molecules and differential adhesion, sections 3B and 4A Physical Chemistry of Biological Morphogenesis a b Plate 6 Gastrulation: (a) Schematic cross-section through a hollow sphere of one layer of cells, showing shape changes and cell movements by which the gut starts to form. Given in many textbooks as typical of processes in the vertebrates, actually seen in a form close to the idealised diagram only in Amphioxus and echinoderms. (Reproduced by permission of McGraw-Hill Book Co. from Gilchrist, A Survey of Embryology j (bj In nematode worms, two cells differentiate and move inward in a manner like the sorting out in Plate 5.From these cells, the gut starts to form. Turing regarded (a) as symmetry breaking from a sphere to be explained by reaction-diffkion ....:E' Division... bi-polarity.' A. B. C. Plate 7 Initiation of branches or leaves from aplant stem depends on changes in direction of celldivision. A two layers of cells lying along the stem. B, C: Growth of d branch upwards needs all three possible directions of cell division, here called E, H, and T because line of division completes the shape of one of those letters on top or front of diagram of cell. (Reproduced by permission from ref. 68) Reaction-diflusion control may lie in tubidin precursors, section 4C Harrison Plate 8 Hydra is about 1 mm long and has about lo6cells of about 15 di’erent types.H: hypostome; 1, 2, 3, 4: gastric segments; B: budding area; P: peduncle; D: basal disc. Grafting experiments using pieces from two specimens show that a grafr of 1 to 1234 does not grow an extra ring of tentacles, but a graft of 12 to 1234 does form ientac~es at the graft G. Gierer-Meinhardt reaction-diffusion theory, section 2C, accounts for this by producing a large activator peak out of the discontinuity in source gradient p. a ;activator;h: inhibitor (Parts of diagram reproduced by permission from refs. 47 and 6) Physical Chemistry of Biological Morphogenesis it ransplant Plate 9 The stump of an amputated left leg of a cockroach hasgraftedon to itan amputated right leg, rotated through 180".The parts fuse, andat the next moult, two supernumerary limbs, both left, grow from the graft. Clockface gradient which may need either reaction diflusion or diflerential adhesion of both to eAplain it, Figure 6 (Reproduced by permission from ref. 57) Plate 10 Four stages in growth of vertebrate limb (chick wing) front a limb bud, showing early origin of several regions within which parts of skeletal structure form by diflerentiation of cartilage cells (black in last stage shown). In some amphibia (newts), limb graft behaviour closely parallels that in insects, Plate 9. Positive feedback in cartilage formation and glycosaminoglycans, section 4B (Reproduced in modified form by permission from ref. 57) Harrison a manner which is not cancelled by the next pattern, leading to an increasingly complicated compartmentalisation of the region.He related this to experi- mental observations on insect development, first to the formation of some boundaries known as ‘clone restriction lines’ in the development of an insect wing, and second to the origin of segmentation at an early stage of embryonic development. These examples serve to indicate that, if the hypothetical morphogens exist at all, they are likely to be very diverse both in respect of chemical nature and in respect of the location of their morphogenetic activity. For the single-celled plants, this activity must be near to the cell surface and concerned with either addition of lipids to the cell membrane or addition of cellulose to the wall.For multicellular animals, they may be concerned with nuclear DNA and inhibitors of gene expression attached to it at specific points. C. Hypothetical Reaction Mechanisms and Non-linear Models.-Great diversity is possible in chemical reaction mechanisms that yield systems of non-linear rate equations equivalent, upon linearisation around equilibrium, to the Turing equations. Only a few schemes have been studied extensively by mathematical analysis and ~omputation.5~6J~~~O~~~ From these, however, it is already evident that different models have quite different characteristics in the regions of non-linear behaviour. The Prigogine ‘Brusselator’ model is the reaction : A+B+D+E (21) proceeding with the aid of morphogen intermediates X and Y according to: A+X (22d B+X+Y+D Wb) 2x + Y + 3x (22c) X+E (22d) Mathematical analysis has been mainly for time-independent A, B, D, and E, as fixed by supply and removal, and diffusible X and Y with, as in Turing’s model, 9y> 9x.The presence of two reactants, one of which converts into X while the other removes X, is an important feature of this model. As usual, there are threshold conditions for the development of kinetically maintained structure: A must exceed a threshold value, but B must be less than a threshold value. This makes it rather easy to devise models for control of A and B that would allow interesting things to happen over only part of a system, as quite commonly happens in morphogenesis. For instance, if B diffuses into a system from its boundaries, and is used up in an overall first-order decay [apart from its destruction in reaction (22b)], a steady state can arise in which B is higher at the boundary than at the *l J.D. Murray, ‘Lectares on Nonlinear-differential-equation Models in Biology’, Clarendon Press, Oxford, 1977. Physical Chemistry of Biological Morphogenesis centre of the system. Morphogenetic action may then be ‘switched on’ only in some central region. This could be relevant, for example, to tip growth of some algae, fungal hyphae, and root hairs of higher plants, in which the growth action is concentrated in a roughly hemispherical tip of a cell, leaving behind it a cylinder which grows not at all or very slowly.Sometimes, more complicated patterns can form at the tip (Plate 3). Variants of the Brusselator scheme have been devised by Tyson and co- w0rkers,4~~~3for example : Reactants + Y (23a) Y +x (23b) 2X+Y +3x (234 X +-Products (23d) For this class of models, computations show another important feature that is not shared by some other types of non-linear model (e.g.,the model of Gierer and Meinhardt discussed below). Morphogen peaks can rather easily arise, decay, and move around as pattern develops, in a manner somewhat reminiscent of Ostwald ripening of a precipitate, but of course with better spatial control and with the spatial heterogeneity sustained only by kinetic effects. La~alli~~ has shown by computations using the rate equations equivalent to equations (23), for a two- dimensional region with random input and no-flux boundaries, that a very irregular pattern of X peaks at first arises and gradually changes into a perfect hexagonal array. Not all models have the adaptability to find the kinetic route to this structure from an irregular one.Reaction schemes containing three intermediates X, Y,and Z are capable of generating both spatial pattern and time-oscillatory behaviour without the need for bimolecular autocatalysis in a single step. In some instances, the distinction is trivial, depending simply on the degree of approximation made in writing down the kinetic scheme. Consider, for example, the sequence: C+X+Y (24d X+Y+Z (24b) A+Z+X+Z (2W X+B (244 If the first two steps are fast, so that equilibrium is maintained in them at all times, they are merely a long-winded way of writing the addition of 2X to a catalytic site C to give an activated site CX2 which is Z.The first three steps are then equivalent to: A + 2X = 3X (25) A more complicated example of a three-intermediate system is the scheme of IaJ. J. Tyson and J. C. Light, J. Chem. Phys., 1973, 59, 4164. 43 J. J. Tyson and S. A. Kauffman, J. Math. Biol., 1975, 1, 280. T. C. Lacalli, Philos. Trans. R. SOC.London, Ser. B, 1981, 294, 547. Harrison Field and Noyes2l for the Belousov-Zhabotinski reaction. This is the cerium- catalysed oxidation of malonate by bromate, overall : 3CH2(C02H)2+ 4Br0,---f 9C02 + 4Br-+ 6H,O (26) Spatial pattern and temporal oscillations are made visible in this reaction by adding an Fe2+/Fe3+ couple with an indicator such as ferroin, giving orange and blue colours.For a stirred solution, one sees oscillations, in which the colours alternate in time. For an unstirred system in a tall cylinder, stripes of alternating colour are seen moving vertically. In a Petri dish, concentric rings of alternating colour expand from several centres, and interact where they meet in a manner not characteristic of interference of It remains unclear whether this reaction is closely analogous to important phenomena in biological systems, or whether it is a chemical curiosity. Stationary spatial order is not a common feature of the reaction, except in flow systems.45 Some doubt has also been cast on the existence of diffusive coupling in the system. In a tall cylinder, the coloured stripes con- tinue to move uninterrupted when the cylinder of solution is cut by a horizontal Plexiglas ~late.~6 The Field and Noyes scheme involves bromous acid, bromide, and cerium(rv) as the three intermediates.Out of an overall mechanism with many steps, the significant ones, with their X, Y,Z designations as used in the ‘Oregonator’ scheme, are probably: BrO9-+ Br-+ 2H+ -+ HBr02 + HOBr A+Y +X HBrO, + Br-+ H+ + 2HOBr X+Y +P BrO,-+ HBr02 + 2Ce3++ 3H+ -+ 2HBr0, + 2Ce4++ H20 (27c)X+B +2x + z 2HBr0, -+ Br0,-+ HOBr + H+ (27a2X --f products Q 4Ce4+ + BI-CH(CO~H)~+ HzO + HOBr -+ 2Br-+ 4Ce3++ 3C02 + 6H+ -+ 2Y (27e) In contrast to the rather changeable behaviour of this reaction, many biological systems show evidence for the existence of remarkably persistent gradients.Much of this evidence arises from cutting organisms and grafting so as to juxtapose pieces which nature would not have thought of putting together. (Plates 8, 9, Figure 6). The model of Gierer and MeinhardPs gives a good account of some of these situations, and has probably attracted more attention among experimental biologists than any other model of the reaction-diffusion type (this whole field of modelling being still regarded with much scepticism among biologists). The model envisages a gradient, fixed in time or changing only very slowly, of sources for the morphogens in an activator-inhibitor scheme. The latter is such that it tends to amplify small differences between regions, e.g.to O6 M. Marek and E. Svobodova. Biophys. Chem., 1975,3, 263. 46 N. Kopell and L. N. Howard, Science, 1973, 180, 1171. 513 Physiral Chemistry of Biological Morphogenesis Figure 6 The clockface angular co-ordinate model of French, Bryant, and which generalises results of insect leg transplant experiments (Plate 9). Both reaction-diffusion and diferential adhesion are being used in attempts to account for this behaviour. Here, super- numerary limbs grow wherever there is a complete circle of values, 1 to 12, and clockwise speciJes left leg. Solid and broken lines are contours of positional information change a linear gradient into a curved one with a large peak at the high end, and to stabilise the result of this amplification fairly strongly against disturbing influences.Thus it contrasts markedly with the adaptability above-mentioned for the Brusselator, and is not a likely model for growing a two-dimensional hexa- gonal array out of chaotic input. The source gradient might be something on the multicellular scale of organisation, such as variation in the fractions of two types of cell in a tissue. Hydra has a greater proportion of nerve cells at the ‘head’ end, where the tentacles form, than at the base.47 Gierer and Meinhardt actually proposed a number of related models, but the one they used most extensively uses two morphogens a and h (activator and inhibitor), corresponding roughly to Turing’s X and Y.The symbols a and h in the following equations will, however, signify complete concentrations, not *? P.Grant, ‘Biology of Developing Systems’, Holt, Rinehart, and Winston, 1978, p. 457; L. Wolpert, J. Hicklin, and A. Hornbruch, Symp. Soc. Exp. Bid., 1971, XXV, 391. 5 14 Harrison deviations from equilibrium. As usual, gh > ga.The parameters p and p’, both functions of distance s, represent the source gradients for a and h; PO,c, c’, p and v are rate constants. aaJat = pop + cpa2/h -pa + 9/a2a/as2 (2W ahJat = c’p‘a2 -vh + Ona2h/W (28b) This model was first applied, with notable success, to the results of grafting experiments in Hydra (Plate 8).6~4~More recently, it has been applied to insect morphogenesis.D. Temperature Sensitivity of Spacing in a Pattern.-If the spacing between repeated parts in a pattern is indeed to be regarded as a chemical wavelength of the form 27~(LB/k)+,it follows that spacing is a combination of chemical rate para- meters and should itself show the attributes of a rate parameter, among them its well known temperature dependence in the Arrhenius form. An activation energy of the order 10kJ moIbl is likely for 9;that of k may be much more variable, but for processes occupying a time scale of a few hours something like 50 kJ mol-1 is the most likely value. The apparent activation energy of the spacing, from a plot of In h versus 1/T, should then be EA = (1/2)(E~-Ek)-(1/2)(10 -50) = -20 kJ mol-l (29) In studies of the single-celled alga Acetabularia in my lab~ratory,~~ spacings have been measured, as a function of temperature, between hairs in the whorls that are from time to time formed through a large part of the growth of the organism (one whorl every few days for some months; Plate 3).Figure 7 shows plots of this temperature dependence, which is just as expected from the above very general- ised argument. This good agreement is probably fortuitous. The simplest expression for a properly controlled spacing is 27~[9~9~/(-kzk3)]* in which the diffusivity and rate constant have been replaced by geometric means of two such quantities, and uncertainties in the estimate of what EAshould be increase with all such increases in complexity of the theoretical expression.Nevertheless, I hope that this example may serve to encourage further studies along these lines, which have the potential to give fairly clear indications of whether or not a reaction-diffusion mechanism is operating. 3 The Equilibrium Approach : Phase Transitions and their Cell-as-molecule Analogues A. Inhibitory Fields.-Various scattered structures, both on the surface of a single cell (Plate 1) and in lines or sheets of cells, form arrays that are neither random nor fully ordered. Statistical analysis of the distribution of such struc- tures usually shows that they pack as if each structure were much larger than it appears to be, so that around each visible structure there is a region (a circle, in L.G. Harrison, J. Snell, R. Verdi, D. E. Vogt, G. D. Zeiss, and B. R. Green, Protoplasma, 1981, 106,211. Physica I Chemistry of Biobgical Morphogenesis -30 A, IJm 25 -20 -1 -y’\ I I I Figure 7 Spacing A between parts of a pattern (Acetabularia hairs, Plate 3) has an Arrhenius-type temperature dependence with a slope corresponding to an apparent acti- vation energy of -20 kJ mol-1 (Reproduced by permission from ref. 48) the two-dimensional cases) within which the formation of a similar structure is forbidden. This region is referred to as an ‘inhibitory field’. Attempts to account for the inhibitory field have usually invoked a diffusible substance, either an inhibitor moving out from the structure or, more simply, a substance needed for the structure moving towards it and consequently being depleted in the surround- ing region below some threshold for formation of similar structures.In such a ‘depletion model’, the possible relevance of classical ideas of phase transitions is obvious, since we are concerned with nucleation of a structure above some Harrison threshold concentration, which might be analogous to a critical supersaturation. Among the methods of statistical analysis used for partly ordered patterns, one of the simplest and most popular is the Clark and Evans R ~arameter.4~For each pattern point in a two-dimensional array, the distance to its nearest neighbour is found. The average value of this distance is divided by p-*/2, where p is the area density of points.The result, R, is unity for a random array, 2 for a perfect square array, and 2.1491 for a perfect hexagonal array. It is striking that R values between 1.62 and 1.70 are found for structures as diverse as: hair follicles on Australian ~heep,~O cone cells in a monkey's retina,s1 and pores in the cell wall of a single-celled alga.l4 (See Figure 8 and Plate 1). C0mputation5~ for an inhibi- tory field of fixed radius around each pattern point yields R =1.757. a R= 1.643 b R=1-757. 0. 0.1 0 .. .. * 0. .. .. !!".... . ..0 * . . . ...*.. b4 2 I OO 246 points Figure 8 Partly-ordered patterns: (a) Pores in the cell wall of a desmid (cf.Plate 1, for a Fferent species but almost identical pattern); (b) computed pattern for an invisible inhibitory field' of$xed size around each pattern point. R is Clark and Evans' measure of order (see text); (c) for division of pattern into 16 smaller squares (4 x 4 grid), Poisson distribution of number of points in each square, i.e. random distribution; (d) actual distri- bution for pattern (a), showing that it is far from random (Reproduced by permission from ref. 14) There is a serious physiochemical problem in reconciling the concept of a fixed- radius field with the suggestions that the field is generated by inward or outward diffusion. One might expect such diffusion to be time dependent, with any particular concentration contour probably expanding in radius with t*.Lacalli and I1* computed patterns on this basis, and obtained very low order: R =1.352. 4s P. J. Clark and F. C. Evans, Ecology, 1954,54445. 6oJ. H. Claxton, J. Theor.Biol., 1964, 7, 302. 61 H. Wade and H. J. Riemann, Proc. R. SOC.London, Ser. B, 1978,200,441. 51'7 Physical Chemistry of Biological Morphogenesis A second difficulty concerns the nature of the critical concentration condition for nucleation of new pattern points. The computations just mentioned involve a constant rate of nucleation in all parts of the system not covered by inhibitory fields. In classical nucleation theory, the nucleation rate varies extremely rapidly with concentration above the critical supersaturation. We concluded that this model of the critical concentration would not work.Another type of critical concentration commonly found in solutions is the critical micelle c0ncentration.5~ Detergent solutions commonly contain mono- meric species A up to a concentration Cc at which a polymerisation of some fairly definite number m of species A to form micelles M occurs: mA = M (30) The ideal chemical equilibrium equation for this process shows that, if rn is a 3 / 0 /C 2 1 0 1 2 3 Figure 9 Ideal micelle equilibrium for 100 monomers per micelle. This is a type of ‘critical concentration’ efect in which the system is well controlled above rhe critical concentration, in contrast to critical supersaturation effects. Ao, total concentration (as monomers);CMC,critical micelle concentration (Reproduced by permission from ref. 14) Ks L.R. Fisher and D. G. Oakenfull, Chern. SOC.Rev., 1977, 6, 25. 518 Harrison large and constant number, if A is added to solution until Cc is reached, any further addition of A leads to micelle production while the monomer concentra-tion stays at Cc (Figure 9, for m = 100). This constancy of A concentration beyond the critical value at which micelles start to form is exactly what is needed to give uniform behaviour in the whole system outside the inhibitory fields. Our14 model for cell wall pores is shown in Figure 10. As the cell grows to its full size, !” mon I I I I I I II I I II I I II I I II I I I I I I cC I Y-------M d II I 1 I‘ rC Figure 10 The ‘micelle’modelfor the boundary of an inhibitoryfield at a plant cell surface.N, nucleus of pore initial; mem, cell membrane (interior of cell is above); wall, primarycellulosic cell wall; mon, solution of monomers: mic, solution of micelles; C,,critical micelle concentration; M, monomer concentration; solid line is micelle concentration (Reproduced by permission from ref. 14) the cytoplasm is bounded, as usual, by a lipid bilayer membrane. Outside (below, in the diagram) there is a thin layer of solution bounded by the primary cellulosic cell wall. As the cell reaches full size, there is a period of some minutes before the thicker secondary cell wall forms between membrane and primary wall. In this short period, plugs of an unknown material (N) are laid down to form the ‘pore initials’, which later disappear to leave the pores in their place.We envisage the unknown material as being present in the solution layer as monomers A and micelles, or polymers, M, with fraction f of all the material (calculated as monomers) in the polymerised state. From time to time, a micelle may attach to the membrane and change into a state in which attachment of additional material takes place at an equilibrium concentration CI 4 Cc.N has a definite radius a (N 50nm) and additional material goes to thicken N downwards without increas-ing a. This diffusion of material towards N depletes the surrounding region Physical Chemistry of Biological Morpkogenesis below Cc,so that there are no micelles present up to some radius rc, of the order of 103nm but changing with time.By considering diffusion of both monomers and micelles, we were able to show that, if the micelle diffusivity is 9~and that of monomers is roughly equal : rc = a(3.02L@~f/a~)(’-f)/~ (31) [From equations (23) and (24) of our paper14 with the approximations indicated below those equations.] This model allows for rCto vary with time very much more slowly than t+,and hence is capable of accounting for the observed degree of order in the pore pattern. B. Sorting Out :Cells as Molecules.-In the embryonic development of animals, cells differentiate into a number of different types, and each type ends up occupy- ing a different spatial region to constitute the various tissues.(This description is oversimplified; a tissue may contain more than one cell type.) A major question, still incompletely resolved, is whether some chemical influence, e.g. a reaction-diffusion pattern of concentration, directs the cells in a particular region to differentiate in a particular way, or whether individual cells can differentiate any- where, followed by migration of like cells to the same region. It has long been known that, if two types of embryonic tissue are taken apart into separate cells, and the cells are intimately mixed, they tend to ‘sort out’ into two aggregates, each of one cell type. Steinberg16 studied this phenomenon in such a way as to place it on a basis comparable to the sorting out of two types of molecule in a pair of immiscible liquids.Steinberg took six tissues from chick embryos (A, B, C, D, E, F, respectively: back epidermis, pigmented epithelium of the eye, heart ventricle, liver, cores of limb cartilage, and neural tube) at early enough stages to ensure that each contained only one cell type. He showed that these tissues, taken in pairs, tended to arrange themselves as a sphere of one tissue entirely enclosing a sphere of the other, in a reproducible order (e.g.A always goes inside B). Essentially the same final configuration is reached in two types of experiment : ‘engulfment’, in which two pieces of tissue are skewered in adjacent positions on one skewer, and ‘sorting-out’ as described in the preceding paragraph (Plate 5).This suggests that the final configuration is an equilibrium one. He showed that if two pair combinations yield A > B and B > C (where > means ‘goes inside’), the transitive property is present, i,e. the A/C experiment yields A > C. From such study of all 15 possible pair combinations, it was possible to prove the existence of a hierarchy A > B > C > D > E > F. The chance of this occurring ‘by accident’ for a list of n items is r1!/2~(n-l)/2= 6!/215 = 0.022 for n = 6. Hence the existence of the hierarchy is established to about 98 % confidence. Such a series suggests the existence of a quantifiable property measuring position in the series. (As, for example, one might find a replacement series for metals in solution, and assert the existence of a quantifiable property, which is of course oxidation potential.) For the corresponding phenomenon in a series of immiscible liquids, this property would be surface tension.(A question Harrison of relative volumes comes in here. If the two assemblies of cells have the same volume, the contact surface between A and B has the same area whether A goes inside B or the reverse. The interfacial tension YAB then makes the same contri- bution to the free energy of A-inside-B and B-inside-A, and cancels out of the free energy difference between those configurations. One need then think only of the relative surface tensions YA and YB between each aggregate and the medium in which they are suspended.) By placing a piece of each tissue on a flat plate, Steinberg” essentially carried out a ‘sessile drop’ surface tension determination He did not quantify the result, but showed simply that the final shape was flatter as the tissue lay lower in the hierarchy.This led to the concept of cohesive forces between adjacent cells, or ‘differential adhesion’ between cells of different types. Many kinds of model have been proposed for this. In the simplest (homophilic), a molecule attached to the exterior of one cell surface adheres to a like molecule on the surface of another cell. The same adhesive molecule might be present throughout the hierarchy, with the cell types differing only in the fraction of the surface covered by these molecules.Suppose that these fractions are a and b for cell types A and B. If the molecules are in fixed positions, the adhesive fraction of any contact area between two cells will be a2,b2,and ab for the contacts AA, BB, and AB. Thus, if the cells are all geometrically similar so that contact areas are the same for all pair combinations, the binding energy for AB is the geometric mean of those for AA and BB. This, curiously enough, is the same rule suggested by Berthelot in 1898for van der Waals forces in mixtures of fluids, and later justified in the theory of London dispersion forces, appro~imately.~3 Quantitative aspects of adhesion between cells remain rather obscure. If cells, suspended in an aqueous medium, had bare lipid membranes, they would have actual van der Waals attractive forces at the contact of adjacent cells. The resulting binding energy has been estimated at 330 kTpm-2 of contact area.For contact area of only 10pm2, this gives a binding energy of 3300kT. Colloidal particles are considered to form stable aggregates if binding forces exceed about lOkT between particles. Thus cells with bare lipid surfaces should be bound together into very rigid masses; neither sorting out nor the suspension of cells in media such as the blood should be possible.54 Evidently the glycoprotein cell coat of animal cells must, among other functions, cancel out the membrane-to- membrane adhesions, leaving it possible for specific molecules to re-establish much smaller adhesions. To avoid the same problem of excessive adhesion, these specific interactions must be quite weak or quite sparsely distributed.This may be illustrated by considering the quantities involved in the immiscibility criterion for two types of cell. Two pieces of tissue, each about 1 mm3,would each contain of the order of N = 106 cells. If each cell is regarded as a ‘molecule’, the unmixing of m J. H. Hildebrand and R. L. Scott, ‘Regular Solutions’, Prentice-Hall, 1962. 64 D. E. Brooks, personal communication, using data from V. A. Parsegian and D. Gingell,J. Adhesion, 1972, 4, 283; ‘Recent Advances in Adhesion’, ed. L.-H. Lee, Gordon and Breach, New York, 1972; D. Gingell and S. Vince, ‘Adhesion and Motility of Cells’, ed A. S. G. Curtis, Cambridge University Press, 1979.Physical Chemistry of Biological Morphogenesis an intimate mixture of these cells into two separate aggregates involves an unfavourable entropy change : AS = -2kNln2 = -1.4 x 106k (32) Immiscibility therefore requires a favourable energy change for unmixing of the order of a million times kT for the whole assembly, or about kT per cell. More precisely, if a cell-cell contact leads to binding energy: WAA = ckTd2;WBB = ckTb2; WAB = ckTab (33) then for 10pm2 contact, one adhesive molecule per nm2 at a = 1 (i.e. 107 as maximum number of molecule-to-molecule adhesions per contact), and adhesive energy of order kT (about 2.5 kJmol-l) between molecules, c = 107. For close packing of molecules (12 contacts) in both mixed and unmixed assemblies, the energy of unmixing would be: dE = ~NWAA-/-6NWBB -3N(WAA t WBB + 2wAB) = -3cNkT(a -b)’ (34) This, together with equation (31) indicates that separation is favoured for: la -bl > (21n2/3c)*= 2 x lo-* (35) i.e.the discussion concerns parts in ten thousand of the surface covered with adhesive molecules. The above considerations are on the basis of a static picture of the cell as a rigid object. This is clearly incorrect, because Brownian motion of rigid objects of size of the order of lOpm would be much too slow to account for sorting out on a time scale of hours. The cell surface must be seen as continually changing in shape as a result of (i) molecular collisions, in the manner of Brownian motion, producing temporary small-scale deformations of the cell surface, and (ii) similar deformations being produced from inside the cell by, for example, the action of contractile microfilaments, which might still be random in relation to any directional effect on motion of the cell~.~5 Such motions would of course tend to diminish the effective contact area between cells at any instant, so that much greater surface coverages of adhesive molecules would be needed to produce time-average interaction energies as discussed above.One way to express this kind of effect might be to use rigid-cell geometrical pictures, but introduce a fictitious temperature, much higher than the real temperature, to represent the enhanced random motion. Computer modelling of sorting out is an active field.56 Some remarkable phenomena occur in insect and amphibian (newt) limb regeneration, especially in grafting experiments, indicating the existence of persistent gradients which can be represented by an angular co-ordinate around the limb and a linear co-ordinate along it (Plate 9, Figure 6).s7 These are un- 55 M.S. Steinberg and L. L. Wiseman, J. Cell Biol., 1972, 55, 606. 56 R. Gordon, N. S. Goel, M. S. Steinberg, and L. L. Wiseman, ‘Mathematical Models for Cell Rearrangement’, ed. G. D. Mostow, Yale Univ. Press, 1975, p. 196; N. S. Goel and G. Rogers, J. Theor. Biof.,1978, 71, 103 and 141 ;R. J. Matela and R. J. Fletterick, J. Theor. Biol., 1980,84673. 67 V. French, P. J. Bryant, and S. V. Bryant, Science, 1976, 192, 969. Harrison explained, but there is definite evidence for gradients of adhesiveness of cells in animal embry0s.~8 4 The Molecular Basis of Morphogenesis This review does not include a thorough presentation of the structural approach for two reasons.First, the review is intended to indicate the areas of specific physicochemical interest, and much structural information is of more purely biological or organochemical interest. Second, the balance of space allotted to different topics in this review reflects my bias on the extent to which the various types of concept are likely to turn out to be important in the crucial control steps in morphogenesis: kinetic > equilibrium > structural. The chemical reader should recognise that this is probably precisely the opposite order to that which the majority of experimental biologists would currently give.I have selected a few problems for brief discussion below, because I believe that they give a perspective on how structural aspects may fit in with the other matters discussed above. A. Heterophilic or Lock-and-key Adhesion.-Adhesion between two cells might involve bonding between two different molecules. This is often referred to as the lock-and-key model, probably with a geometry in mind similar to that of a sub- strate fitting into a suitably shaped infold of an enzyme; except that here the sub- strate will not be a free-moving small molecule, but probably held as terminal to some larger structure as the key is held in the hand. A cell with 100 %’ locks would bond best to a cell with 100%keys.This model has been used particularly in relation to some features of the assembly of the vertebrate nervous system, especially the joining of the optic nerve to the brain. In the lower vertebrates, the junction is made at the optic tectum, which can be thought of very roughly as a square of a million cells, 1.000 x 10oO. Each retinal axon seems to find precisely the correct cell to connect to, so that there is a specificity of about one part in 1000 in each of two rectangular co-ordinate directions. Rival theories for how this happens are numerous and di~erse,~~*~O and include an activation-inhibition model (Willshaw and von der Malsburg) with interesting correspondences to the Turing model.RothGO has proposed a lock-and-key model in which there is a gradient from 100 % locks to 100 % keys across each direction on the tectum, and matching gr?dients across the array of axons advancing to meet the tectum. Accounts of this model have generally failed to point out, however, that, if the locks and keys are in fixed positions on the cell surface and cannot adjust to find each other, a cell with 50 % of each will join up equally well with an axon regard- less of the fractions of locks and keys on the latter. The model is not fully specified without a precise account of how far the locks and keys are able to migrate to find their partners, and the model doesn’t work without some limited mobility. 58 M. S. Steinberg, in ref.37. 5g R. W. Sperry, ‘Organogenesis’, ed. R. L. DeHaan and H. Ursprung, Holt, Rinehart and Winston, 1965, p. 161; R. A. Hope, B. J. Harnmond, and R. M. Gaze, Proc. R. Soc. London, Ser. B, 1976, 194, 447; D. J. Willshaw and C. von der Malsburg, Proc. R. Soc. London, Ser. B, 1976. 194, 431 ; Piiilos. Trans. R. Soc. London, Ser. B, 1979, 287, 203. soR. B. Marchase, A. J. Barbera, and S. Roth, in ‘Cell Patterning’, (CIBA Foundation Symposia, new series, no. 29), Elsevier, Amsterdam, 1975, p. 315. Physical Chemistry of’Bialogical Morphogenesis Roth, in addition to proposing the model and finding some experimental evidence for an adhesive gradient (using tecta and retinal cells in vitro), has devised plausible biochemical models for the nature of the locks and keys.The animal cell coat, outside the lipid membrane, consists of glycoproteins, which are long polypeptide chains carrying a number of oligosaccharide side-chains, each having perhaps ten monosaccharide molecules in it. The side chains are built up with the aid of glycosyl transferase enzymes, which can attach to the end of the growing chain, wait for the next required sugar to come along, in the form of a sugar nucleotide, and attach it to the chain, at which stage the enzyme is released from attachment to the chain. Roth’s model is that the glycoprotein is attached to one cell, the enzyme to the other, and when the two have joined it happens that the next sugar substrate is absent and the enzyme is never released. This gives the lock-and-key junction.Roth devised various forms of this model, and among them pointed out that, if there were three types of side chain, each of which could exist in ten different stages of partial completion, 103 chemically different arrangements could arise from just a few monosaccharides, quite enough to account for a specificity of one part in 1000. To my mind, this sort of model is very useful but perhaps merely pushes the crucial question back one stage. The production of the required gradients of incomplete side chains would require some gradients of sugar substrates, and how do those gradients arise? I am led back towards reaction-diffusion. B. Positive Feedback on the Multicellular Scale.-The model of Gierer and Meinhardt described in Section 3C above used autocatalysis (positive feedback) on the molecular scale, with a fixed source gradient which might involve relative numbers of two types of cells.If any substance is found which is produced by a particular type of cell and which tends to make other cells differentiate into that same type, then there is a positive feedback direct into the source gradient. Such a substance is not a morphogen in the strict Turing sense. It might, however, give the differentiation process some partial mathematical analogy to reaction-diffusion theory, depending upon how closely the subsequent movements of differentiated cells resemble diiFusion. Schaller61 (in the same institute as Gierer and Meinhardt) found an oligo- peptide hormone, produced by nerve cells of Hydra, which when added to Hydra tissue gave it an increased tendency to form ‘heads’, i.e.rings of tentacles. The ‘head’ contains a higher fraction of nerve cells than the rest of the animal. There is certainly a positive feedback loop here, but this substance is not regarded (Meinhardt, personal communication) as the morphogen a in equations (28). Glycosaminoglycans (GAGS) are polysaccharides composed of alternating units of a sugar acid (e.g. glucuronic acid) and an amino-substituted sugar. They are widespread in animals, but are found particularly as constituents of thc extra- cellular matrix of connective tissue.62 Hyaluronate (1) and the chondroitin 61 H. C. Schaller, J. Embrvol. Exp. Morph., 1973, 29, 27 and 39.62 J. E.Scott, Chem. Br., 1979, 15, 13. Harrison sulphates (2) and (3) are especially prevalent in cartilage. In the embryogenesis of vertebrates, differentiation into cartilage precursors is the first stage in formation of the skeleton, determining its complicated geometry. The structure of a limb, for example, is controlled mainly by activity in the tip region of a limb bud, in which CH,OH \ OH OH a mass of primary mesenchyme cells is source material for differentiation towards cartilage and other tissues, and is controlled by adjacent structures such as the apical ectodermal ridge and zone of polarising activity (Plate Urist et al.64 showed that muscle tissue from newborn rats could be made to differentiate into cartilage by the influence of bone matrix gelatin, and that this change was enhanced by chondroitin sulphate but depressed by hyaluronate.Both substances are produced by the differentiated cells, so that there is some evidence here for both self-activation and self-inhibition. These authors, however, mention Turing’s theory and beiieve that a morphogen is present but that it is not to be identified with either of these polysaccharides. They write: ‘Morphogens are 13~L. Wolpert, J. Lewis, and D. Summerbell, in ref. 60,p. 95. 13* M. S. Urist, Y. Terashima, M. Nakagawa, and C. Stamos, In Vim, 1978, 14, 697. Physical Chemistry of Biological Morphogenesis short-lived, low-molecular mass, rapidly diffusable hydrophobic proteins that have not yet been isolated, purified, and identified but might be found in and on differen tiat ing cell surfaces.’ Cyclic adenosine monophosphate (CAMP) (4) is a small molecule best known because its production just inside the surface of liver cells is the immediate response to an extracellular signal from the hormone epinephrine (adrenaline) and the start of a chain of reactions inside the cell, which ends with greatly enhanced release of glucose to the blood.CyclicAMPalsoappears, however, in themorphogenesis of cellular slime moulds (Plate 4). At the end of the stage of independent amoeboid single cells, some of these cells send out pulses of cAMP which act as signals to all the others to gather into aggregate^.^^ The aggregates then differentiate into two types of cells, which rearrange into a stalk and a mass of spores.It is believed that, during the final rearrangement (culmination stage) cAMP may be produced by stalk cells and act as a chemotactic signal for the movements of these cells leading to stalk formation.66 This is a kind of positive feedback; but on another level, the relation of cAMP to certain enzymes (adenylate cyclase, which produces CAMP, and phosphodiesterase, which destroys it), there is a possibility of finding a morphogen pair X and Y,consisting of a small molecule and a large one, quite likely to show the required imbalance of diffusivity. C. Growth of Cell Surfaces.-Plant cell morphogenesis (Plates 2 and 3) involves expansion in area of both the lipid membrane and the cellulosic cell wall outside it. The lipids are neither formed at the surface nor added to it molecule by IF,G. Gerisch, D.Hulser, D. Malchow, and U. Wick, Philos. Trans. R. SOC.London, Ser. B, 1975, 272, 181. (A paper in a discussion on the physics and chemistry of biological recog- nition, which is all relevant to the present topic.) P. C. Newell, Endeavour (new series), 1977. 1,63. IsM. Sussman and R. Brackenbury, Annu. Rev. Plant Physiol., 1976,27, 229. Harrison molecule. They are conveyed from the interior of the cell in the form of vesicles up to lOOnm in diameter.67 These vesicles carry, as the electron microscope indicates, rosette-shaped structures from each of which a cellulose microfibril of a definite length grows after incorporation of the vesicle into the cell membrane.Thus the amount of cellulose produced goes in lock-step with the amount of material added to the membrane. The key to morphogenetic control is therefore, to my mind, the control rate of addition of vesicles. This still leaves a very wide field, chemically. Movement of vesicles through the interior of the cell is likely to be related to cytoplasmic streaming and could be controlled by the cytoskeleton of protein microfilaments. On the other hand, it may be that vesicles are always present at the surface in excess, and that most of them bounce off the membrane but some stick and fuse to it because of chemical differences in the surface of the membrane and the vesicles in different regions.One must then look for mor- phogens among the substances which act to set up gradients of these surface modifiers. The latter could be something as simple as Ca2+, which is known to have a strong influence on electrical potentials at membranes and to affect, for example, the fusion of the sperm to the egg, and the fusion of neurotransmitter- filled vesicles to synaptic membranes in nerve cells. But the surface modifiers could also be something as complicated as a glycoprotein. In multicellular plants, because of the rigidity of their cell walls and inability of the cells to sort out, control of direction of cell division is the primary morpho- genetic control in development of branches, leaves, etc. (Plate 7).6* The plane of division of a cell is first marked, before anything significant has happened-to the geometry of the nucleus, by the appearance of a ‘pre-prophase band’ of micro- tubules, girdling the equator which is to be the plane of division.12 This suggests that morphogenetic control mechanisms may reside at the cell surface rather than the nucleus, and leads me again to look a stage or more back from the micro- tubules and to enquire what substances and reactions determine the positions of nucleation sites for tubulin polymerisation.Chemicals that control plant growth were described in an earlier review in this series.69 They include the cytokinins, which are purine and adenine derivatives, and which promote cell division and differentiation to the extent that an entire tobacco plant can be grown from a pith segment with the aid of 6-benzyloxypurine.Such compounds are promising candidates for the name of morphogen. In general, structural studies by electron microscopy and other techniques such as immunofluorescence are providing extensive and spectacular evidence for the occurrence of microtubules and microfilaments in important places during development. I am inclined to believe, however, that these structures are mani- festations of morphogenetic control, rather than the controllers themselves. But they are important as pointers towards the control mechanisms, which will probably ultimately be found in the biosynthesis of nucleation sites for these macromolecules. Either or both of nucleation-depletion theory and reaction- 67 L. A. Staehelin, in ref.37. 6a P. B. Green, Annu. Rev. Plant Physiol., 1980, 31, 51. s*R. L. Wain, Chem. Soc. Rev., 1977, 6, 261. Physicul Chemistry of Biological Morphogenesis diffusion theory of the Turing type could turn out to be the proper explanation of control. Note added in proof: A recent discussion of the Royal Society on ‘Theories of biological pattern formation’ contains the most recent ,information on several of the topics in this review.’* Philos. Trans. R. SOC.London, Ser. B, 1981, 295, 425-617

ISSN:0306-0012    

DOI:10.1039/CS9811000491

出版商:RSC    

年代:1981

数据来源: RSC