摘要:

14 Biological Chemistry Part (i) Insect Chemistry By R. BAKER and D. A. EVANS Department of Chemistry University of Southampton Southampton SO9 5NH 1 Introduction The interdisciplinary research of biologists and chemists has established the impor- tance of chemical factors in many aspects of animal behaviour. The insect world has attracted the greatest attention and many facets of behaviour such as mating foraging and egg-laying are known to be regulated by discrete chemical signals in the form of naturally occurring chemosensory substances. This Report covers significant advances in this field largely over the past two years. The earlier literature has been the subject of several reviews and articles.’ Coverage is limited to compounds which are ‘releasers’ of some behavioural response at the time of perception.‘Primer’ effects which cause longer-term physiological changes and other hormonal effects such as the interference with metamorphosis by juvenile hormones are not considered. 2 Sex-attractant Pheromones Sex pheromones of the Lepidoptera (moths and butterflies) have received the widest attention and a large number of structure determinations have been successfully completed.* This is partly due to the structural uniformity of such pheromones which are most often found to be straight-chain alkenyl alcohols acetates or the corres- ponding aldehydes e.g. see Table. One of the few recent examples of structural diversity is provided by the Douglas Fir Tussock Moth Orgyiupseudotsugutu,which employs (2)-6-heneicosen- 11-one as a sex pher~mone.~ Increasingly pheromone complexes in which synergistic effects play a specific part are being elucidated; e.g.in the case of the Oriental Fruit Moth (Grupholithu rnolestu) it has been shown that a small proportion of a geometrical isomer increases the potency of synthetic pher~mone.~ Attention has also been paid to inhibitors of J. G. MacConnell and R. M. Silverstein Angew. Chem. Intenrat. an. 1973,12,644;D.A. Evans and C. L. Green Chem. SOC.Rev. 1973 2 75; ‘Chemicals Controlling Insect Behaviour’ ed. M. Beroza Academic Press New York 1970; ‘Control of Insect Behaviour by Natural Products’ ed. D. L. Wood R. M. Silverstein and M. Nakajima Academic Press New York 1970; ‘Advances in Chemoreception’ Volume 1 ed.J. W.Johnston jun. D. G.Moulton and A. Turk Appleton-Century-Crofts New York 1970; ‘Frontiers of Biology Series Pheromones’ ed. M. C. Birch North Holland New York 1974; A. W. Johnson Proc. Roy. Inst. Great Britain 1974,41 163. K.Eiter Pure Appf. Chem. 1975,41,201. R. G.Smith and G. E. Daterman Science 1975,188,63. M. Beroza and G. M. Muschik Nature New Biol. 1973,244 149; J. A. Klun 0.L. Chapman K. C. Mattes P. W. Wojtkowski M. Beroza and P. E. Sonnet Science 1973,181,661. 347 R. Baker and D.A.Evans Table Some sex pheromones of Lepidoptera species Parent Terminal chain length functional group Unsa tura tion Species Reference ClO Cl2 alcohol acetate alcohol acetate (E1-5 (E)-5(E,E)-8,lO - acetate 03-9 11 (E)-9,ll c13 C14 acetate alcohol (z)-9,11 (E,z)-7,9 (Z)-11 (2,2)-7,11(?) (E)-11 acetate - (21-9(Z)-lO (E)-13 c16 aldehyde alcohol acetate aldehyde (a-11(2,E)-9,11 (2,E)-9,12 (2)-9(Z)-11 (2)-11 (2,E)-7,ll (Z,Z)-7,11 (2)-11 c18 acetate (E,z)-3,13(ZZ)-3,53 Species A Anarsia lineatella Peach Twig Borer Moth; B Laspeyresia pomonella Codling Moth; C Dipopsis castunea Red Bollworm Moth; D Argyrotaeniu velutinana Red Banded Leaf Roller; E Archips argyrospilus Fruit Tree Leaf Roller; F Lobesia botrana Grape Vine Moth; G Phthorimaea operculella Potato Tuberworm Moth; H Platynota sultana Omnivorous Leaf Roller; I Spodoptera littoralis Cotton Leafworm Moth; J.Spodoptera exempta Armyworm Moth; K Archips semiferanus Oak Leaf Roller; L Archips podana Fruit Tree Tortrix Moth M Argyrotaenia citrana Orange Tortrix Moth; N Heliothis virescens Tobacco Budworm Moth; 0,Mamestra configuruta Bertha Armyworm Moth; P Pectinophora gossypiella Pink Bollworm Moth; Q Sitotroga cerealella Angoumois Grain Moth; R Synanthedon pictipes Lesser Peachtree Borer; S Sanninoidea exitiosa.a W. Roelofs J. Kochansky E. Anthon R. Rice and R. Carde Environ Entomol. 1975 4 580; M. Beroza B. A. Bierl and H. R. Moffitt Science 1974,183,89; B. F. Nesbitt P. S. Beevor R. A. Cole R. Lester and R.G. Poppi Nature New Biol. 1973,244 208; B. F. Nesbitt R. A. Cole P. S. Beevor R. Lester and R. G. Poppi,J. InsectPhysiol. 1975,21,1091;e W. L. Roelofs A. S. Hill and R. Carde,J. Chem. Ecol. 1975,1,83;f W. Roelofs A. Hill R. Carde J. Tette H. Madsen and J. Vakenti Environ.Entomol. 1974,3,747; 8 H. R. Buser S. Rauscher and H. Am 2.Naturforsch. 1974,29c 781; H. G. Fouda J. N. Seiber and 0.G. Bacon J. Econ. Entomol. 1975,68,423; i A. S. Hill and W. L. Roelofs J. Chem. Ecol. 1975,1,91;J Y. Tamaki and T. Yushima J. Insect Physiol. 1974,20 1005; P. S. Beevor D. R. Hall R. Lester R. G. Poppi J. S. Read and B. F. Nesbitt Experientia 1975 31 22; I L. B. Hendry R. J. Gill A. Santora and R. 0.Mumma Entomol. Exp. Appl. 1974,17,459; C.J. Persoons A. K. Minks S. Voerman W. L. Roelofs and F. J. Ritter J.InsectPhysiol. 1974,20,1181; A. S. Hill R. T. Carde H. Kido and W. L. Roelofs J. Chem. Ecol. 1975 1,215; J. H. Tumlinson D. E. Hendricks E. R. Mitchell R. E. Doolittle and M. M. Brennan J. Gem. Ecol. 1975,1,203; p M. D. Chisholm W. F.Steck A. P. Arthur and E. W. Underhill Gznad. Entomol. 1975,107,361; 4 H. E. Hummel L. K. Gaston H. H. Shorey R. S. Kaae K. J. Byrne and R. M. Silverstein Science 1973,181,873; B. A. Bierl M. Beroza R. T. Staten P. E. Sonnet and V. E. Adler J. Econ. Entomol. 1974,67,211;* K. W. Vick H. C. F. Su L. L. Sower P. G. Mahany and P. C. Drummond Experientia 1974,30,17; J. H. Tumlinson C. E. Yonce R. E. Doolittle R. R. Heath C. R. Gentry and E. R. Mitchell Science 1974,185,614. Biological Chemistry -Part (i) Insect Chemistry pheromonal responses in certain species (e.g.ref. 5) and such effects suggest possible uses in pest control. Butterflies have attracted less study than the more economically damaging moth species. Research has continued on hairpencil secretions some components of which are known to play important roles in courtship as mating aphrodisiacs.Nine species from the Danaus and Amauris genera have been shown to employ the pyrrolizine derivative (1) and/or (2) in this The postulate that such pyr- rolizines and related substances [(3) (4)] produced by Danaid butterflies are biogenetically related to alkaloids [e.g. (5)]of plants on which they feed has been strengthened by the detection of (5)itself in a hairpencil The Monarch butterfly Danaus plexippus produces a hairpencil secretion consisting of benzyl 0 CHMe, II I CH2O -C -C -CHMe I IOHOH OMe OMe (2) (3) R = H (4) R = OH caproate and a bicyclic ether possessing an ionone skeleton possibly (6) or an isomer.' A sex attractant of the Furniture Carpet Beetle Anthrenusflavipes has been shown to be (2)-3-decenoic acid." Females of Trogoderma glabrum produce a multicom- ponent pheromone consisting largely of n-hexanoic acid and methyl (2)-7-hexadecenoate in addition to (7) (8) and (9)." The Khapra beetle Trogoderma (7) (8) R = CH,OH (9) R = CO,Me granarium responds strongly to (10) in comparison to related derivatives and this has been suggested as a component of the sex pheromone.12 H.Am C. Schwarz H. Limacher and E. Mani Experientia 1974,30 1142. 6 J. Meinwald C. Boriack D. Schneider M. Boppre W. F. Wood and T. Eisner Experientia 1974,30 721. J. A.Edgar and C. C. J. Culvenor Nature 1974,248 614. J. A.Edgar and C. C. J. Culvenor Experientia 1975,31,393. T. E.Bellas R.G. Brownlee and R. M.Silverstein Tetrahedron 1974 30 2267. H.Fukui F.Matsumura M. C. Ma and W. E. Burkholder Tetrahedron Letters 1974,3563. R.G. Yarger R. M. Silverstein and W. E. Burkholder J. Chem. Ecol. 1975,1,323. H.Z.Levinson and A. R. Levinson Naturwiss. 1974,61,685. 350 R.Baker and D.A.Evans Sex pheromones of fly species which are economically important or health hazards have been inve~tigated'~ and both (2)-14- and (2)-13-nonacosene and (2)-13- heptacosene have been implicated in the sex attraction of the face-fly Musca uut~mnaZis.'~The German cockroach (Bluffellagermanica) responds to 3,ll- dime thyl-2-nonacosanone. Is 3 Aggregation Pheromones and Population Attractants Interest in the chemosensory communication complexes of the economically damag- ing bark-boring beetles (Coleopteru Scolytidae) has continued.Increasingly the interplay between aggregation pheromones and volatile compounds present in host-trees is being established.16 Studies of the Smaller European Elm Beetle Scolytus multistriatus have suggested 2,4-dime t hyl-5-e thyl-6,8 -dioxa bicyclo[ 3,2 lloctane (11) as the aggregation pheromone in combination with 4-methyl-3-heptanol and cubebene (12).l7 Another Scolytid beetle Gnathotrichus sulcatus has been shown to employ sulcatol (13) in aggregation behaviour. l8 OMe QoH :H /J (12) (13) (14) (S)-(+):(R)-(-)=65:35 The guaiacol derivative (14) has been suggested as a gregarization pheromone implicated in phase transformation in the migratory locust Locusta rnigratoria.l9 4 Pheromones of Social Insects The pheromone systems which maintain social order and dictate behaviour in colonies of social insects are exceedingly complex. Alarm pheromones which serve to alert a colony to prepare defence against an intruder and trail pheromones which are used by successful foragers to mark a path between the nest and a food-source have been most studied. Pyrazine derivatives (15a-e) are used as alarm pheromones by several species of Odontornachus ants," and the presence of 13 P. A. Langley R. W. Pimley and D. A. Carlson Nature 1975,254,51;E. C. Uebel P. E. Sonnet B. A. Bierl and R. W. Miller J. Chem. Ecol, 1975,1,377. 14 E. C. Uebel P. E. Sonnet R. W. Miller and M. Beroza J. Chem. Ecol. 1975,1 195. I5 R.Nishida H. Fukami and S. Ishii Experientia 1974,30 978. 16 J. A. Rudinsky M. E. Morgan L. M. Libbey and T. B. Putnam Enuiron. Entomob 1974,3,90;J. A. A. Renwick P. R. Hughes and J. P. Vite J. Insect Physiol. 1975,21 1097; J. P. Vite A. Bakke and P. R. Hughes Natunviss. 1974,61 365. 17 G. T. Pearce W. E. Gore R. M. Silverstein J. W. Peacock R. A. Cuthbert G. N. Lanier and J. B. Simeone J. Chem. Ecol. 1975,1 115; W. E. Gore G. T. Pearce and R. M. Silverstein J. Org. Chem. 1975,40,1705. IS K. J. Byrne A. A. Swigar R. M. Silverstein J. H. Borden and E. Stokkink J. InsectPhysiol. 1974,20 1895. 19 D. J. Nolte S. H. Eggers and 1. R. May J. Insect Physiol. 1973 19 1547. 20 J. W. Wheeler and M. S. Blum Science 1973,182 501. Biological Chemistry -Part (i) Insect Chemistry (15e-g) inter alia in the Argentine ant Iridomynnex humilis has been reported.‘l Alarm pheromones of Atta species of leaf-cutting ants consist of aliphatic alcohols R2 (15a) R’ = Me; R2 = C2H (15e) R’ = iso-C5H11;R2 = Me (15b) R’ = Me; R2= n-C3H (150 R’ = (2)-styryl; R2 = Me (15c) R’ = Me; R2= n-C,H9 (15g) R’ = (Etstyryl R2 = Me.Me R’ (15d) R’ = Me R2 = n-C5Hl1 and ketones22 whereas (16) functions similarly in the Ponerine ant Gnamptogenys pleurod~n.~~ The Azteca ant utilizes cyclopentyl ketones [e.g. (17) (18)] in this The African weaver ant Oecophylla longinoda is notoriously aggressive in defending its territory and an intricate multicomponent alarm pheromone system has been uncovered. Hexanal l-hexanol and 3-undecanone combine in attracting ants to a point of disturbance and (19) acts as a ‘biting marker’ which provides a target for mass-attack on an intr~der.~’ H The trail pheromone of the ant Lasius fuliginosus consists of alkanoic acids,26 whereas the indolizine derivative (20)has been proposed as a trail pheromone in the Pharaoh’s ant Monomorium pharaonis.” Chemical examination of the secretions of bee species has continued and the use of combinations of terpenoids and other substances in scent-marking by bees has been described.28 A substance which elicits brood-tending behaviour (i.e.brood pheromone) in the Fire Ant Solenopsis invicta a serious North American pest has been shown to be the glyceride tri~lein.’~ 21 G.W. K. Cavil1and E. Houghton,Austral.J. Chem. 1974,27,879;J. Insect Physiol. 1974,20,2049. 22 R. G. Riley R. M. Silverstein and J. C. Moser J. Insect Physiol. 1974 20 1629. 23 R.M.Duffield and M. S. Blum Experientiu 1975,31,466. 24 J. W.Wheeler S.L. Evans M. S. Blum and R. L. Torgerson Science 1975,187,254. 2s J. W. S. Bradshaw R. Baker and P. E. Howse Nature 1975,258,230. % S. Huwyler K. Grob and M. Viscontini J. Insect Physiol. 1975,21 299. 27 E.Talman F. J. fitter and P. E. J. Verwiel ‘MassSpectrometry in Biochemistry and Medicine’ ed. A. Frigerio and N. Castagnoli 1974,Raven Press New York p. 197. 28 J. Tengo and G. Bergstrom J. Chem. Ecol. 1975 1 253 and earlier references in this series. z9 W. S. Bigley and S. B. Vinson Ann. Entomol. Soc. Amer. 1975,68 301. R.Baker and D.A.Evans 5 Host Food and OvipositionAttractants The involvement of volatile compounds in host selection either as a food-source or for oviposition has now been established for numerous species.Host volatiles eliciting attraction of bark beetles have continued to receive much attenti~n.~’ Little has been reported on oviposition pheromones although there is considerable interest in synthetic oviposition attractant^.^^ A study of chemical communication in the House Longhorn Beetle Hylotrups bajulus has shown that (-)-verbenone present in the frass of this timber pest acts as an oviposition attractant and that p- cymen-8-01 is a synergi~t.~~ 6 Repellents Phytorepellents and Antifeedants The chemical basis for the resistance of particular plants to insect attack is slowly being unravelled and in some cases the presence of antifeedants or phytorepellents has been demonstrated.The structure of Nic-2 a constituent of the insect-repellent plant Nicandra physaloides has been shown to be (21).33 Azadirachtin from the plant Azadirachta indica (Indian neem) has structure (22) and is active as a very effective phagorepellent and as a synthetic growth di~ruptor.~~ Piperenone (23) a constituent of the plant Piper futokadzura acts as an antifeedant towards larvae of Spodoptera lit~ra.~’ 30 H. J. Meyer and D. M. Norris J. Insect PhysioL 1974 20 2015; M. Sumimoto T. Kondo and Y. Kamiyama ibid. 1974,20,2071; E. C.Levy,I. Ishaaya E. Gurevitz R.Cooper,and D. Lavie J. Agric. Food Chem. 1974,22,376. 31 B. S.Fletcher and C.A. Watson Ann. Entomol. SOC.Amer. 1974,67,21. 32 D. A. Evans and M. D. Higgs Tetrahedron Letters 1975,3585. 33 R.B. Bates and S. R.Morehead J.C.S. Chem. Cbmm. 1974,125. 34 P. R. Zanno I. Miura K. Nakanishi and D. L. Elder J. Amer. Chem Soc. 1975,97 1975. 35 K. Matsui and K. Munakata Tetrahedron Letters 1975 1905. 353 Biological Chemistry -Part (i) Insect Chemistry 7 Defence Secretions If structural uniformity characterizes some classes of pheromones then mention must be made of the diversity encountered in compounds used in defence mechan- isms by insects. Secretions ofmillipedes (non-insects!) range from simple q~inones~~ to (24) and (25) which are present in Polyzonium r~salbum.~’ Another nitro- compound 1-nitro-1-pentadecene has been demonstrated as a defensive com- pound of the termite Prorhinotermes simplex.38 A close ecological relationship exists between various species of ants and termites.In the Tropics ants frequently prey upon termites and the latter develop selectivity toxic secretions for specific defensive roles. An example is provided by the termite Amitermes euuncifer the soldiers of which which produce the formicidal epoxyeudesmane (26) in the frontal gland.39 In contrast studies of the defensive and offensive behaviour of ants have revealed the venoms and poison-gland constituents. Fire Ant venom contains piperidines (e.g. ref. 40) whereas terpenes are commonly encountered in this role e.g. in the poison gland of Myrmicaria natalen~is.~’ The frequently spectacular defence mechanisms of beetles have attracted much attention,42 but special mention must be made of a fascinating series of nitrogenous bases employed in defensive secretions in ladybird species (C~ccinellidae).~~ Myr-rhine (27) is reported in a Myrrha species,43a whereas the diastereoisomeric hip- podamine and its N-oxide (convergine) are described as defensive secretions in the American Ladybug Hippodamia conuergen~.~~~ Rove beetles produce the alkaloid actinidine(28) in this context together with related monoterpenoids such as iridodial (29).44 The latter is used in the defensive behaviour of the related Staphylinus olens together with 4-methylhexan-3-0ne.~~ 36 W.F. Wood J. Shepherd B. Chong and J. Meinwald Nature 1975 253 625. 37 J.Meinwald J. Smolenoff A. T. McPhail R. W. Miller T. Eisner and K. Hicks Tetrahedron Letters 1975 2367; see also Science 1975 188 734. 38 J. Vrkoc and K. Ubik Tetrahedron Letkrs 1974,1463. 39 L. J. Wadhams R. Baker and P. E. Howse Tetrahedron Letters 1974,1697. 40 J. G. MacConnell R. N. Williams J. M. Brand and M. S.Blum Ann. Entomol. SOC.Amer. 1974,67 134. 41 J. M. Brand M. S. Blum H. A. Lloyd andD. J. C. Fletcher Ann. Entomof.Soc. Amer. 1974,67,525. 42 W. R. Tschinkel J. Insect Physiol. 1975 21 659 753; T. Eisner D. Aneshansley M. Eisner R. Rutowski B. Chong and J. Meinwald Psyche 1974,81 189 (Chem. Ah. 1975,82 19948). 43 (a) B. Tursch D. Daloze J. C. Braekman C. Hootele A. Cravador D. Losman and R. Karlsson Tetrahedron ktters 1974 409; (b) B.Tursch D. Daloze J. C. Braekman C. Hootele and J. M. Pasteels Tetrahedron 1975,31 1541; (c) R. D. Henson A. C. Thompson P. A. Hedin P. R. Nichols and W. W. Neel Experientia 1975,31,145. 44 T. E. Bellas W. V. Brown and B. P. Moore J. Insect Physiol. 1974,20,277. 45 L. J. Fish and G. Pattenden J. Insect Physiol. 1975 21 742. R. Baker and D.A.Evans H HnH)f-J yH0 CHO \I (28) (29) (27) The Whirligig beetle (a Gyrinid species) produces (30) together with related norsesquiterpenoids as defensive toxins.46 Fish prey upon this species and it is noteworthy that these compounds have pronounced narcotic and toxic effects towards fish. The Staphylinid beetle Stennus comma produces the base stenusin (31) together with 1,8-cine01.~’ Stenusin has large spreading effect on water and is implicated as a surfactant for flotation.Me 8 Insect Waxes Much interest is evident in the chemical composition of the surface of the insect Apart from the functions of cuticular waxes in waterproofing other suggestions of action as ‘keepers’ for pheromones and other secretions are under study. The sesterterpene (32) is a wax-constituent of Ceropfastes afbofineatus,49 and high molecular weight keto-esters have been shown to be present in the waxes of Dactylopius pr~ciphilus.~~ 46 J. R. Miller L. B. Hendry and R. 0.Mumma J. Chem.Ed. 1975,1,59. 47 H. Schildknecht D. Krauss J. Connert H. Essenbries and N. Orfanides Angew Chem. Internat. Edn. 1975,14427. 48 R. H. Hackman ‘Chemistry of the Insect Cuticle’ in ‘Physiology of the Insecta’ Academic Press New York 2nd edn.1974 Vol. 6 p. 469. 49 T. Rios L. Quijano and J. Calderon,J.C.S. Chem. Cornrn. 1974,728. 50 J. Meinwald J. Smolenoff A. C. Clubnall andT. Eisner J. Chem. Ed. 1975 1 269. 355 Biological Chemistry -Part (i) Insect Chemistry 9 Biosynthesis and Biotransformation Much thought has been directed at the intriguing question of how insects elaborate pheromones. Is de nouo biosynthesis occurring or biotransformation of a close precursor in the diet? Perhaps the greatest effort has been expended in investigation of the production of oxygenated monoterpenoid bark-beetle pheromones presum- ably by oxidation of host-wood monoterpene hydrocarbon^.^' One school of thought suggests haemolymph oxidation processes whereas another favours oxida- tion by symbiotic bacteria in the digestive tract.The latter view is supported circumstantially by the isolation of a Bacillus bacterium from the gut of an Ips species and its ability to oxidize a-pinene to verbenol in vifm5* The epoxide sex pheromone of the Gypsy Moth Porthetria dispar 7,8-epoxy-2-methyloctadecane has been shown to be produced in vivo by oxidation of the corresponding alkene.53 Labelling experiments corroborate the hypothesis that the hairpencil secretions of Danaid butterflies are biogenetically related to alkaloids in the plants on which they feed,54 whereas preliminary feeding experiments using radiolabelled acetate suggest that the ladybird defensive compounds [e.g. (27)]have a polyketide biogene~is.~~’ Methionine has been suggested as the biosynthetic precursor of alkyl sulphides secreted by the Paltothyreus ant.55 10 Olfaction The mode of perception of chemosensory substances by insects is the subject of much speculation.Although detailed discussion is outside the scope of this review a number of articles indicate recent progress in this area.56 11 Techniques of Microscale Structure Elucidation Microscale methods of isolation separation and structure elucidation of compo-nents of insect secretions have been continuously improved. Gas chromatography remains the key separative technique and is most powerful in pheromone studies when used in combination with mass spe~trometry.~~ Attention has been paid to the critical problem of isolation of target substances from biological material particu- larly using methods which avoid the problems of solvent extra~tion.~~ Notably the 51 P.R. Hughes J. Insect Physiol. 1975,21 687; also Naturwiss. 1973,60 261. 52 J. M. Brand J. W. Bracke A. J. Markovetz D. L. Wood andL. E. Browne Nature 1975,254,136. 53 G. Kasang D. Schneider and M. Beroza Natunviss. 1974,61 130. 54 D. Schneider M. Boppre H. Schneider W. R. Thompson C. J. Boriack R. L. Petty and J. Meinwald J. Comp. Physiol. 1975 97 245. 55 R. M. Crewe and F. P. Ross Nature 1975 254,448. 56 R. H. Wright Ann. N.Y. Acad. Sci. 1974,237 Odors Eval. Util. Control Conf. 1973,p. 129;W. A. Kafka ibid. p. 115; W. A. Kafka and J. Neuwirth 2.Nuturforsch. 1975,3Oc 278; D. Schneider Sci.Amer. 1974 231 28; G. Kasang J. Insect Physiol. 1974 20 2407; G. Kasang B. Knauer and M. Beroza Experientia 1974,30,147; M. S. Mayer Experientia 1975,31,452; G. Singer J. M. Rozental and D. M. Norris Nature 1975,256 222. 57 M. Beroza J. Chromatog. Sci. 1975,13,314; S. Stallberg-Stenhagen and G. Bergstrom &on. Suppl. 1973,p. 77; D. E. Games A. H. Jackson D. S. Millington and B. W. Staddon,Adu. MassSpectrometry 1974,6 207; H. R. Buser and H. Am J. Chromatog. 1975,106 83. 5* G. Bergstrom Chem. Scripta 1973,4,135; L. L. Sower J. A. Coffelt and K. W. Vick J.Econ. Entomol. 1973,66,1220; K. J. Byrne W. E. Gore G. T. Pearce and R. M. Silverstein J. Chem. Ecol. 1975,1,1 149. R. Baker and D.A.Evans improvement in Fourier Transform n.m.r.spectrometers now allows routine deter- mination of spectra at a microgram level (e.g. 20 pg for a mon~terpene~~) and we may expect an increasing dependence of pheromone studies upon this technique. 12 Synthetic Studies Acydic Derivatives.-The sex pheromone of the female pink bollworm moth Pectinophora gossypiella (Saunders) [ 1 1mixture of the (2,Z)-7,11(39) and (2,E)-7,11 (40)] has been prepared.59"' In one via (37) and (38) the controlled partial equilibration of the Wittig intermediate formed from (35) and (36) was used (Scheme 1). A convenient preparation of (35) was reported from (Z,Z)-1,5-cyclo- octadiene via the epoxide (33) and the a-ketol (34). 03 (-=J - -* OHC& C0,Et - 0 0 OH (35)1 -6% (36) (33) (34) R C0,Et (39) R = (CH2),0Ac (37)+ R C0,Et (40) R = (CH,),OAc (38) Reagents i Bu'OOH Mo (CO),; ii DMSO air 110°C;iii Pb(OAc), EtOH.Scheme 1 The sex pheromone of the female Angoumois grain moth (E,2)-7,11-hexadecadienyl acetate and its three geometric isomers have been prepared from 3-octyn-1-01and 6-chlorohexan- 1-01.~~ A synthesis of (Z,E)-9,ll-tetradecadien-l-y1acetate a major component of the sex pheromone of both Spodoptera lit~ra~~ and the female Egyptian cotton leafworm S. littoralis (Boisd) and (Z,E)-9,12-tetradecadienylacetate a sex attractant for a number of insect species has been The 92-configuration of the former pheromone was obtained with good stereoselectivity by using sodium methylsul- phinylmethide in DMSO as base in the Wittig The coupling of Grignard reagents with allylic halides has been utilized for the synthesis of the sex pheromones of the codling moth (Laspeyresia pornonella) (43) 59 R.J. Anderson and C. A. Henrick J. Amer. Chem. SOC. 1975,97,4327. 6o P. E. Sonnet J. Org. Chem. 1974 39 3793. 61 K. Mori M. Tominaga and M. Matsui Tetrahedron 1975,31 1846. 62 H. C. Su and P. G. Mahany J. Econ. Entomol. 1974,67 319. 63 G. Goto T. Shima and H. Masuya Chem. Letters 1975 103. 64 H. J. Bestmann 0.Vostrowsky and A. Plenchette Tetrahedron Letters 1974 779. 65 D. R. Hall P. S. Beevor R. Lester R. G. Poppi and B. F. Nesbitt Chem. and Znd. 1975 216. Biological Chemistry -Part (i) Insect Chemistry and the red bollworm moth (46).66 The former pheromone (E,E)-8,lO- dodecadien-1-01 (43) was prepared by reaction of sorbyl bromide (41) and (42).Similarly the coupling of (44) with the Grignard complex of 7-bromoheptan- 1-01 tetrahydropyranyl ether (45) gave 9E 11-dodecadien-1-yl acetate (46) after hy- WBr + BrMgwOTHp (41) (42) OH (43) (46) drolysis and acetylation. Compound (46)has also been prepared by an interesting general method for the conversion of an allylic alcohol into a 1,3-diene (Scheme 2).67 OH 'R i ii AOSiMe, R- R = (CH&OH jiii iv Reagents i V(acac),-Bu'OOH; ii (Me,Si),NH-Me,SiCl-pyridine; iii v PBr,; vi Zn. Scheme 2 The sex pheromone of the European grapevine moth Lobesia botrana (Schiff) proposed as (E,Z)-7,9-dodecadien- 1-yl acetate has been synthesized (Scheme 3).68 A route involving boron in this case the reductive alkylation of acetylene through a lithium trialkylalkynylborate has been employed in a synthesis of pr~pylure.~' 66 K.Mori Tepahedron 1974,30,3807. 67 S. Tanaka A. Yasuda H. Yamamoto and H. Nozaki J. Amer. Chem. SOC.,1975,97,3252. J. N. Labovitz C. A. Henrick and V. L. Corbin Tetrahedron Letters 1975,4209. 69 K. Utimoto M. Kitai M. Naruse and H. Nozaki Tetrahedron Letters 1975,4233. R. Baker and D.A.Evans -f-=-/CO,Me + Reagents i MeC(OMe), EtC0,H; ii Li voAoA Li,CuCI,; iii CCl,CO,H; iv (Me,CHCHMe),BH; v CH,CO,H; vi CCI,CO,H; vii Ac,O-pyridine. Scheme 3 Wittig reactions have also been used to prepare 1-substituted (2)-9-alkene~,~' with particular attention to the synthesis of the sex attractant (muscalure) of the housefly Musca domestica (2)-9-tricosene." This compound has also been pre- pared by the interesting metathesis reaction between 1-decene and 1-tetradecene in the presence of (Ph,P),Mo(NO),Cl and ethyl aluminium dichIoride7 and the reaction of oleic acid with n-pentyl-lithium followed by Huang-Minlon red~ction.~ A method for a stereoselective synthesis of (E)-olefins has been applied to (E)-ll-tetradecenal(51) the sex attractant of the eastern spruce budworm Choris-toneura fumiferana Clemen~.'~Alkylation of y-substituted allylphosphonates (47) occurs exclusively at the a-position with alkyl halides such as (48) resulting in formation of (49).Reductive fission with lithium aluminium hydride yields the (E)-olefin (50). 0 (47) (49) I 70 H.J. Bestrnann W. Stransky 0.Vostrowsky and P. Range Chem. Ber. 1975 3582. 71 H. J. Bestrnann 0.Vostrowsky and M. Hatz Chern.-Ztg. 1974,98 161. '*L. Di Nunno and S. Horio Chimica e Industria 1975 57 242. 73 T. L. Ho and C. M. Wong Canad.J. Chem. .1974,52,1923. 74 K. Kondo A. Negishi and D. Tunernoto Angew. Chem. 1974,86,415. Biological Chemistry -Part (i)Insect Chemistry The photochemical cycloaddition of carbonyl compounds and olefins recently applied to carbonyl-olefin metathesis" has been used to synthesize (E)-non-6-en- 1-01 (55),a sex attractant of the Mediterranean fruit fly.76 Photolysis of propional- dehyde and cyclohexa-1,3-diene in acetonitrile gave (52) which on hydrogenation and treatment of (53) with [Rh(CO),Cl] in refluxing benzene gave (54) and hence (55).(53) (54) R = CHO (55) R = CH,OH The major sex attractant of the oak leaf roller (Archips semiferunus Walker) (2)-10-tetradecenyl acetate has been synthesized from azelaic A sulphenylation-dehydrosulphenylation procedure has provided an interesting route to the queen substance of honey bees (56) from methyl-9-oxodecanoate (Scheme 4).78A 14R-stereochemistry has been assigned to (-)-14-methylhexadec-8-cis-en-1-01 and 0 0 . i ii ~ HO OMe OMe iii-v1 0 0 0 vi vii' fiOMe OMe (56) $Me Reagents i SOCl,; ii Me,CuLi ether -78 "C; iii HOCH,CH,OH TsOH benzene; iv LiNPr;; v MeSSMe HMPA; vi NafO,; vii CaCO, PhMe. Scheme 4 methyl (-)-14-methylhexadec-8-cis-enoate,components of the sex attractant of the female Dermestid beetle Trogoderma inclusum Le Conte by synthesis of their an tipodes from (S)-(-)-2-me thylbutan- 1-01.79 Both enantiomers of disparlure cis-7,8-epoxy-2-methyloctadecane,the sex attractant emitted by the female gypsy moth (Porthetria dispar L.) have been prepared." In preliminary biological tests the (7R,8S)-(+)-disparlure was more active than an authentic specimen of racemic disparlure. The epoxide has also been prepared from a Wittig reaction followed by epoxidation.*' Treatment of the p-silyl 75 G. Jones S. B. Schwartz and M. T. Marton J.C.S. Chem. Comm. 1973 374. 76 G. Jones M. A. Acquadro and M. A. Carmody J.C.S. Chem. Comm. 1975,206. 77 L. B. Hendry S. H. Koneniowski D. M. Hindenlang Z. Kosarych R. 0.Mumman and J. Jugovich J.&em. EEol. 1975,1,317. 78 B. N. Trost and T. N. Salmann J. Org. Chem. 1975,40 148. 79 K. Mori Tetrahedron 1974,30,3817. S. Iwaki S. S. Marumo T. Saito M. Yamada and K. Katagiri J. Amer. Chem. SOC.,1974,% 7842. H. J. Bestmann and 0.Vostrowsky Tetrahedron Letters 1974 207. 360 R.Baker and D.A.Evans oxyanion adduct obtained by reaction of carbonyl compounds with a-silyl carban- ions with thionyl chloride or acetyl chloride has been used in the preparation of alkenes and applied to the synthesis of cis-7 ,8-epoxy-2-me thyloctadecane .82 A procedure for formation of a-branched methyl ketones has been employed for the synthesis of the mixture of diastereoisomers of 3,1l-dimethyl-2-nonacosanone (57) a sex pheromone of the German cockroach Blattella germanicu (L.) (Scheme 5).83 A Wittig procedure has also been used in an alternative A Me Me I I OHC(CH,),OTHP + C1 SH,,C=PPh -+ C1gH,,C=CH(CHZ),OTHP 1 i ii .c Me Me Me I I I C,gH37CH(CH2)7CHCCH=PPh3 3 CI8H,,CH(CHz),Br II ivl 0 Me Me I I C H,,CH(CH ,),CHCMe II 0 (57) 0 II Reagents i H,-Pd; ii PPh,Br,; iii EtCCH=PPh,; iv -OH.Scheme 5 vinylcopper intermediate has been utilized in the synthesis of bombyk01.~~ The courting hormone of the Douglas Fir Tussock moth (Orgyia pseudotsugata) (2)-6-heneicosen-11-one (59)has been synthesized from the dithian (58) (Scheme 6).86 A Highly selective procedure for the dehydration of P-hydroxy-esters via p-alanoxy-esters has been developed and applied to the synthesis of (E)-4,6-dimethyl-4-octen-3-one (manicone) (60),a component of the mandibular secretion of the ant Manica (Scheme 7).87The alarm pheromone of Attu texanu (S)-(+)-4-methyl-3-heptanone and its enantiomer have been prepared in high optical purity.88 A number of syntheses of 2-methyl-6-methylene-7-octen-4-ol(6 l) the aggrega- tion factor of the bark beetle Ips confusus have appeared.A new synthesis of 1-substituted cyclobutenes has been described and their thermal opening to form 1,3-dienes utilized.89 Metallation of methylenecyclobutane with n-butyl-lithium and tetramethylethylenediamine in hexane gives the anion (62). Reaction of this with electrophiles yields the two anticipated products (63) and (64),and optimum conditions were found for the formation of,(63).The addition of iso-valeraldehyde 82 T. H. Chan and E. Chang J. Org. Chem. 1974,39,3264. 83 M. Schwarz J. E. Oliver and P. E. Sonnet J. Org. Chem. 1975,40,2410. 84 A. W. Burgstahler and L. 0.Weigel J. Org. Chem. 1975,40 3456. 85 J. F. Normant A. Commercon and J. Villieras Tetruhedron Letters 1975 1465. 86 R. G. Smith G. D. Davies and G. E. Daterman J. Org. Chem. 1975,40 1593. 87 J. A. Katzenellenbogen and T. Utawanit J. Amer. Chem. Soc. 1974,% 6153. R. G. Riley and R. M. Silverstein Tetrahedron 1974 30 1171. 89 S. R. Wilson and L. R. Phillips Tetrahedron Letters 1975 3047. Biological Chemistry -Part (i) Insect Chemistry (58) OH ClOH21 +.HlL -C*oH21 L-g5H' -1 HH HH (59) Reagents i BuLi THF; ii CI(CH,),C~CC,H,,; iii CuO CuCl, H,O ; iv LiAIH Et,O; v P-2Ni-H,.Scheme 6 C02Et 2,/yyC02Et 4 Reagents i Al(OEt),Cl; ii LiNPr,; iii NaOH; iv (COCI,),; v Et,CuLi. Scheme 7 to (62) gave (65) which was separated from the isomer by chromatography; a smooth conversion into 2-methyl-6-methylene-7-octen-4-ol(6 1)was obtained by heating at 150°C. The diene (61) has also been prepared by reaction of 3-methyl-2-butenal with 2-bromomethyl-1,3-butadiene in the presence of zinc in refluxing THF," and from myrcene by initial epo~idation.~' The absolute configuration of natural (61) OH I (65) R. G. Riley and R. M. Silverstein J. Org. Chem. 1974,39 1957. 9' K.Mori Agric. and Biol. Chem. (Japan) 1974,38,2045. R.Baker and D. A.Evans has been established as (S)-(-) by a synthesis beginning with L-(+)-leucine and involving an optically active a-methyIene-y-lact~ne.~* An elegant Norrish tw I a-cleavage of the acy -disubstituted cyclopentanone (66) has been used as the key step in the preparation of 7-methy1-3-n-propyl-(Z7Z)-2;6-decadien-1-01 (67).93A similar approach has been used in the synthesis of dihydro- terpenediol one of the major components of the hairpencil of the African Monarch butterfly Danaus chrysipp~s.~~ &4/\I\\/GCHO + LCHO (66) 1Pr"MgBr Alicydic Derivatives.-Grandisol(70) a component of the pheromone released by the male boll weevil Anthonomus grandis has attracted the attention of a number of groups and the synthetic studies have resulted in the development of valuable new methods for the formation of cyclobutyl derivatives.Cyclization of (68)in the presence of potassium hexamethyldisilazane in refluxing benzene led to formation of (69) which was converted by a series of reactions into (70).95Base-promoted cyclization of the 6-chloro-ester (71) in THF solution containing a catalytic amount of hexamethylphosphoramide gave a mixture of (72)and (73).96Addition to the ester mixture of an excess of methyl-lithium gave (74)as the major product which R' ''7 HCOH LiNPr 4-~2-4 _-_ THF-HMPA __-CO,Et \ \ Me (71) (72) R' = H R2 = CO2Et (74) (73) R' = C02Et R2=H 92 K. Mori Tetrahedron Letters 1975,2187. 93 J. P. Morizur G. Muzard J. J. Basselier and J. Kossanyi Bull. SOC. chim. France 1975 257. 94 J. P. Morizur G. Bidan and J.Kossanyi Tetrahedron Letters 1975,4167. 95 G. Stork and J. F. Cohen J. Amer. Chem Soc. 1974 %,5270. % J. H. Babler Tetrahedron Letters 1975,2045. Biological Chemistry -Part (i) Insect Chemistry was converted into (70). A new and novel fragmentation reaction of an ozonide has been applied to the synthesis of (*)-grandis01 uia (75) (Scheme 8). H (75) Reagents i MeLi; ii O, -70 "C CH,Cl,; iii NaHCO, H,O. Scheme 8 A further reaction sequence for the synthesis of grandisol has involved the addition of 1-lithiocyclopropyl phenyl sulphide to the aldehyde (76) followed by acid-catalysed rearrangement to cyclobutanone (77).98 Repetition of this sequence gave (78) which was transformed by bromination ring cleavage and silver-catalysed solvolysis into (79) and finally by a sequence culminating in a sulphoxide elimina- tion into grandisol (70).4 Phs7 phsl ACHO phs%? (76) (78) (77) 1 PhS > C0,Me ('O) -53 (79) The acid-catalysed cyclization reactions of y-geraniol and methyl y-geranate have been examined as possible routes to the cyclohexyl constituents of the Boll Weevil pher~mone.~~ Cembrene-A the trail pheromone of the termite Nasutitermes exitiosus has been synthesized by an elegant anion-induced cyclization of an acyclic terpene.lm trans trans-Geranyl-linalool (80) was converted into all-trans-geranylgeranyl phenyl 97 R. L. Cargill and B. W. Wright J. Org. Chem. 1975,40 120. 98 B. M. Trost and D. E. Keeley J. Org. Chem. 1975,40,2013. 99 R.H.Bedoukian and J. Wolinsky J. Org. Chem. 1975,40,2154. loo M.Kodama Y. Matsuki and S. Ito Tetrahedron L.emrs. 1975,3065. R.Baker and D.A.Evans thioether followed by terminal epoxidation to yield (81). Intramolecular cyclization took place on reaction of (81) in THF in the presence of butyl-lithium and 1,4-diazabicyclo[2,2,2]octane with formation of (82). Desulphurization with lithium-ethylamine and dehydration with thionyl chloride in pyridine gave (83). tPhS Cembrene (83)has also been prepared by a route in which the significant step was a nickel carbonyl coupling of terminal allylic bromides. lo' The synthesis of the four stereoisomers of 3-methyl-5-butyl-octahydroindolizine a reported trail pheromone of the Pharaoh's ant Monomorium pharaonis has been described.lo2 Both enantiomers of frontalin the pheromone of the bark beetle Dendroctonus frontah have been synthesized from (R)-(+)-2-hydroxy-2-methylpentane-1,5-dioic acid 5,2-lactone and its antip~de."~ (1R,7R)-(+)-exo-Brevicominand its antipode one of which is an attractant in the frass of Dendroctonus brevicomis have been synthesized from (2s,3s)-D-( -)-tartaric acid.lo4 A Kolbe electrolysis has been used to form (84),which on oxidation to the diol and acid-catalysed cyclization yields brevicomin (85)(Scheme 9).lo5 A homologue of brevicomin has been prepared by acid-catalysed cyclization of a pyranmethanol derivative.13 Biological Control of Insect Pests Many of the insect species mentioned in this review are pests which are economically important because of the damage they cause.Consequently in addition to the furtherance of our basic knowledge of chemosensory communication there exists the possibility of the use of this information in novel pest-control methods. Because of the inherent specificity of pheromones and related phenomena and the minute Iol W. G. Dauben G. H. Beasley M. D. Broadhurst B. Muller D. J. Peppard P. Pesnelle and C. Suter J. Amer. Chem. Soc. 1974,% 4724; ibid. 197597 4973. Io2 J. E. Oliver and P. E. Sonnet,1.Org. Chem. 1974,39,2662; P. E. Sonnet and J. E. Oliver J. Heterocyclic Chem.,1975,12,289. 1°3 K. Mori Tetrahedron,1975 31 1381. Io4 K. Mori Tetrahedron 1974 30 4223. *05 J. Knolle and H. J. Schafer Angew Chem. Internut.Edn. 1975,14 758. lo6 B.P. Mundy L. B. Lipkowitz and G. W. Dirke Synth. Comm. 1975,5,7. Biological Chemistry -Part (i) Insect Chemistry 0 (84) 1 0 0 HO Scheme 9 concentration threshold above which they operate it should be possible to avoid the problems of pollution and indiscriminate action associated with synthetic pesticides. Many pheromone-based treatments are currently undergoing field evaluation and recent reviews indicate the progress achieved.’” Io7 J. L. March Science 1973,181 736;W. Roelofs Enuiron. Letters 1975,8,41;E. R. Mitchell Bioscience 1975,25 493; H.Z.Levinson Natunviss. 1975,62,212.

ISSN:0069-3030    

DOI:10.1039/OC9757200347

出版商:RSC    

年代:1975

数据来源: RSC

摘要:

14 Biological Chemistry Part (ii) Steroids By D. N. KIRK Chemistry Department Westfield College Hampstead London NW3 7ST 1 Introduction This Report presents a selection of the work published during 1974 and 1975. The fourth and fifth annual volumes of the Chemical Society’s Specialist Periodical Report on Terpenoids and Steroids have appeared with comprehensive coverage of the literature from September 1972 to August 1973 and the corresponding period in 1973-1974 respectively. Sections on ‘Steroid Properties and Reactions’ appear in both volumes. ‘Microbiological Reactions with Steroids’ and ‘Steroid Conforma- tions from X-Ray Analysis Data’ are reviewed in Volume 4 and ‘Steroid Synthesis’ in Volume 5. The sixth volume of the series is expected during 1976.2 Synthetic Transformations The discovery that ‘vitamin D’ is transformed in vivo into its la,25-dihydroxy- derivative (l) the active form of the vitamin has prompted intensive studies in synthesis. The 1a-hydroxy-group has been introduced by reactions of suitable 1a,2a-epoxy-derivatives with lithium-ammonia’*2 or aluminium amalgam,3 or by selective hydroboration of a 1’5-dien-3 p-01.~The 5,7-diene system required for photoisomerization to the ‘previtamin’ [9( lO)-seco-5( 10),6,8-triene (2)] has been HO‘ (1) D. H. R. Barton R. H. Hew M. M. Pechet and E. Rizzardo J.C.S. Chem. Comm. 1974,203. D. Freeman A Acher and Y. Mazur TetrahedronLetters 1975,261. 3 T. A. Narwid J. F. Blount J. A. Iacobelli and M. R. Uskokovic Helu. aim. Acta 1974,57,781.C. Kaneko S. Yamada A. Sugimoto Y. Eguchi M. Ishikawa T. Suda M. Suzuki S. Kakuta and S. Sasaki Steroids 1974,23 75. 366 Biological Chemistry -Part (ii) Steroids introduced either before5 or after4 the 1a-hydroxylation sequence (Scheme 1). Protection of the sensitive 5,7-diene was achieved by formation and subsequent reductive cleavage of the adducf with 4-phenyl-1,2,4-triazoline-3,5-dione. \ 0 HO viii xii i iiJ 1 7 0hi Reagents i K0Bu'-DMSO; ii H+ (kinetic control); iii NaBH,; iv B,H, H,O,-HO-; v H,O,-HO-; vi Li-NH,-NH,Cl; vii Al-Hg NaBH, LizC03-DMF; viii Ca(BH,) ;ix 4-phenyl-1,2,4- triazoline-3,5-dione;x m-CIC6H4C03H;xi LiAIH4 ;xii dibromodimethylhydantoln,(MeO)JP. Scheme 1 The 25-hydroxycholestane side-chain is usually obtained by Grignard methylation of a 27-norcholestan-25-one which was originally obtained as a by-product of cholesterol oxidation.The 25-ketone is now available by novel routes from either an androstan- 17-one6 or a pregnan-20-one.' An alternative synthesis of 25-hydroxycholesterol employs a Gzintermediate (3) obtained from stigmasterol the rest of the side-chain being added by use of the lithium acetylide derivative (4).* Some vitamin D derivatives with additional hydroxylation at C-24 have also been synthesized.' Androstane" and pregnane'' analogues of 1a-hydroxy-vitamin D are almost devoid of biological activity. C. Kaneko A. Sugimoto Y.Eguchi S.Yamada M. Ishikawa S. Sasaki and T. Suda Tetrahedron 1974 30,2701. J. Wicha and K. Bal J.C.S.Chem. Comm. 1975,968. T. A. Narwid K. E. Cuoney and M. R. UskokoviE Helv. Chim. Acra 1974,57,771. 8 J. J. Partridge S.Faber and M. R UskokoviE Helv. Chim. Ada 1974,57,764. N. Ikekawa M. Morisaki N. Koizumi Y.Kato and T. Takeshita Chem.and Phrm. Bull. (Jupan),1975 23,695; M. Seki N. Koizumi M. Morisaki and N. Ikekawa TerrahedronLetters 1975,15; N. Koizumi M. Morisaki N. Ikekawa A. Suzuki and T.Takeshita ibid. p. 2203; N. Ikekawa M. Morisaki N. Koizumi M.'Sawamura Y.Tanaka and H. F. DeLuca Biochem.Biophys. Res. Comm. 1975,62,485. lo H. Sakamoto A. Sugimoto C. Kaneko T.Suda and S. Sasaki Chem. and Pharm. Bull. (Japan),1975 23 1733. H.-Y. Lam H.K.Schnoes I-l. F. DeLuca and L. Reeve Steroids 1975,26,422. 368 D.N. Kirk (5) OMe (3) The first satisfactory syntheses are reported for 18’2 l-dihydroxypregn-4-ene- 3,20-dione [‘18-hydroxy-deoxycorticosterone’; as hemiacetal (6)] a steroid impli- cated in hypertension.The 18-hydroxy function was introduced into a pregnan-20- one via the 2Op-01 either by the ‘hypoiodite’ sequence” or by photolysis of the 20-nitrite in the presence of oxygen,13 which affords the 18-nitrate. 18-Hydroxypregn-4-ene-3,20-dione(in 18-+20-hemiacetal form (7)] gives the 21- acetoxy-derivative (8) in a single step by reaction with lead tetra-acetate.” The intermediate 18,20-epoxypregn-20-ene (9) can be obtained if required by treating (6) R = OH (7)R = H (8) R = OAC the hemiacetal(6) with aluminium isopropoxide in toluene,14 but is very sensitive to acids. A new synthesis of compounds of the aldosterone series proceeds via the Barton reaction (photolysis of an 11p-nitrite) to the 18-oxhe and thence the nitrone (10).15 Choice of the 1,4-dien-3-one as the starting material prevented competing attack on the C-19 methyl group.Either labelled or unlabelled aldosterone could be produced in a final selective hydrogenation of the 1,2-ethylenic bond. The nitrone function in compounds of type (10) provides the necessary activation for acetoxyla- tion at C-21 through acetylation and rearrangement leading to the 18,21-diacetate (1 1). Oxidation of the nitrone with Jones’ reagent can be controlled to give either the hemiacetal (12) or the lactone (13).15 New five-step carbonyl transpositions (Scheme 2) convert an androstan-17-one efficiently into the 16-0~0-i~0mer,’~ and enones into isomeric enones [e.g.3-oxo-A4 +4-0x0-A’ (Scheme 2) or 3-0x0-Ah’ -+2-0xo-A~I.’~ Beckmann cleavage and l2 D. N. Kirk and M. S. Rajagopalan J.C.S. Chem. Comm. 1974 145; J.C.S. Perkin I 1975 1860. I3 D. H. R. Barton M. J. Day R. H. Hesse and M. M. Pechet J.C.S. Perkin I 1975 2252. l4 M. Biollaz J. Kalvoda and J. Schmidlin Helo. Chim. Acta 1975,58 1425. D.H. R. Barton N. K. Basu M. J. Day R. H. Hesse M. M. Pechet and A. N. Starratt J.C.S. Perkin I 1975,2243. l6 B. M. Trost K. Hiroi and S. Kurozumi J. Amer. Chem. Soc. 1975,97,438. 17 M. K. Patel and W. Reusch Synth. Comm. 1975 5 27. Biological Chemistry -Part (ii) Steroids 0-R' 0'&ocH2R2 0& (11) R' = H OAC; R2 = OAC (12) R' = H OH; R2 = H (13) R' = 0;R2 = H Reagents i Li N-cyclohexyl-N-isopropylamide, THF -78 "C;ii PhSSPh HMPA 0°C;iii NaBH,-MeOH; iv MeSO,CI-py; v K0Bu'-DMSO; vi HgCI, MeCN-H,O; vii H,O,-NaOH ; viii MeONa-MeOH ; ix TsNHNH ; x 2MeLi ; xi H + Scheme 2 re-cyclization (Scheme 3) provides a route from a 17-0x0-steroid to its 18-nor analogue.18 New methods for building up steroid side-chains include reactions of 17-0x0-groups with tosylmethyl isocyanide l9 the a-1ithio-derivatives of ethyl diazoacetate lnM.M. Coombs and C. W. Vose J.C.S. Chem. Comm. 1974,602. l9 J. R. Bull and A. Tuinrnan Tetrahedron 1975 31 2151; J. R. Bull .I.Floor and A. Tuinrnan ibid.,p. 2157. 370 D.N.Kirk 1iii Reagents :i Dicyclohexylcarbodi-imide,CF,CO,H DMSO ;ii m-CIC,H,CO,H ;iii BF ,hydrolysis.Scheme 3 or isocyanoacetate,20 propen-2-yl-lithi~m,~' or tri-n-butyl(1-methoxycarbonyl-prop-2-ylidene)pho~phorane~~ (Scheme 4). A novel side-chain degradation Reagents i TsCH,NC; ii MeLi; iii CNCHC0,Et Li' ; iv CH,=CMe Li'; v PhSCI; vi Li+ NEt; ; vii PhSSPh; viii HgCl ;ix Bu,P=CMeCH,CO,Me Scheme 4 2o U. Schollkopf B. BBnhidai H. Frasnelli R. Meyer and H. Beckhaus Annalen 1974 1767. B. M. Trost and J. L. Stanton J. Amer. Chem. Soc. 1975,97,4018. 22 A. Scettri E. Castagnino and G. Piancatelli Gazzerta 1974 104,437. Biological Chemistry -Part (ii) Steroids 371 involves oxygenation of tne phenyl ketone (14) in alkaline solution the resulting bisnorcholan-22-oic acid (15) was further degraded by lead tetra-acetate in benzene-pyridine to the 20-acetoxypregnane (16).23 3 Oxidation and Reduction A new variation on the principle of 'remote oxidation' employs an iodo-aryl- substituted 3a-ester (see p.372) of 5tr -cholestan-3a-01.~~ Photolysis of the derived iododichloride (17) results in radical-induced chlorination at whichever tertiary centre is best placed for attack by the free radical generated at the iododichloride site (14a when the p-iodophenyiacetate is used or 9a with the rn -iodobenzoate). Alternatively iodobenzene dichloride may be photolysed in the presence of the steroid iodo-aryl ester which acts as a radical carrier with similar results.2s Dehydrochlorination of the 14a-or 9a-chlorinated steroids gave A14-or A9(")-enes respectively.9a -Chlorination of a suitable pregnane derivative by this method opened the way to a novel synthesis of A variant using the 3a-(4'- iodobiphenyl-3-carboxylate(18) permits attack at C- 17 leading to a procedure for removal of the entire side-chain and generation of a 17-0xo-group.~~ 1' (17) (18) The Oxford group continue to publish details of their studies on microbiological hydroxylation of varied steroid Lewis acids catalyse the oxygenation of ergosteryl acetate to give the 5a,8a-epidioxide oxygenation in the dark appears to involve triplet oxygen.28 Cholesteryl acetate can be nitrated safely at C-6 by using 23 M. Feiizon F. J. Kakis and V. Ignatiadou-Ragoussis Tetrahedon 1974,30 3981. 24 R. Breslow R. Corcoran J. A. Dale S. Liu and P.Kalicky J. Amer. Chem. SOC.,1974?-96 1973. 25a R. Breslow R. J. Corcoran and B. B. Snider 1 Amer. Chem. Soc. 1974,% 6791. 25b R. Breslow B. B. Snider and R. J. Corcoran J. Amer. Chem. SOC.,1974,96,6792. 26 B. B. Snider R. J. Corcoran and R. Breslow J. Amer. Chem. Soc. 1975,97 6580. 27 Sir E. R. H. Jones G. D. Meakins J. 0. Miners and A. L. Wilkins J.C.S. Perkin I 1975 2308 and references therein. 28 D. H. R. Barton R. K. Haynes P.D. Magnus and I. D. Menzies J.C.S. Chem. Comm. 1974,511;D. H. R. Batton R. K. Haynes G. Leclerc P. D. Magnus and I. D. Menzies J.C.S. Perkin I 1975,2055. 372 D.N.Kirk modified reaction condition~.~~ Iodine-potassium iodate in acetic acid3’ is a cheap and effective alternative to iodine-silver acetate for the conversion of 5a-cholest-2- ene into 2~-acetoxy-3a-iodo-5a-cholestane (Prevost reaction).The 2-ene with phenylselenenyl acetate unfortunately gives a mixture of the 2P-acetoxy-3a- phenylseleno- and the 3a -acetoxy-2~-phenylseleno-derivatives, leading to a mix- ture of two allylic acetates after oxidative elimination of selenium.31 Reduction [LiAlH(OBu‘),] of a series of 5a-cholestan-3-ones with C-5 sub- stituents gave product mixtures which are considered to imply dissymmetry of the carbonyl *IT* orbital depending upon the nature of the 5a-s~bstituent.~~ Sodium cyanoborohydride in acidified THF reduces 3-0x0-steroids selectively in the pres- ence of 17-0x0- or 20-0xo-functions.~~ 5a-Cholestan-3P-01 is among alcohols deoxygenated in useful yield by treating the thiobenzoate with trib~tylstannane.~~ 4 Substitution and Elimination Sa-Cholestan-3a-yl esters [e.g.(18)] required for ‘rtmote oxidations’ (see above) are available in a single step from the 3P-01 by the ‘substitution-inversion’ proce- dure using diethyl azodicarboxylate triphenylphosphine and the appropriate car- boxylic acid.26 3a-Phenoxy-derivatives result when a phenol is used in place of the carboxylic acid.35 Cholesterol with benzoic acid under these conditions gave a complex mixture including 3a,5a-cyclocholestanyl derivative^.^^ Potassium superoxide (KO,) with 18-crown-6 to capture potassium ions converted cholesteryl tosylate directly into cholest-5-en-3a-01 in an unusual direct substitution with inversion.37 Fluoro-derivatives are obtained from steroidal or their trimethylsilyl by the use of phenyltetrafluorophosphorane.Acetate ion converts a 4P-bromo-3-oxo-5P -steroid initially into the 20 -acet~xyketone,~’ which subsequently inverts to the more stable 2P-isomer. The implied mechanism (Scheme 5) includes allylic substitution by attack on the A2-enol with a trans relationship of the entering and leaving groups which is contrary to earlier evidence in favour of cis stereochemistry in comparable reactions but which may be compati- ble with recent orbital-symmetry consideration^.^^ Unexpectedly large effects of remote polar substituents (at C-17) on the rates of solvolysis of 5a-androstan-3-yl tosylates suggest that charge-dipole interactions between the reaction site and the substituent are modified by delocalization of negative charge into the 29 A.T. Rowland Steroids 1975 26 251. 30 L. Mangoni M. Adinolfi G. Barone and M. Parrilli Guzzeitu 1975,105,377. 31 K. B. Sharpless and R. F. Lauer J. Org. Chem. 1974 39 429. 32 C. Agarni A. Kazakos and J. Levisalles Tetrahedron Letters 1975 2035. 33 M.-H. Boutigue and R. Jacquesy Compt. rend. 1973 276 C 437. 34 D. H. R. Barton and S. W. McCornbie J.C.S. Perkin I 1975 1574. 35 M. S. Manhas W. H. Hoffman B. Lal and A. K. Bose J.C.S. Perkin I 1975,461. 36 R. Aneja A. P. Davies and J. A. Knaggs Tetrahedron Letters 1975 1033. 37 E. J. Corey K. C. Nicolaou M. Shibasaki Y. Machida and C. S. Shiner Tetruhedronhtters,1975,3183. 38 Y. Kobayashi I. Kumadaki A. Ohsawa M. Honda and Y. Hanzawa Chem.undPharm. Bull. (Jupun) 1975,23 196. 39 N. E. Boutin D. U. Robert and A. R. Cambon Bull. Soc. chim. France 1974 2861. 40 T. T. Takahashi and J. Y. Satoh Bull. Chem. Soc. Japan 1975,48,69. 41 R. L. Yates N. D. Epiotis and F. Bernardi J. Amer. Chem. SOC.,1975,97 6615. 42 P. E. Peterson and D. M. Chevli J. Org. Chem. 1974 39 3684. Biological Chemistry -Part (ii) Steroids -* ACO HO HO 0@-Br H 1 Scheme 5 5 Molecular Rearrangements The hyperacidic solvent system HF-SbF induces some remarkable carbocation rearrangements involving the steroid ‘backbone’. Oestr-4-ene-3,17-dione (19) is isomerized to the more stable 14p configuration (20) through migration of a cationic centre from C-5 to C-14 and back to C-5;43the carbocation can be intercepted mainly at C-8 by an added cycloalkane as reducing agent to give 5ar,8a,14@- and Sa,8p,14P-oestrane-3,17-diones(21).Gaseous hydrogen gives an alternative H0 0’ (19) 14a-H (20) 14p-H reduction product the rearranged ‘anthra-steroidal’ dione Oestrone (23) similarly affords the ‘anthrasteroid’ (22),44 although in the absence of a reducing agent oestrone and some of its isomers afford the de-aromatized oestra-4,9-diene- 3,ll-diones (24).45 The trienone (25) aromatizes in ring A as well as being 43 J.-C. Jacquesy R. Jacquesy and G. Joly Tetrahedron Letters 1974,4433;Bull. SOC. chim. France 1975 2281,2289. * 44 J.-c. Jacquesy R. Jacquesy and G. Joly Tetrahedron 1975,31 2237. 45 J.-P. Gesson J.-C. Jacquesy R. Jacquesy and G.Joly Bull. SOC.chim. France 1975 1179. 374 D.N. Kirk epimerized at C- 14 to give the phenolic product (26).46 Pregnan-20-ones isomerize in hyperacidic media to give a mixture of the four isomers resulting from equilibra- tion of configurations at C-13 and C-17 through a 13,17-~eco-intermediate.~' 'Backbone' rearrangements [e.g. (27) +(28)] induced by sulphuric acid-acetic anhydride apparently proceed through a concerted mechanism,48 for no isotope (27) (28) incorporation occurred in deuterium-labelled solvents. The amine holamine (29) and related compounds however rearrange (in H2S04)to give the 13(17)-ene (30) with extensive loss of a deuterium label from C-8,49implying that this reaction involves a deprotonation-reprotonation mechanism via an olefinic intermediate.Backbone isomerizations of androst-5-ene (31) and ~-homoandrost-5-ene (32) give A8-olefins (33) with equilibrated configurations at C-5 C-10 C-14 and (in the D-homo-compound) also at C-13; trifluoroacetic acid promotes these and related rearrangements under very mild c~nditions.~'The ease of rearrangement even in the D-homo-compound (32) contradicts the earlier view that strain at the trans-C/D 46 J.-C. Jacquesy R. Jacquesy and Ung Hong Ly Tetrahedron Letters 1974,2199. 47 J.-C. Jacquesy R. Jacquesy and J.-F. Patoiseau Bull. SOC. chim. France 1974 1959. 48 E. T.J. Bathurst and J. M. Coxon,J.C.S. Chem. Comm. 1974,131; E. T. J. Bathurst J. M. Coxon and M. P. Hartshorn Austral. J. Chem. 1974,27 1505. 49 J.Thierry F. Frappier M. Pais and F.-X. Jarreau Tetrahedron Letters 1974 2149. 5O D. N. Kirk and P. M. Shaw J.C.S. Perkin I 1975,2284. 375 Biological Chemistry -Part (ii) Steroids & 8 H H2In 10 \ l4H H2)n 5 \ Me (31) n = 1 (32) n = 2 (33) ring junction provided the ‘driving force’ for backbone rearrangements which are now seen as merely olefin equilibrations via cationic intermediates. The rearrange- ment of androst-5-en-17-one (34) stops at the intermediate 5-methyl-5P-oestr- 9(11)-en-17-one (35) in trifluoroacetic acid but proceeds further to give the two 8-enes (36) in sulphuric acid-methanol. (34) (35) (36) 5,104s Hydrogen fluoride induces extensive rearrangements in suitable cholestanes [e.g. (37) +(38)J; both the ‘backbone’ and the side-chain are in~olved.’~ HO.-9% HH HO..H OH (37) (38) The 9a,l la!-epoxide (39) was rearranged by boron trifluoride with lop-methyl migration to give in part the 9P-methyl product (40).52 AcO AcO (39) (40) 51 A. Ambles C. Berrier and R.Jacquesy Bull. SOC.chim. France 1975,835. 52 A. C. Campbell C. L. Hewett M. S. Maidbent and G. F. Woods J.C.S. Perkin I 1974 1799. 376 D.N. Kirk 6 Miscellaneous Reactions Prolonged heating in acetic anhydride-pyridine converts ketoximes into enimides through a radical mechanism. 5a -Cholestan-3-one oxime (41) gave the enimide (42) which afforded the enamide (43) after chromatography on alumina. When treated with lead tetra-acetate7 the enamide gave the a-acetoxy-ketone (44).53 (42) R = AC (43) R = H The oxime (45) of a 17-0x0-steroid gave the enamide (46) with the 13a-configuration; this efficient method of inversion of configuration at C-13 appears to be preferable to the earlier photoisomerization of a 17-0x0-steroid but like the (45) (46) latter is thought to proceed by a radical mechanism through a 13,17-seco-intermediate.54 A 20-oximinopregnane (47) was converted by an ingenious ‘one- pot’ application of the enamide-lead tetra-acetate sequence into the 17a,21- diace toxypregnan-20-one (48).55 Me CH,OAc I (47) (48) Tosylhydrazones afford the parent ketones on reaction with N-bromosuccinimide followed by sodium hydrogen s~lphite,~~ Diethylene or with titanium tri~hloride.~’ orthocarbonate converts ketones into ethylene acetals under very mild acidic 53 R.B. Boar J. F. McGhie M. Robinson D. H. R. Barton D. C. Horwell and R. V. Stick J.C.S.Perkin I 1975,1237. 54 R. B. Boar F. K. Jetuah J. F. McGhie M. S. Robinson and D. H. R. Barton J.C.S. Chem. Comm. 1975 748. 55 R. B. Boar J. F. McGhie M. Robinson and D. H. R. Barton J.C.S. Perkin I 1975 1242. 56 G. Rosini J. Org. Chem. 1974,39,3504. 57 B. P. Chandrasekhar S. V. Sunthankar and S. G. Telang Chem. and Ind. 1975,87. Biological Chemistry -Part (ii) Steroids conditions;'$ methyl thio trime t h ylsilane affords bis(me t h yl t hio)ace tals even without acid catalysis and attacks 5cu-androstane-3,17-dioneselectively at C-3.59 Glycoside formation from steroids,60 and the controlled trimethylsilylation of steroidal alcohols and enolizable ketones,61 have been reviewed.The absolute configurations of steroidal alcohols a-glycols and vicinal amino-alcohols can be determined by c.d. measurements on metal complexes.62 Steroidal ketones provided the majority of compounds used in a new and wide-ranging empirical analysis of c.d. data (n+ =*) for decalone ana10gues.~~ 58 D. H. R. Barton C. C. Dawes and P. D. Magnus J.C.S.Chem. Comm. 1975,432. 59 D. A. Evans K. G. Grimm and L. K. Truesdale J. Amer. Chem. Soc. 1975,97,3229. 6o G. Wulff and G. Rohle Angew. Chem. Internat. Edn. 1974 13 157. 61 H. Gleispach J. Chromatog. 1974,91,407. 6* J. Dillon and K. Nakanishi J. Amer. Chem. Soc. 1974,% 4055,4057,4059; 1975,97 5409. 63 D. N. Kirk and W. Klyne J.C.S.Perkin I 1974 1076.

ISSN:0069-3030    

DOI:10.1039/OC9757200366

出版商:RSC    

年代:1975

数据来源: RSC

摘要:

14 Biological Chemistry Part (iii) Enzyme Mechanisms By A. D. 6. MALCOLM and J. R. COGGINS Department of Biochemistry University of Glasgow Glasgow G12 800. 1 Introduction A list of recent and relevant reviews’ and books2 is included in this Report. The methods used to study enzymes are now sufficiently sensitive that it is no longer necessary to restrict experiments to enzymes which are merely easy to prepare or particularly stable and this is reflected in the selection of enzymes for this year’s Report. 2 Nitrogenase The importance of nitrogenase in the fixation of nitrogen is obvio~s,~ but since it is oxidized rapidly and irreversibly to an inactive form it has not been easy to study un ti1 recently. Nitrogenase has now been isolated from a variety of sourcess and appears to have very similar structure and properties irrespective of the source.The molecular weight is approximately 3X lo5Daltons and the molecule comprises two proteins the Mo-Fe protein and the Fe protein. The properties of the enzyme6 are sum- marized in Table 1. Although nominally the reaction catalysed is N + 6e + 6H’ -+ 2NH the enzyme can utilize a variety of substrates such as N20 CN- N3-,and GH,; the reduction of acetylene to ethylene is commonly used to assay the enzyme’s activity. (a)R. E. Amstein M. Harvey and H. R. V. Amstein F.E.B.S. Letters 1975,55,Supplement pp. 26,42; (b) H. Gutfreund Progr. Biophys. Mol. Biol. 1975,29,161;(c) D. S. Sigman and G. Mooser Ann. Reu. Biochem. 1975,44,889; (d) R. L. Soffer Adv.Enrymol. 1974,40,91; (e)E. E. Snell ibid. 1975,42 287; (f) T. L. Rosenberry ibid. 1975,43,103;(8) W. P. Jencks ibid. p. 219; (h) R. L. Hill and K. Brew ibid. p. 411; (i) I. A. Rose ibid, p. 491; (j)R. E. Feeney G. Blankenhoern and H. B. F. Dixon Adu. Protein Chem. 1975,29 136; (k) I. Fridovich Ann. Rev. Biochem. 1975 44 147. * (a)J. T. F. Wong ‘Kinetics of Enzyme Mechanisms’ Academic Press London 1975; (b) 1. H. Segel ‘Enzyme Kinetics’ John Wiley London 1975; (c) ‘Isoenzymes’ ed. C. L. Markert Academic Press London 1975 Vols. 14; (d) ‘The Enzymes Vol 11 Oxidation-Reduction Part A’ ed. P. D. Boyer Academic Press New York 1975. R. W. F. Hardy and U. D. Havelka Science 1975,188,633. 4 (a)R. H. Burns Methods EnzymoL 1972,24B 415; (b) L. E. Mortenson ibid. p.446. 5 R. R. Eady and J. R. Postgate Nature 1974 249,805. 6 R. R. Eady B. E. Smith K. A. Cook and J. R. Postgate Biochem. J. 1972,128,655. 378 Biological Chemistry -Part (iii) Enzyme Mechanisms Table1 Some propertiesof nitrogenasea Mo-Fe protein Fe protein Molecular weight 229 000 66 700 Subunits 2 X 51 300 +2 X 59 600 2 X 34 000 g atom per mole of Mo Fe S2- 1(or 2') 18 (or 24-32') 18 0 4 3.8 ti in air 600 s 45 s Other abbreviations Component IKP1 KP2Component 11 a R. R. Eady B. E. Smith K. A. Cook and J. R. Postgate Biochem.J. 1972,128,655;bV.K. Shah L. C. Davis and W. J. Brill Biochim. Biophys. Acta 1975,384,353; 'M. G. Yates and K. PlanquC European J. Biochem. 1975,60,467. The ratio of the rates of reduction of nitrogen and acetylene depends on the molar ratios of the two proteins which comprise nitr~genase.~ For example when the Mo-Fe protein :Fe protein ratio is 1 1 the ratio of C2H2:N2 reduction is a minimum at 3.4 1,but when the protein ratio is 20 1the ratio C,H2 N2reduced is 5.3 1.Since the Fe protein is particularly labile it is often difficult to use acetylene reduction for the estimation of nitrogenase in crude extracts. A side-reaction the reduction of protons to hydrogen gas is always present and a correction for this must be made in experiments where total electron flow is measured. In spite of the fact that the above reaction is exergonic (AGig8=-53.5 kJ mol-') nitrogen fixation is accompanied by hydrolysis of ATP to ADP and inorganic phosphate and it therefore seems possible that either photophosphorylation8 or oxidative phosphorylation is required to provide this ATP.There is as yet little information on the role of this ATP. The high bond strength of the nitrogen molecule results in an endergonic reaction for the reduction involving the first two electrons and a priori it seems most likely that at least some of the energy from the ATP hydrolysis is utilized in lowering the activation energy of the early steps in reduction.' One problem is that a variety of values for the ratio of ATP hydrolysed :pair of electrons transferred has been reported. This has been clarified" by Watt et al. who have shown that the ATP :2e ratio depends on the temperature being 7.5 at 10"C,dropping to a minimum of 4.0 at 20 "C before rising to 6.0 at 35 "C.It is difficult to see how hydrolysis of ATP can be coupled to substrate reduction and yet vary by as much as this. There is considerable evidence however that nitrogenase undergoes a conformational change close to 20 "C.Burns'' showed that there was a break in the Arrhenius plot for nitrogenase from Azotobacter at V. K. Shah L. C. Davis and W. J. Brill Biochim. Biophys. Acta 1975 384 353. (a)C.-Y. Huang J. S. Boyer and L. N. Vanderhoeff PlantPhysiol. 1975 56 222; (b) ibid. p. 228. (a)C. A. Appleby G. L. Turner and P. K. Macnicol Biochim. Biophys. Am 1975,387,461;(b)M. Ci. Yates and C. W. Jones Adv. Microbial Physiol. 1974,11,97. lo G. D. Watt W. A. Bulen A. Burns and K. L. Hadfield Biochemistry 1975,14,4266.R. C. Burns Biochim. Biophys. Acta 1969,171 253. A.D.B.Malcolm and J. R. Coggins 21 "C leading to a value for A€$# of 61 kJ mol-' above this temperature but 163kJ mol-' below it. Kulikov et a1.l' have used spin labels based on iodoacetamide (1)and maleimide (2) to label the sulphydryl groups and amino-groups of nitrogen- ase. The rotation frequency v of the labels may be calculated from the e.p.r. (2) spectra and when log v is plotted against the reciprocal of the absolute temperature a break is observed for both labels at 19 "C. It is particularly significant that both labels give the same answer since about six moles of (2) may be incorporated per mole of nitrogenase but only between 1 and 3 moles of (1). These data suggest that the conformational change at 19 "Cis not a localized one but probably involves the entire enzyme molecule.This is important in view of the finding of Thorneley et ~l.,'~ who studied the association of the Mo-Fe protein with the Fe protein and showed that there was a break in the Arrhenius plot for the components from Klebsiella pneumoniae at 17 "C. Above this temperature the enthalpy of association is zero but below it has the large value of 418 kJmol-'. The most attractive conclusion however arises from their calculation of the rate of acetylene reduction per mole of complex. When this is plotted in an Arrhenius plot a single straight line is obtained (AH= 80 kJ mol-') which suggests that the biphasic plot obtained earlier" arises entirely from the association phenomenon of the proteins.In the temperature region where this reaction makes no contribution to enthalpy measurements (above 17"C) the value of 63 kJ mol-' obtained by Burns agrees reasdnably with the 80 kJ mol-' found by Thorneley et al. However it is not clear what the effect of the findings of Shah et aL7 on the relative acetylene and nitrogen activities as a function of protein ratio may be. As with all enzymes which utilize ATP and a bivalent ion the question arises of exactly what is the substrate. Mg2' is the best cation followed by Mn" Co" Fe2' and Ni"." Since binding constants for Mg" to ATP4- are known the concentration of (MgATP)'- in a solution may be calculated. The dependence of the activity on this follows simple Michaelis-Menten kinetic^,'^ with a K of 0.4mmol 1-I suggesting that this is the substrate hydrolysed by the enzyme.It is surprising that the kinetics are so simple since at least four moles of ATP are hydrolysed for each mole of ethylene produced. High concentrations of Mg'+ are inhibitory and this appears to be the result of the formation of complexes such as (Mg2ATP).14 ATP itself is also an inhibit~r.'~ The usual Lineweaver-Burk or Woolf plots show that ATP4-is competitive with (MgATP)'- but a secondary plot of the primary slopes only produces a straight line whefi plotted against [ATP4-]' and not when plotted vs. [ATP4-]. The simplest l2 A. V. Kulikov L. A. Syrtsova G. 1. Likhtenshtein and T. N. Pisarskaya Molekul. Biol. 1975,9,162. l3 R. N. F. Thorneley R. R.Eady and M. G. Yates Biochim. Biophys. Acfa 1975,403 269. l4 R. N. F. Thorneley and K. R. Willison Biochern. J. 1974 139 211. Is R. N. F. Thorneley Biochim. Biophys. Acfa 1974 358 247. Biological Chemistry -Part (iii) Enzyme Mechanisms interpretation of these data is that ATP4- not only binds to the (MgATP)2- site but also co-operatively to a second site. In a short note Peeters et al. have found a Hill coefficient n as high as 2.9 for ATP when reduction of HCN is measured.16 An attempt to determine whether all the substrates are reduced at the same site (both topologically and electronically identical) has been made by studying the effect of one substrate on the reduction of a different one." For example C2H2 is a non-competitive inhibitor of N2 reduction whereas N inhibits C2H2 reduction competitively; GH increases the reduction of CN- but CN- is a non-competitive inhibitor of GH4production.The authors suggest that there are several different sites on the enzyme. Since substrates such as acetylene only require one electron pair for reduction the authors speculate that the enzyme needs to be more highly reduced for it to act on dinitrogen which requires three pairs of electrons. This is consistent with the failure to observe intermediates such as N2H2 (corresponding to reduction by one electron pair) in the reduction of nitrogen although other explanations are possible. Since the number of unpaired electrons on the metal ions changes during catalysis e.p.r. and Mossbauer spectroscopy have been used.The Wisconsin group" have studied the Mo-Fe protein from Azotobacter vinelandii and find that it contains 2 atoms of Mo and about 23 of Fe per molecule. The Mossbauer spectrum suggests that there are four different chemical environments for the iron atoms. Only two of these sites give rise to e.p.r. signals and each is probably occupied by four spin-coupled iron atoms. During reduction of nitrogen the e.p.r. signals disappear and it seems that each centre is reduced by only one electron. The appearance of an e.p.r. signal when the Fe protein from Azotobacterchroococ-cum is reduced with dithionite is very rapid (7;< 1ms)I9 and corresponds to the faster of the two phases observed at 425 nm in a stopped-flow spectrophotometer whereas the slow phase (7;> 100ms) observed by spectrophotometry is not accom- panied by a change in the e.p.r.spectrum. This slow phase is unlikely to be important in the enzymic mechanism since it is also observed in the oxygen-inactivated protein. The fast step is inhibited by MgADP but not affected by (MgATP)2-. At least some of the ATP hydrolysed in the overall reaction seems to be associated with electron transfer between the two-component proteins. Stopped-flow studies2' show that (MgATP)2- binds rapidly (k >2.5 X lo61 mol-' s-') to either the Fe protein or to the intact enzyme and in the latter case this is followed by a slower (k = 200 s-') redox reaction in which up to three equivalents of the Fe protein can be oxidized by one equivalent of the Mo-Fe protein.Even this latter rate is some lo2times faster than the overall turnover of the enzyme. Since each molecule of Fe protein requires two electrons for reduction it appears that the total of six electrons required for reduction of N to NH is being observed here. Figure 1summarizes some of these nitrogenase reactions. J. F. M. Peeters A. R. van Rossen and K. A. H. Heremans Arch. Infernat. Physiol. Biochim. 1975,83 200. J. M. Rivera-Ortiz and R. H. Burris J. Bacreriob 1975,123 537. l8 E. Miinck H. Rhodes W. H. Orme-Johnson L. C. Davis W. J. Brill and V. K. Shah Biochim. Biophys. Acfa,1975 400 32. l9 M. G. Yates R. N. F. Thorneley and D. G. Lowe F.E.B.S.Letters 1975,60 89. *O R. N. F. Thorneley Biochem. J. 1975 145 391. A.D.B.Malcolm and J.R. Coggins Electron donor (S204*-,Ferredoxin erc.) SLOW inhibited by ADP Substrates Products 0%'GHz,etc.) Figure1 The variety of substrates for nitrogenase is the more surprising when it is remembered that in molecules such as C2H2 and CO the highest occupied orbitals are T-orbitals and such molecules readily form T-complexes with a variety of transition metals. However in dinitrogen the highest occupied orbital is of the cr type and it has therefore been more difficult to prepare metal-N compounds as possible models for nitrogenase. Because of the CT nature of the highest energy electrons most suggestions for the binding of nitrogen to nitrogenase involve a dinuclear complex (3). Mn -k ....N=N ....Mm -+ (3) However Chatt's group21 have shown that dinitrogen can be reduced to ammonia in mononuclear complexes MeOH [M(N2)2(PR3)4] + H2S04--+ 2NH + N + ...21 J. Chatt A. J. Pearman and R. L. Richards Nature 1975,253 39; 1976,259 204. Biological Chemistry -Part (iii) Enzyme Mechanisms where M is molybdenum or tungsten {since the oxidation state of the metal changes from zero to six) and R may be alkyl or aryl. A dinuclear complex which will accept electrons from a model ferredoxin (4)and use these to reduce acetylene2' is the 0x0-bridged Mo" complex of cysteine (5). L J (4) (5) The same groupz3 have found an interesting model for the role of ATP in the reduction process. When complex (5) is used to catalyse the reduction of substrates such as C,H or CH2CHCN by NaBH, ATP stimulates reduction and is itself converted into ADP.Other acids also have an effect but not nearly such a large one. For example under standard conditions ATP produces 192 nmol H,PO produces 36 nmol and acetic acid produces 7 nmol of propane from vinyl cyanide. It seems likely that efficacy of ATP in this reaction is related to the ability of molybdenum complexes to hydrolyse ATP. For a recent comprehensive review of nitrogenase see ref. 24. 3 Glycogen Phosphorylase Glycogen phosphorylase (E.C. 2.4.1.1 .) catalyses the breakdown of glycogen to glucose- 1-phosphate (glycogen) +Pi +(glycogen),-l + a-D-glucose-1-P This reaction is important as one of the early stages in energy production. The enzyme is also important since it was the first for which a mechanism of hormone modulation was elucidated.The membrane of the muscle has a receptor for epinephrine (adrenalin) (6) which then activates adenylate cyclase (which converts (6) adenosine-5'-triphosphateinto cyclic 3'-5'-adenosine monophosphate). The CAMP then activates a protein kinase which catalyses the phosphorylation by ATP of inactive phosphoryiase kinase to produce active phosphorylase kinase. The phos- phorylase kinase utilizes more ATP in order to phosphorylate low-activity phos- phorylase b to high-activity phosphorylase a. This metabolic sequence is illustrated 22 K. Tan0 and G. N. Schrauzer J. Amer. Chem. SOC.,1975,97,5404. 23 G. N. Schrauzer G. W. Kiefer K. Tano and P. R. Robinson J. Amer. Chem. SOC.,1975,97,6088.24 (a)W. G. Zumft and L. E. Mortenson Biochim. Biophys. Acta 1975,416 1; (b)T. Ljones in 'The Biology of Nitrogen Fixation' ed. A. Quispel North Holland Amsterdam 1974 Ch. 13; (c) G. N. Schrauzer Angew. Chem. Internat. Edn. 1975,14 514. A. D.B.Malcolm and J. R. Coggins Adrenalin t Adenyl cyclase ATP CAMP n Protein kinase J A*y-ADp Phosphorylase Phosphorylase kinase kinase (inactive) (active) ADP Phosphorylase 6 Phosphorylase a (low activity) (high activity) Figure 2 in Figure 2. An inspection of this cascade shows how even a single molecule of hormone can give rise to millions of molecules of phosphorylase. Both phosphorylase b (Pb) and phosphorylase a (Pa) contain bound pyridoxal-5'- phosphate at a stoichiometry of 1mole per subunit (Pb contains two identical subunits and Pa contains four).The activation of phosphorylase 6 not only involves the phosphorylation of a unique serine in each of the twoidentical subunits but also an aggregation. Both Pa and Pb exist in a dimer-tetramer equilibrium but the tetramer is greatly favoured for Pa while the dimer predominates in the case of Pb. The three-dimensional structure of PbZ5 at 6A resolution has been determined in the presence of inosine-5'- monophosphate and confirms that the two subunits are structurally identical. The overall structure is roughly ellipsoidal but there is a cleft on one side of the molecule and it is tempting to speculate that this may be the polysaccharide-binding site. 2s L. N. Johnson N.B. Madsen J. Mosley and J. S. Wilson J. Mol. Biol. 1974,90,703. Biological Chemistry -Part (iii) Enzyme Mechanisms 385 Although the complete sequence of phosphorylase is not yet known the sequences of various peptides have been determined. The peptide containing the phosphory- lated residue is readily identifiable by using 32P and its sequence is Ser-Asp-Gln-Glu-Lys-Arg-Lys-Gln-Ile-Ser(P)-Val-Arg-Gly-Leu in rabbit muscle,26 which is surprisingly different from Leu-Thr(P)-Gly-Phe-Leu-Pro-Gln-Glu-found in ye as t . This sequence has been extended in the case of the rabbit muscle enzyme and has been shown to lie close to the N-terminus (which is in fact acetylated):28 1234 14 AcSer-Arg-Pro-Leu.. . . ... .. . Ser(P).. . . .. . .. The enzyme from dogfishz9 is identical in residues 10-18.The peptide containing PLP is easily detected since reduction of the -C=N-bond with NaBH will give a fluorescent pyridoxamine derivative while reduction with NaB3H4 will give a radioactive peptide. The rabbit3’ and sequences are Rabbit Ile-Ser-Thr-Ala-Gly-Thr-Gln-Ala-Ser-Gly-Thr-Gly-Asp-Met-Lys-Phe-Met-Gly Yeast Ile-Ser-Thr-Ala-Gly-Thr-Glu-Ala-Ser-Gly-Thr-Ser-Asn-Met-Lys-Phe-Val-Met The role of the pyridoxal phosphate moiety is a mystery the more so since a majority of the muscle cell’s content of vitamin B is bound to phosphorylase and also because phosphorylase retains full activity after reduction of the imine bond with NaBH4.31 Studies of chemical analogues of PLP3’ have shown that the 5’-phosphate group is necessary but it seems more likely to be involved in conformational changes than directly in catalysis.A study of the n.m.r. properties of this phosphorus nucleus33 has shown that it experiences two different environments in phosphorylase 6. It was encouraging that the activator AMP and the inhibitor glucose-6-phosphate altered the position of this equilibrium in opposite directions. Unfortunately on measuring the spectrum of Pa (i.e. after phosphorylation of Ser-14 on Pb) both phosphorus resonances now occur at the same chemical shift as in Pb +G6P thus preventing any correlation being made between the enzyme’s activity and the 31Pn.m.r. Further evidence for the action of PLP as an influence on the conformation of phosphorylase comes from a study of the effects of various enzymes on phosphoryl- a~e.~~ The presence of PLP enhances both the phosphorylation by phosphorylase kinase and dephosphorylation by phosphorylase phosphatase while a non-specific 26 C.Nolan W. B. Novoa E. G. Krebs and E. H. Fischer Biochemistry 1964,3,542. 27 K. Lerch and E. H. Fischer Biochemistry 1975,14 2009. 28 K. Titami P. Cohen K. A. Walsh and H. Neurath; F.E.B.S. Letzers 1975 55 120. 2y P. Cohen J. C. Saari and E. H. Fischer Biochemistry 1973,12 5233. 30 A. W. Forrey C. L. Sevilla J. C. Saari and E. H. Fischer Biochemistry 1971,10 3132. 31 E. H. Fischer A. B. Kent E. R. Snyder and E. G. Krebs J. Amer. Chem. Soc. 1958,80,2906. 32 S. Shaltiei J. L. Hedrick A. Pocker and E. H. Fischer Biochemistry 1969 8 5189.33 S. J. W. Busby D. G. Gadian G. K. Radda R. E. Richards and P. J. Seeley F.E.B.S.Letters 1975,55 14. 34 T. H. Huxley ‘Collected Essays’ Macmillan London 1894 Vol. 8 p. 244. 35 D. J. Graves G. M. Carlson J. R. Skuster R. F. Parrish T. J. Carty and G. W. Tessmer J. Biol. Chern. 1975,250,2254. 386 A. D. B.Malcolm and J. R. Coggins protein kinase and trypsin both react more slowly with the holoenzyme than with the apoenzyme. Evidence in favour of some interaction between PLP and the phos- phoserine comes from experiments in which phosphorylase a is treated with trypsin to remove the N-terminal phosphoserine-containing,peptide. The resultant enzyme phosphorylase b’ is still active but on treatment with cysteine loses PLP and hence activity three times faster than does untreated phosphorylase b.The same lab~ratory~~ has also made the remarkable discovery that if the tetradecapep- tide containing phosphoserine (from Ser-5 to Leu-18 in the sequence) is isolated and added either to phosphorylase b or to b’ it will induce in Pb the properties of Pa. As the authors state the activation of b’ by this peptide is analogous to the reactivation of subtilisin-cleaved ribonuclease with the so-called S~eptide,~’ but the activation of Pb which already contains this tetradecapeptide (albeit unphosphorylated) is both astonishing and unique. The sulphydryl groups of the enzyme are reactive and have been used both to introduce labels into the enzyme38739 and also by measuring their rate of reaction with 5,5’-dithiobis(2-nitrobenzoate),to study changes in the enzyme’s structure The conformational change induced in Pa by AMP has been studied by labelling the enzyme with N-( 1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (l).38 The e.p.r.spectrum of the label can be used to follow the extent of the conforma- tional change. The introduction of fluorescence labels39 into phosphorylase b has allowed the rate of conversion into tetrameric Pa to be measured. It seems that the conforma- tional change which accompanies activation precedes the formation of the tetramer by a minute or so. The rate of photo-oxidation of histidine residues by Rose Bengal is also altered by the presence of effectors of the enzyme’s It can be difficult to determine precisely the relative amounts of Pa and Pb in a mixture.However assays in the presence of caffeine (which inhibits Pb) should allow Pa to be determined while assays in the presence of AMP (which stimulates Pb) and sulphate (which inhibits Pa) allow the total Pa +Pb concentration to be estimated.43 Since the more highly branched an oligosaccharide the better it binds to the enzyme,44 it seems likely that the enzyme binds ends of polysaccharide chains particularly well and of course it is here that the phosphorolytic reaction occurs. 4 Kinases The three-dimensional structural homology between phosphoglycerate kinase and adenylate kinase and the pyridine-nucleotide-linked dehydrogenases was described last year.45 A detailed comparison of these structures containing the dinucleotide 36 T.J. Carby J.-I. Tu and D. J. Graves J. Biol. Chem. 1975 250 4980. F. M. Richards Proc. Nut. Acad. Sci. U.S.A. 1958,44 162. 38 J. R. Grifliths N. C. Price and G. K. Radda Biochim. Biophys. Acta 1974,358,275. 39 D. J. Brooks S. J. W. Busby and G. K. Radda EuropeanJ. Biochem. 1974,48,571. M.-H. Buc-Caron and H. C. Buc European J. Biochem. 1975,52,575. 41 T. Sanner and L. Tron Biochemistry 1975,14,230. 42 A. Kamogawa and T. Fukui Biochim. Biophys. Acta 1975,403 326. 43 W. Stalmans and H.-G. Hers European J. Biochem. 1975 54 341. 44 H.-Y. Hu and A. M. Gold Biochemistry 1975,14 2224. 45 A. D. B. Malcolm and J. R. Coggins Ann. Reports (B) 1974,71 539. Biological Chemistry -Part (iii)Enzyme Mechanisms 387 binding fold and an appraisal of the evolutionary significance of their similarity has appeared.46 It has been suggested that affinity chromatography on Cibracon Blue- Sephadex columns may be a general method for recognizing and purifying proteins containing this super-secondary structural element.47 During the past year X-ray crystallography n.m.r.and chemical modification have contributed to our understanding of the mechanism of kinases. Adenylate Kinme.-Recent work on adenylate kinase (EC 2.7.4.3) illustrates the value of comparative studies on enzymes from different biological sources and the necessity of using a number of different physical and chemical techniques in the elucidation of enzyme mechanisms. The three-dimensional structure of pig adenylate kinase has been determined.49 The active site was believed to contain both a cysteine and a histidine but because all attempts to bind substrates or substrate analogues to the crystals failed there was some uncertainty about the position of the active site since the pig enzyme contained two cysteines and two histidines.The probable position of the active site was suggested by work on the carp enzyme which contains only one cysteine and one histidine.” The cysteine- and histidine-containing tryptic peptides were isolated and their amino-acid compositions showed them to be homologous with peptides containing Cys-25 and His-36 from the pig enzyme. The presence of Cys-25 in the active site of the pig enzyme has been confirmed by chemical modification with the fluorogenic reagent 7-chloro-4-nitrobenz0-2,1,3-oxadiazole (NBD-C1).51 At pH 7.9 this reagent reacted 40 times faster with Cys-25 than with Cys-187.Complete reaction of Cys-25 led to complete inactivation of the enzyme; substrates such as MgATP2- which bind to the triphosphate site of the enzyme slowed the reaction considerably but the reactivity of Cys-187 was scarcely affected. The mu1 tisubstrate analogue P’,P’-di(adenosine- 5’)pen taphosp hate (Xp,A) decreased the rate of reaction of Cys-25 by a factor of 300. In the presence of Ap5A adenylate kinase was selectively modified at Cys-187; the introduction of the fluorescent group at this position did not effect the activity of the enzyme. A slow transfer of the NBD group from the thiols to Lys-3 1was also observed.This transfer occurred most easily between Cys-187 and Lys-31. These residues are only 48 apart in the crystalline state and this result therefore provides good evidence that the enzyme has a similar structure in dilute solution and in the crystalline state. The thiol groups of pig adenylate kinase have also been modified with a spin-label derivative of iodoacetamide although it was not possible to obtain enzyme predo- minantly labelled at only one of the cysteine residues.s1 Two superimposed spectra were observed for the modified enzyme corresponding to a strongly immobilized and a weakly immobilized spin label. A study of the effect of MnATP2- on the spectrum showed that the manganese was not more than 18 8 from the enzyme- bound nitroxide group which gave rise to the immobilized spectrum.46 W. Eventoff and M. G. Rossmann Crit. Rev. Biochem. 1975,3 111. 47 S. T. Thompson K. H. Cass and E. Stellwagen Bm. Nat. Acad. Sci. U.S.A.,1975,72,669. 48 M. Cohn in ‘Energy Transformations in Biological Systems’ Ciba Symposium 31 (new series) Associated Science Publishers Amsterdam 1975 p. 87. 49 G. E. Schulz M. Elzinga F. Marx and R. H. Schirmer Nature 1974,250 120. L. Noda G. E. Schulz and I. von Zabern European J. Biochem. 1975,51 229. 51 N. C. Price M. Cohn and R. H. Schirmer J. Biol. Chem. 1975 250 644. A. D. B.Malcolm and J.R. Coggins In the 220 MHz n.m.r. spectrum of pig adenylate kinase the C-2-proton peaks are well resolved and they have been individually assigned to His-36 and His-189 by comparison with the carp enzyme which has only Hi~-36.~~ The chemical shift of the C-2-H of His-36 in both enzymes has a normal titration curve (pK,=6.3).The C-2-H of His-189 does not titrate in the pH range 5.8-8.1. Occupation of the monophosphate site by AMP or the triphosphate site by ATP or GTP causes a downfield shift of the C-2-H of His-36 but no shift in the C-2-H of His-189. MnATP2- causes broadening of the C-2-H peak of His-36. These data together with the results obtained after chemical modification that are described ab~ve,~’ suggest that the active site is in the cleft of the molecule between the two structural domain^;^' the phosphate groups of ATP and AMP must bind close to His-36. N.m.r. evidence also suggests that there is no direct interaction of the adenine of AMP with an aromatic residue but that there may be a stacking of the adenine of ATP with T~r-153;~~ this is also consistent with the crystallographic In addition there was evidence from the n.m.r.study for the possible involvement of arginine residues in the binding of MgATP2-.52 The crystal studies show that there are several arginine residues near the cleft,52 and chemical modification with phenylglyoxal has established that a single arginine is essential for activity although its position in the sequence remains to be determined.53 Creak Kinase.-Creatine kinase (EC 2.7.3.2.) a dimeric enzyme that catalyses the phosphorylation of creatine in mammalian muscle has been extensively although crystallography and primary sequence analysis are not well advanced.It is known that creatine kinase is strongly inhibited by nitrate ions in the presence of the dead-end complex ~reatine-enzyme-MgADP-.~~.~~ The planar anion mimics the transferable phosphoryl group in a transition state in the reaction and so locks the two substrates together on the enzyme (Figure 3).”,” N.m.r. studies Me O\ /O y-’Mg. I R-N-C-NH,--P--O-P-O-AMP 4 IINH 0II (4 Me I R-N-C-NH2II+NH2 O\ /O y-..Mg. N O-P-O-AMPII II0 0 (b) Figure 3 (a) The postulated transition-state complex of the creatine kinase reaction and (b) the quaternary dead-end complex showing how this can simulate the transition state (adapted from ref. 55). (using Mn instead of Mg) have established that there is a series of conformational changes accompanying the binding of MgADP- to the enzyme the binding of 52 G.G. McDonald M. Cohn and L. Noda J. Bid. Chem. 1975,250,6947. 53 J. Berghauser Biochim. Biophys. Acta 1975,397 370. 54 D. C.Watts in ‘The Enzymes’ ed. P. D. Boyer Academic Press New York 1973,Vol. 8,p. 383. 55 E. J. Milner-White and D. C. Watts Biochem. J. 1971,122,727. 56 W.R.Clegwidden and D. C. Watts Biochim. Biophys. Acta 1975,410,99. Biological Chemistry -Part (iii)Enzyme Mechanisms 389 creatine to the enzyme-MnADP- complex and the binding of nitrate to the creatine-enzyme-MnADP-complex.48357This is a particularly good example of the use of n.m.r. spectroscopy to demonstrate that the structure of an enzyme active site changes sequentially as the various intermediate complexes and the transition state are formed.48 Similar results were obtained for adenylate kinase” and arginine kina~e.~~ Earlier studies had shown that each subunit of creatine kinase contained one reactive and probably essential (active site) thiol group which could be readily modified with alkylating reagents such as iodoa~etamide.~~’~’ It has now been shown that stoicheiometric modification of one thiol per subunit does not always lead to inactivation of the enzyme.60-62 Titration of creatine kinase with the thiol-specific chromophoric reagent 2-chloromercuri-4-nitrophenol (CNP) led to the incorpora- tion of 2 moles of reagent without loss of enzymatic activity.60761 The rate of the reaction depended on the liganded state of the enzyme; addition of creatine or MgADP- or nitrate separately decreased the rate two-to six-fold while the addition of creatine MgADP- and nitrate together (which gives the transition-state analogue complex) caused a reduction in the rate by 200-f0ld.~’ These differences in chemical reactivity suggest that creatine kinase undergoes progressive conformational changes as the complexed state of the enzyme changes; this is entirely consistent with the n.m.r.results mentioned above.48 The failure of the transition-state analogue to protect the enzyme against modification and the observation that the mercurinitrophenol-enzyme was slowly inactivated on treatment with iodoacetamide led to speculation that the thiol that had been modified by CNP was different from that modified by most other thiol reagents6’ Protein chemistry will be necessary to resolve this question.The reaction of creatine kinase with methyl methanethiolsulphonate led to the incorporation of one equivalent of MeS per subunit (measured by 14C The stoicheiometrically modified enzyme was 20% active which suggests that the reactive thiol group of creatine kinase is not essential for activity. The MeS-group is relatively non-bulky neutral and non- hydrogen-bonding which distinguishes it from most other thiol reagents and proba- bly accounts for its failure to inactivate the enzyme. However until the exact site of modification is established two questions remain open. They are (i) whether there are 1or 2 modifiable thiols per subunit in creatine kinase and (ii) whether 1thiol is essential for activity.Treatment of creatine kinase with the arginine-specific reagents butanedione or phenylglyoxal resulted in a decrease of enzymic activity which correlated with modification of a single arginine residue per The modified enzyme could no longer bind nucleotides; MgATP2- and MgADP- afforded protection against inactivation. It was concluded that an arginyl residue plays an essential role in the mechanism of creatine kinase probably as the recognition site for the negatively charged oligophosphate moiety. Arginine may function in this way throughout the 57 G. H. Reed and M. Cohn J. Biol. Chem. 1972,247,3073. 5R N. C. Price G. H. Reed and M. Cohn Biochemistry 1973,12,3322.59 D. H. Buttlaire and M. Cohn J. Biol. Chem. 1974 249 5733 5741. 6o F. A. Quiocho and J. W. Thomson Prm. Nut. Acad. Sci. U.S.A. 1973,70,2858. 61 F. A. Quiocho and J. S. Olson J. Biol. Chem. 1974 249 5885. 62 D. J. Smith and G. L. Kenyon J. Biol. Chem. 1974,249 3317. C. L. Borders and J. F. Riordan Biochemistry 1975,14,4699. 390 A.D.B.Malcolm and J. R. Coggins kinase family of enzymes since preliminary studies on hexokinase phosphofruc- tokinase phosphoglycerate kinase and pyruvate kinase indicated that each of these enzymes was rapidly inactivated by b~tanedione.~~ Two other ATP-binding enzymes glutamine synthetase and carbamoyl phosphate synthetase are also inacti- vated by arginine-directed reagents as is adenyla^ie kina~e,~~ suggesting a general role for arginine in ATP-recognition A study of the iodination of creatine kinase has established that tyrosine residues are involved in nucleotide binding.65 Arginine Kinwe.-The muscles of invertebrates contain a number of phosphoryl-ated guanidino-compounds (phosphagens) and there is a specific kinase concerned with each.66 Arginine kinase (EC 2.7.3.3.) is the best known of these enzymes; it occurs in monomeric dimeric and tetrameric forms.Most work has been carried out on the monomeric enzyme from Crustacea. Primary sequence studies are well ad~anced,~~’~~ and crystallographic work has begun.69 Most of the phosphagen kinases seem to have cysteine residues at the active site and active-site tryptic peptides have been isolated from a number of them and compared with similar peptides isolated from the closely related mammalian enzyme creatine kina~e.~’ and chemical evidence72773 There is spectral evidence71772 for the involvement of tyrosine at the active site.It is likely that the mechanism of arginine kinase is similar to that of creatine kinase; for example the dead-end complex arginine-enzyme-MgADP- in the presence of nitrate ions strongly inhibits both the monomeric lobster enzyme and the dimeric enzyme from the sea cucumber.72 N.m.r. studies also suggest that there are mechanistic similarities between arginine kinase and creatine kina~e.~’.~~ There are some interesting differences between the monomeric lobster enzyme and the dimeric enzyme from sea cucumber.The former shows barely detectable co-operativity between the sites for nucleotide and arginine whereas the latter shows significant ~o-operativity.~~ Arginine kinase and the other phosphagen kinases have one substrate-binding site per oligomer. Two exceptions to this are lombricine kinase from earthworms which is a dimer of non-identical subunits only one of which binds and the tetrameric arginine kinase from the polychaete worm Sabelfapavonina which has only two binding sites for These species differences should prove useful in further studies. 64 S. G. Powers and J. F. Riordan Roc. Nat. Acad. Sci. U.S.A. 1975,72,2616. 65 A. Fattoum R. Kassab and L. A. Pradel Biochim. Biophys. Acta 1975 405 324. 66 J. Morrison in ‘The Enzymes’ ed. P. D. Boyer Academic Press New York 1973 Vol.8 p. 457. 67 F. Regnouf R. Kassab and A. Fattoum EuropeanJ. Biochem. 1974,44,67. 68 B. Debuire K. K. Han M. Dantrevant and G. Biserte Internat.J. Peptide Protein Res. 1975,7,69. 69 J. Berthou C. RCrat B. RQat A. Gadet R. Fourme M. Renaud C. Dubord L. A. Pradel C. Roustan and N. Van Thoai J. Mol. Biol. 1975,95 331. 70 A. Brevett Y. Zeitoun and L. A. Pradel Biochim. Biophys. Acta 1975,395 1. 71 E. 0.Anoiske and D. C. Watts Biochem. J. 1975 149 387. 72 A. Fattoum R. Kassab and L. A. Pradel Biochim. Biophys. Acta 1975,405 324. 73 A. Fattoum R. Kassab and L. A. Pradel Biochemie 1975 57 859. 74 W. J. O’Sullivan E. Smith B. E. Chapman and K. M. Marsden Biochim. Biophys. Acta 1974,370,153. 75 E. H. Anoiske B. H. Moreland and D.C. Watts Biochem. J. 1975,145,535. 7h E. Terrosian L. A. Pradel R. Kassab and G. Desvages European J. Biochem. 1974,45 243. 77 Y. Robin A. Guillou and N. Van Thoai European J. Biochem. 1975 52 531. Biological Chemistry -Part (iii) Enzyme Mechanisms 391 3-Pbosphoglycerate Kinme.-There are generally considered to be two possible mechanistic pathways for enzyme-catalysed phosphoryl group The sequential mechanism involves the transfer of a phosphoryl group between sub- strates within a ternary complex while the ping-pong mechanism requires a free phosphoryl-enzyme intermediate. The kinases considered above are believed to have sequential-type mechanism^.^^ The kinetic data for yeast phosphoglycerate kinase (EC 2.7.2.3) also support a sequential mechanism,78 although two groups have reported isolating a phosphoryl enzyme with the phosphate covalently attached to a glutamic acid ~ide-chain.~~'~' This conflict between the chemical and kinetic evidence has been partly One group has reported that highly purified enzyme cannot be phosphorylated." Another group has shown that the phosphoryl enzyme is almost certainly a complex between enzyme and 1,3-biphosphoglycerate." The partial exchange reactions between ADP and ATP (also cited as evidence for the ping-pong me~hanisrn~~.~') are probably artifacts due to the presence of small amounts of cosubstrate82 or a contaminating kinase." The reported isolation of diaminobutyric acid from the phosphoryl enzyme after treat- ment with hydroxylamine and Liken rearrangement remains unexplained." A preliminary n.m.r.study using lanthanide-ATP complexes promises to allow the geometry of the active site of phosphoglycerate kinase to be dete~mined.'~ Chemical modification suggests that there may be a tyrosine at or near the active site.84 A study of the liver enzyme shows that it may contain a di~ulphide,'~ an unusual feature for an intracellular enzyme. The completion of the primary structure of phosphoglycerate kinase is eagerly awaited as this will allow the crystallographers to proceed from their backbone models46 to the calculation of a complete three- dimensional structure. Pyruvate Kinwe.-A review on the mechanism of pyruvate kinase (EC2.7.1.40) has appeared recently.86 The enzyme is ideally suited for n.m.r.and e.p.r. studies since it requires two metal ions K' and Mg2-t,R7 which can be readily substituted by Tl' Mn2' Ni2+ and a2+, and the substrate can contain 31Pand I3C nuclei.86 As a result the geometries of various enzyme-M'-M"-phosphoenolpyruvate complexes have been studied in detai1.86*88-89 Circular dichroism and kinetics have shown that in the absence of univalent cations the pyruvate kinase-Co"-phosphoenolpyruvate com-plex exists in an inactive conformati~n.~~ The function of the univalent ior is to cause a conformational change to give the active form. This is consistent with an earlier n.m.r. finding that the TI'-Mn" distance is 8 A in the absence and 5 A in the presence 'I3 J. F. Morrison and E. Heyde Ann. Rev. Biochem. 1972,41,29.79 C. T. Walsh and L. B. Spector J. Biol. Chern.,1971,246 1255. A. Brevet C. Roustan G. Desvages L. A. Pradel and N. Van Thoai European J. Biochem. 1973,39 141. M. Larsson-Raznikiewicz and B. Schierbeck Biochem. Biophys. Res. Comm. 1974.57 627. 82 P. E. Johnson S. J. Abbott G.A. Orr M. Stmiriva and J. R. Knowles Biochern. Biophys. Rcs. Cornm. 1975 62 382. P. Tanswell E. W. Westhead and R. J. P. Williams F.E.B.S. Letters 1974,48 60. 84 A. Fattoum C. Roustan L. A. Pradel and N. Van Thoai F.E.B.S. Lenen. 1975.51 18. .W K.D. Kulbe,M. Bojanovski and W. Lamprecht EuropeanJ. Biochern. 1975,52 239. R6 A. S. Mildvan Ann. Rev. Biochern. 1974,43 357. 87 F. J. Kane in 'The Enzymes' ed. P. D. Boyer Academic Press New York 1973 Vol. 8 p. 353. RR T.L. James and M. Cohn 1.Bid. Chem. 1974,249 3519. 89 E. Melamud and A. S. Mildvan. J. Biol. Chern. 1975,250,8193. 90 C. Y. Kwan. K. Erhard and R. C. Davis 1.Bid. Chem.. 1975.250. 5951. 392 A.D.B.Malcolm and J. R. Coggins of phosphoenolpyruvate (PEP).87 Difference spectroscopy has also been used to characterize the substrate binding site in pyruvate kinase and the spectral properties of the binary complexes formed between several different kinases and their sub- strates have been compared." It has been reported that pyruvate kinase catalyses the hydrolysis of PEP at the active site that is effective in catalysing the physiologically important kinase reaction. There are specific requirements for univalent and bivalent metal ions and the pH-rate profiles of the hydrolase and kinase reactions in the presence of Ni" and Co'' are similar suggesting that there are common features in the mechanism^.^^ The kinetics of pyruvate kinase which are equilibrium random-order can be satisfactorily explained by assuming that the substrates (in presence of K') are free PEP free ADP and free M2'.93 There are two main isoenzyme types of pyruvate kinase.The L-type (liver) shows allosteric activation by PEP fructose- 1,6-diphosphate (F-176-diP) and inhibition by ATP and alanine while the M-type (muscle) shows none of these regulatory proper tie^.^^*^^,^^ A comparison of the structures of the regulatory and non- regulatory forms is essential for understanding these allosteric properties. An electron-density map at 6A resolution has been calculated for the cat muscle enzyme.96 This shows that pyruvate kinase is organized into two structural domains of very different size.There is a cleft between the two domains but the subunit is not as distinctly bi-lobed as in other kinase~.~~~~~ Using the difference Fourier method the binding sites for ADP bivalent metal and PEP have been identified. The M2+ and PEP sites are very close together as first suggested by n.m.r. studies.86 Identifica- tion of the binding site for bivalent cations gives direct structural evidence for the enzyme-metal complex first proposed on the basis of e.p.r. results.86 The active site (assumed to be the M2+-PEP site) is located between the two lobes of the subunits with the substrate-binding sites on the larger domain.Other kinases have their active sites in similar position~.~~-~~ Pyruvate kinase exists in vivo as homogeneous tetramers (M or L,); these can be hybridized together in vitro to give 5 separable tetramers (M, M3L M2L2 ML3 L4).'Oo The L enzyme shows sigmoid kinetics with respect to PEP and is activated by F-1,6-diP to behave like the M enzyme; the latter shows hyperbolic kinetics that are essentially unaltered by the addition of F-1,6-diP. Kinetic characterization of the mixed hybrids provides a useful way of assessing whether there are subunit interac- tions in such a multimeric enzyme. L3M behaves rather like L while LM behaves like M,; however the kinetics are not those expected from equivalent mixtures of L4 and M,.It appears that a preponderance of L-type subunits induces the M-type subunit to spend a larger proportion of its time in the less active state which has sigmoid kinetics. L2Mz shows hyperbolic kinetics indicating that two M subunits 91 A. Brevet C. Roustan L. A. Pradel and N. Van Thoai European J. Biochem. 1975,52 345 92 K. Erhard and R. C. Davis J. Biol. Chem. 1975,250,5945. q3 S. Ainsworth and N. Madarlane Biochem. J. 1975,145 63. 94 T. J. C. van Berkel J. K. Kruijt and J. F. Koster F.E.B.S. Lerfers,1975 52 312. 95 J. M. Cardenas J. J. Strandholm and J. M. Miller Biochemistry 1975,14,4041. 9h D. K. Stammers and H. Muirhead J. Mol. Biol. 1975,95 213. q7 T. N. Bryant H. C. Watson and P. C. Wendell Nature 1974,247 14. q8 C. C. F. Blake and P. R.Evans J. Mol. Biol. 1974,84 585. 99 W. F. Anderson and T. A. Steitz J. Mol. Biol. 1975,92 279. loo D. R. Hubbard and J. M. Cardenas J. Biol. Chem. 1975 250 4931. Biological Chemistry -Part (iii) Enzyme Mechanisms predominate over two L subunits. Hybrids made from inactivated M-type enzyme (prepared by trinitrophenylation of a lysine side-chain in or near the ADP site) and normal L-type enzyme behave in the same way.lo0 Recently evidence has been presented that liver pyruvate kinase is phosphofyl- ated on incubation with [32P]ATP and cyclic 3',5'-AMP-stimulated protein kinaselm with a concomitant decrease in its activity especially at low PEP concentra-tions. This indicates that in the liver the activity of pyruvate kinase may be regulated by phosphorylation-dephosphorylation.The phosphorylated site can be readily removed from the 32P-labelled enzyme without inactivating it by incubation with subtilisin."' The phosphorylated site appears to be near one end of the polypeptide chain (cf.phosphory1asez8) and since PEP does not prevent its proteolytic removal it is probably remote from the active site. The anomeric specificity of the D-F-1,6-diP activation of yeast pyruvate kinase has been investigated by using stopped-flow kinetics and synthetic analogues of D-F-1,6-diP.'" The results suggest that the allosteric site may be non-specific with respect to anomeric configuration but that a C-2-hydroxy-group is required for activation. The conformational changes accompanying the binding of F-1,6-diP are great enough to afford the enzyme dramatic protection against proteolytic degrada- tion.lo3 In the absence of the effector pyruvate kinase is rapidly degraded; sub- strates also protect the enzyme against proteolysis.lo3 The purification and characterization of pyruvate kinase from sturgeon muscle lo4 and Neurospora crassa lo5has been reported. Hexokinase.-Yeast hexokinase B (EC 2.7.1.1) which is a dimer of identical subunits (subunit mol. wt. 52 000) contains four SH groups per subunit. Two are rapidly modified by iodoacetamide with a parallel loss of enzyme activity; the other two subsequently react to completion but at a much lower rate.lo6 The reaction of the two essential thiol groups is inhibited by glucose and less efficiently by An ADP and their Mg complexes.N-Bromoacetyl-galactosamine has been used as an affinity-labelling reagent for yeast hexokina~e.'~' The two essential thiol groups are alkylated but the non-essential groups do not react. Substrates protect against modification and inactivation. Yeast hexokinase B shows negative co-operativity with MgATP2-.'08 Under the conditionsof assay it exists mainly as a monomer,1o9 and there is only one MgATP2- site per monomer which must mean that negative co-operativity is due neither to site-site interactions nor to polymerization.'08 The regulatory properties are proba- bly due to slow substrate-induced conformational changes (hysteresis). The first crystallographic studies on yeast hexokinase B which revealed the overall tertiary and quaternary structure were reported last year.45 The positions of the binding sites for sugar and nucleotide have now been established from difference Iol G.Bergstrom P. Ekman U. Dahlqvist E. Humble and L. Engstrom F.E.B.S. Letters 1975,56,288. Io2 R. Fishbein P. A. Benkovic and S. J. Benkovic Biochemistry 1975 14 4060. C. J. Shern and J. A. Black Arch. Biochem. Biophys. 1975 166,466. lo4 R. F. Randall and P. J. Anderson Biochem. J. 1975 145 569 574. lo5 M. Kapoor Canad. J. Biochem. 1975,53 109. J. G. Jones S. Otieno E. A. Barnard and A. K. Bhargava Biochemistry 1975,14,2396. Io7 S. Otieno A. K. Bhargava E. A. Barnard and A. H. Rarnel Biochemistry 1975 S4,2403. lo8 J. P. Shill and K. E. Neet J. Biol. Chem. 1975 250 2259. J. P. Shill B. A. Peters and K. E.Neet Biochemistry 1974 13 3864. A.D.B.Malcolm and J. R. Coggins electron-density maps calculated at 7 8 res~lution.~~ Sugars (substrates and inhibitors) bind in the deep cleft that divides each subunit into two lobes and the nucleotide substrates bind nearby at one site per dimer which lies between the subunits on the molecular symmetry axis. The inhibitors 0-and p-iodobenzoylglucosamine and o-toluylglucosamine bind equally to the two subunits. In contrast glucose binds strongly to one subunit and xylose to the other. There are extensive structural changes accompanying the binding of both glucose and xylose. The substrate analogue P,y-imido-ATP shows only one strong binding site per dimer. This negative co-operativity in substrate binding almost certainly results from the heterologous or non-equivalent association of the two subunits which provides non-equivalent environments in the two chemically identical There also seems to be positive allosteric interaction between the sugar- and nucleotide-binding sites.Sugar binding is required for nucleotide binding at the inter-subunit site and the binding of nucleotide modifies the binding of sugars. These positive heterotropic interactions seem to be due to extensive substrate-induced structural changes in the enzyme. Crystallographic studies have also been carried out on another crystalline form of yeast hexokinase B which contains only one subunit per asymmetric unit.'" From a 2.7 8 electron-density map it has been possible to build a model of the polypeptide chain.This is folded into three structural domains one of which is mainly a-helical and the other two of which each contain a P-pleated sheet flanked by a-helices. AMP and glucose bind to the crystals and produce changes in the protein structure. Glucose binds in the deep cleft as it does in the dimeric form. AMP however binds in a site quite different from the one in which it is found in the crystalline dimer. Neither of the nucleotide-binding sites found in the monomeric and dimeric yeast hexokinases are similar to the nucleotide-binding domains of the dehydrogenases adenylate kinase and phosphoglycerate kina~e.~~.~~.~ lo Brain hexokinase (probably a monomer of mol. wt. 100 000) has been inactivated by 5,5'-dithiobis(2-nitrobenzoicacid) (DTNB);' l1 inactivation proceeds through prior binding to the enzyme and involves the attachment of 1mole of DTNB per mole of enzyme.At stoicheiometric levels of DTNB the inactivation is accompanied by the formation of a disulphide bond. It is not clear whether this disulphide bond or the mixed disulphide formed first causes the inactivation. Glucose protects against inactivation suggesting that the SH residues involved are in the active site. The differing effects of various substrates and inhibitors on the rate of inactivation indicate that the binding of substrates to the enzyme is interdependent and that glucose and glucose-6-phosphate (G-6-P) produce slow conformational changes in the enzyme. A detailed study of the inhibition by G-6-P of brain hexokinase has appeared,"* and a model has been put forward to account for all the kinetic and binding data.Heart hexokinase exists as an equilibrium mixture of monomer (mol. wt. 97 000) and dimer.l13 In the presence of G-6-P the dimer (inactive) form of the enzyme is favoured. The dimerization is inhibited by MgATP2- but is unaffected by glucose. It ILo R. J. Fletterick D. J. Bates and T. A. Steitz Roc. Nut. Acad. Sci. U.S.A. 1975,72 38. V. D. Redker and U. K. Kenkare Biochemistry 1975,14,4704. W. R. Ellison J. D. Lueck and H. J. Fromm J. Biol. Chem. 1975,250 1864. II3 J. S. Easterby European J. Biochem. 1975,58 231. Biological Chemistry -Part (iii) Enzyme Mechanisms is uncertain whether G-6-P causes dimerization which in turn leads to inhibition or whether it is the quaternary structure change which causes inhibition.Il3 We thank the staff of Glasgow University Library for their help and Dr R.N. F. Thorneley for his advice.

ISSN:0069-3030    

DOI:10.1039/OC9757200378

出版商:RSC    

年代:1975

数据来源: RSC

摘要:

14 Biological Chemistry Part (iv) Nucleic Acids By R. J. H. DAVIES Biochemistry Department Medical Biology Centre The Queen‘s University of Belfast Belfast BT9 7BL 1 Generalsurvey Molecular biologists have for a long time faced a problem with which authors of Annual Reports will be particularly sympathetic how can the cellular DNA of higher organisms which has a physical length of the order of metres be packaged into a nucleus whose volume approximates to m3? Chromosomal DNA is isolated from eukaryotic cells in the form of chromatin which comprises a complex of double-stranded DNA with a number of proteins. In most species the major protein components of chromatin are the five classes of histones denoted H1 H2A H2B H3 and H4. Intensive research during the past year has culminated in some very important conclusions concerning the organization of the DNA and histones in chromatin.’ Evidence drawn principally from electron microscopy,2 neutron diffrac- tioq3 endonuclease dige~tion,~ and protein cross-linking studies’ all supports the so-called ‘beads on a string’ structure for chromatin.In this model repeating globular particles with a diameter of ca. 100 A known as Y bodies or nucleosomes (the beads) are connected by short DNA filaments which are typically 100-250 8 long (the string). The nucleosomes which represent the fundamental subunits of chromatin are believed to contain ca. 200 base pairs of DNA coiled round the outside of an octameric histone unit made up from two of each of the histones H2A H2B H3 and H4.Reconstitution experiments2 suggest tha; the formation of nucleosomes from the histones and DNA is a spontaneous process and does not involve specific DNA sequences. Studies with SV40 closed circular DNA molecules indicate that the formation of each nucleosome creates one superhelical turn6 and leads to the DNA being folded to about one-seventh of its length.7 The topological constraints placed on the duplex DNA by such a structure have inspired Crick and Mug8 to propose a kinked version of the normal B form of DNA to account for its folding in chromatin. While the main features of nucleosome structure now appear S. C. R. Elgin and H. Weintraub Ann.Rev. Biochem. 1975,44 725. * P. Oudet M. Gross-Bellard and P. Chambon Cell 1975 4 281.J. P. Baldwin P. G. Boseley E. M. Bradbury and K. Ibel Nafure 1975,253 245. L. A. Burgoyne D. R. Hewish and J. Mobbs Biochem. J. 1974,143,67. 5 J. 0.Thomas and R. D. Kornberg Roc. Nut. Acad. Sci. U.S.A.,1975,72,2626. 6 J. E. Germond B. Hirt P. Oudet M. Gross-Bellard and P. Chambon Roc. Nut. Acud. Sci. U.S.A. 1975,72 1843. 7 J. D. Griffith Science 1975,187 1202. * F. H. C. Crick and A. Mug Nature 1975,255,530. 396 Biological Chemistry -Part (iu) Nucleic Acids to be established there is still some disagreement over more detailed aspects such as the exact amount of DNA contained in a nucleosome and its arrangement within it. These points may be settled by X-ray diffraction analysis if a recent report,' describing the crystallization of individual nucleosomes as cetyltrimethylammonium salts is substantiated.The nature and significance of the intervening strands connecting the nucleosomes are still in dispute. There is some evidence" to suggest that these 'string' regions may be artifacts which arise during sample preparation and that native chromatin has a compact structure consisting of closely packed nucleosomes in contact with one another. Even so the compression of DNA within chromosomes and cell nuclei is far greater than it is within individual nucleosome units. The nucleosomes must themselves be packaged together into a condensed higher order structure whose nature remains obscure; histone H1 may be implicated in this superpacking process.2 It has recently become possible to separate chromatin into transcriptionally active and transcriptionally repressed fractions.l1 There is some suggestion that the nucleosome subunits are associated only with the repressed fraction and that transcriptionally active chromatin has a different structure. The next few years should see major developments in our understanding of the organization and genetic expression of the DNA in the chromosomes of higher organisms. In favourable circumstances electron microscopy provides a most effective method for genetic mapping and the investigation of sequence homology between different DNA molecules. This is because regions of double-stranded DNA can be readily distinguished from single-stranded regions in electron micrographs. The direct mapping of genes coding for specific RNA sequences has however proved very difficult because contrast between RNA-DNA hybrids and single-stranded DNA is usually poor.Using a new method of sample preparation it is possible to detect RNAeDNA hybrid regions as short as 80 base pairs on a single-stranded DNA molecule.l2 The method has been successfully applied to determining the positions of sequences coding for ribosomal and transfer RNAs on the individual strands of a bacteriophage DNA. A region of weak sequence homology between polyoma DNA and SV40 DNA has been detected by electron microscopy using a novel approach which should be widely app1i~able.l~ Both DNAs are cleaved at a single site by the restriction endonuclease EcoRI. The action of DNA ligase on a mixture of these cleavage products yields SV40 DNA covalently joined to polyoma DNA molecules.Denaturation of these hybrid DNA species followed by annealing gives rise to some intramolecularly renatured 'snapback' molecules in which electron micro-graphs reveal that a region corresponding to 15% of the DNA is largely self- complementary. The construction of recombinant DNA molecules through the use of restriction enzymes which in the opinion of Brenner 'is going to generate the most exciting period I think in biology and is going to last at least 10years' has been the subject V. V. Bakayev A. A. Melnickov V. D. Osicka and A. J. Varshavsky,NucleicAcids Rex 1975,2,1401. lo J. T. Finch M. Noll and R. D. Kornberg Roc. Nut. Acud. Sci. U.S.A.,1975,72 3320.l1 E. M. Berkowitz and P. Doty Proc. Nut. Acud. Sci. U.S.A. 1975,72,3328. l2 M. Wu and N. Davidson Roc. Nut. Acad. Sci. U.S.A. 1975,72 4506. l3 J. Ferguson and R. W. Davis J. Mol. Biol. 1975 94 135. 398 R. J. H. Davies of growing debate. The opportunities which the technique offers for cloning individual genes and transferring them from one organism tr another may in the long term be of tremendous medical and economic importance. For example it may enable genetic disorders of metabolism to be corrected in man or allow genes for nitrogen fixation to be introduced into non-leguminous plants. On the other hand the recombinant organisms produced in such experiments may pose very serious hazards to human and animal health. At present the potential dangers associated with this kind of genetic engineering are extremely difficult to assess.Following a conference at Asilomar,14 which was attended by many leading workers in this field an advisory committee of the National Institutes of Health has now drafted detailed re~ommendations'~ concerning research in this area. In essence it recommends that the construction and propagation of recombinant DNA molecules should be carried out only with enfeebled strains of viruses or bacteria which would be incapable of survival outside an artificial laboratory environment. Experiments are classified according to their potential risk and appropriate levels of physical and biological containment are prescribed. Some experiments are banned altogether. Methods have recently been pioneered for screening bacterial populations carry- ing hybrid plasmids incorporating restriction endonuclease fragments from unfrac- tionated eukaryotic DNA for those bacteria whose plasmids contain specific DNA sequences or genes.'6717 In this way hybrid plasmids containing sea urchin histone genes or the ribosomal RNA genes of Drosophila have been cloned Despite much indirect evidence for their existence human tumour viruses have proved most elusive.Ii1 the past there have been several instances where a virus of animal origin has been mistakenly announced as a human tumour virus. The latest report^^*"^ of the isolation of C-type RNA virus particles from cultures of human leukaemic cells appear however to rest on much firmer ground.Detailed charac- terization of these oncorna viruses by immunological teLhniques strongly supports the contention that they are of human origin and excludes the possibility that they arise from contamination of the cell cultures. One of the viruses18 is infectious for a wide variety of cells and its oncogenic potential is currently being assessed. Oligonucleotide mapping procedures may prove very helpful in understanding the biological function of such viruses. The RNA of other oncorna viruses can be characterized in terms of the large oligonucleotides obtained when it is digested with TIribonuclease. Comparison of the maps of T1digests of the RNA from different strains of an avian tumour virus2' has shown that a segment of RNA close to the 3'-terminus is responsible for its transforming capacity while another segment in the middle of the genome determines its hst range.The published proceedings of three major conferences provide a mine of informa-tion and comment on the following topics the structure and conformation of nucleic 14 P. Berg D. Baltimore S. Brenner R. 0.Roblin and M. F. Singer Science 1975,188 991. '5 Nature 1975 258 561. l6 L. H. Kedes A. C. Y. Chang D. Houseman and S. N. Cohen Nature 1975,255 533. l7 M. Grunstein and D. S. Hogness Proc.Nut. Acud. Sci. U.S.A. 1975,72 3961. N. M. Teich R. A. Weiss S. Z. Salahuddin R. E. Gallagher D. H. Gillespie and R. C. Gallo Nature 1975,256,551. l9 K. Nooter A. M. Aarssen,P. Bentvelzen F. G. de Groot and F. G. van Pelt Nature 1975,256,595. 2o R.H. Joho M. A. Billeter and C. Weissman Proc. Nut. Acad. Sci. U.S.A.,1975,72 4772. Biological Chemistry -Part (iu) Nucleic Acids acids and protein-nucleic acid interactions;21 the chemistry biology and clinical uses of nucleoside analogues;22 the chemistry of nucleic acid 2 Bases Nucleosides and Nucleotides Reactions and Syntheses.-The acetone-photosensitized reaction of 5-fluorouracil with cyclopentenyl acetate gives the adduct (1)as the major On treatment with methanolic NaOH (1)is converted into the ketone (2) in 72% yield. This simple reaction sequence which should be applicable to a variety of enol acetates and 5-fluorouracil derivatives affords a useful new approach to the direct synthesis of 5-substituted uracils. A convenient synthesis of 5-(perfluoroalky1)uracils has been de~cribed.~’ The first instance of the photoaddition of an ether across the pyrimidine 5,6-double bond has been observed.26 Irradiation of 1,3-dirnethyluracil in tetrahydrofuran gives a mixture of the 5-and 6-(tetrahydrofuran-2-~1)derivatives of 1,3-dimethy1-5,6-dihydrouracilin high yield.Treatment of the nucleotides of uracil and cytosine with mercuric acetate under very mild conditions (neutral pH 37-50 “C) introduces a covalently bonded mercury atom into the 5-position of the pyrimidine base.27 These mercurinuc- leotides can be quantitatively converted into the corresponding 5-bromo- 5-iodo- or 5-tritiated compounds by the respective action of N-bromosuccinimide iodine or sodium [3H]borohydride. The mercuration and halogenation reactions can also be carried out effectively with polyribonucleotides.The unusual heterocyclic ring systems in the nucleotides (3) and (4) are produced by the mercuric chloride- catalysed reaction of cyanoacetylene with 5’-CMPand 5’-AMP, respectively.28 Both these compounds are strongly fluorescent. The pyrimidinyl-imidazole nucleotide (5) is obtained by treatment of (4) with boiling alkali. Under autoclaving conditions (120“C in a steam atmosphere) certain 3- substituted adenines including 3-benzyladenine rearrange to give small amounts of the corresponding N6-substituted adenines which are cytokinins (plant growth hormones). In an elegant study involving ‘SN-labelling in conjunction with n.m.r. 21 ‘Structure and Conformation of Nucleic Acids and Protein-Nucleic Acid Interactions’ ea.M. Sun-daralingam and S. T. Rao University Park Press Baltimore 1975. 22 ‘Chemistry Biology and Clinical Uses of Nucleoside Analogs’ ed. A. Bloch Ann. New York Acad. Sci. 1975,255 1-670. 23 ‘Proceedings of the Third Symposium on the Chemistry of Nucleic Acid Components’ compiled by A. Williamson Nucleic Acids Res. 1975 Special Publication No. 1. 24 A. Wexler R. J. Balchunis and J. S. Swenton J.C.S. Chem. Comm. 1975 601. 25 D. Cech R. Wohlfeil and G. Etzold Nucleic Acids Res. 1975 2 2183. 26 M. D. Shetlar J.C.S. Chem. Comm. 1975,653. 27 R. M. IS.Dale E. Martin D. C. Livingston and D. C. Ward Biochemistry 1975,14,2447; Nucleic Acids Res. 1975 2 915. 26 Y. Furukawa 0.Miyashita and M. Honjo Chem.and Phann. Bull. (Japan) 1975,22,2552. 400 R.J. H.Davies spectroscopy and mass spectrometry Leonard and have elucidated the mechanism of this rearrangement. Autoclaving of 3-ben~yladenine-'~N~ gives two products N6-benzyladenine with label distributed equally between N-3 and N-9 and 9-benzyladenine with label distributed equally between N-1 and N6. It is concluded that the rearrangement follows a contortional route in which there is opening and closing of both the pyrimidine and imidazole rings of the adenine nucleus and during which the benzyl group remains attached to its original nitrogen. Labelling with "N has also been used to investigate the Dimroth rearrangement whereby in the presence of base 1-methyladenosine is converted into N6-methyladen~sine.~' Ring-cleavage between N-1 and C-2 followed by 180"internal rotation is unambiguously established as the reaction pathway.An improved mefhod3l for preparing 6-seleno-substituted nucleosides and nucleotides involves direct reaction of the corresponding unprotected 6-amino-compound with H2Se in pyridine adenosine gives the 6-selenoxo-nucleoside (6)in 50% yield. Under acid 0-I I OH OH OH OH (3) (4) Ck! 0 H2N N Ribosyl II I HO-P-0 (6) OH ."3 OH OH conditions the guanine ring of cGMP can undergo homolytic alkylation and acylation reactions32 leading to g-alkyl(7) and 8-acyl(8) derivatives. The latter on 2y N. J. Leonard and T. R. Henderson J. Amer. Chem. SOC.,1975,97,4990. 30 J. D. Engel Biochem. Biophys. Res.Comm. 1975,64 581. " Chyng-Yann Shiue and Shih-Hsi Chu J. Heterocyclic Chem. 1975 12 493. 3* L. F. Christensen R. B. Meyer jun. J. P. Miller L. N. Simon and R. K. Robins Biochemistry 1975,14 1490. Biological Chemistry -Part (iv) Nucleic Acids reduction with sodium borohydride afford 8-(1-hydroxyalkyl) compounds (9). Acyl radicals are generated using a mixture of the appropriate aldehyde with ferrous sulphate and ammonium persulphate and alkyl radicals are produced by the decomposition of alkyl hydroperoxides in the presence of ferrous sulphate. No reaction occurs with CAMP but cIMP undergoes acylation. O=P-O OH I OH (7) X = Alkyl or Benzyl (8) X = Acyl or Benzoyl (9) X = 1-Hydroxyalkyl or 1-Hydroxybenzyl The potential biochemical and clinical applications not to mention financial benefits associated with nucleoside and base analogues continue to inspire their synthesis on a prolific scale.Only the briefest reference to work in this field is possible here. Recently there has been increased interest in the metabolism of guanine nucleotides especially since the broad-spectrum antiviral agent ribovirin (virazole) was found to inhibit the biosynthesis of 5’-GMP. Ring-closure of the imidazole (10)in liquid ammonia33 yields 3-deazaguanine (11). In common with its 0 nucleoside and 5’-ribonucleotide which can be prepared in analogous fashion 3-deazaguanine appears to be a very potent antiviral and antitumour agent of low toxicity. Two independent of 1-deazaguanosine have been reported.A number of 6-amino-8-azapurines have been prepared3’ by condensing 4-amino- 1,2,3-triazole-5-carbonitrilewith amidines. An interesting synthetic route to the previously unknown 6-ribosylpyrimidines has been de~eloped.~~ 33 P. D. Cook R. J. Rousseau A. M. Mian R. B. Meyer jun. P. Dea G. Ivanovics D. G. Streeter J. T. Witkowski M. G. Stout L. N. Simon R. W. Sidwell and R. K. Robins J. Amer Gem. Soc.,1975,97 2917. 34 B. L. Cline R. P. Panzica and L. B. Townsend J. Heterocyclic Chern. 1975,12,603;J. E. Schelling and C. A. Salemink Rec. Trav. chim. 1975,94 153. 35 A. Albert J.C.S. Perkin Z 1975 345. 36 S. Y.-K. Tam F. G. De las Heras R. S. Klein and J. J. Fox TetruahedronLetters 1975 3271. 402 R.J. H.Davies Excellent yields of nucleosides are generally obtained when silylated hydroxy- amino- and mercapto-pyrimidines or silylated 5-and 6-azapyrimidines react with protected 1-0-acyl or 1-0-methyl sugars in the presence of a Friedel-Crafts catalyst.37 The emulsions which are often encountered during the work-up of such reaction mixtures may be avoided3* by using either trimethylsilyl perchlorate or trimethylsilyl trifluoromethanesulphonate as the catalyst.In nitromethane solution mercuric cyanide catalyses the transfer of a purine base from one sugar to Under these conditions 2',3'-O-isopropylidene-inosineand acetobromoglucose (12) react to give a mixture of 9-peracetylglucosyl-hypoxanthine(13) and the N-7 isomer together with the 1,5-anhydro-sugar (14). The hydroxy-groups of some Me Me AcO OAc OAc nucleosides may be protected4' by formation of levulinic esters (15).This protect- ing group is removed under mild conditions by reduction with sodium borohydride which leads to intramolecular lactone formation (16) and concomitant release of free nucleoside (17).A general synthetic procedure has been developed41 for the 0 0 II II ROCCH,CH,CMe NaBH4) O<O)-Me + ROH u (15) (17) (16) preparation of 2'-O-(methoxytetrahydropyranyl)-3'-O-acyl ribonucleosides (18). These function as building blocks in oligoribonucleotide synthesis. The direct phosphorylation of nucleosides to 5'-nucleotides has been achieved42 with greater than 90% selectivity and in greater than 80% yield using phosphoryl chloride in the presence ofwater and pyridine in acetonitrile.Mononucleotides react almost quantitatively with di-n-butylphosphinothioyl bromide to give mixed anhyd- rides (19)which are quite stable even in aqueous These may be converted in high yield into the nucleoside diphosphates and triphosphates by treatment with silver acetate and inorganic phosphate or pyrophosphate in dry pyridine. Using a 37 U. Niedballa and H. Vorbriiggen J. Org. Chem. 1974,39 3654 and the following four papers. 38 H. Vorbriiggen and K. Krolikiewiu Angew. Chem. Internat. Edn. 1975,14,421. 39 F. W. Lichtenthaler and K. Kitahara Angew. Chem:Internat. Edn. 1975,14 815. 40 A. Hassner G. Strand M. Rubinstein and A. Patchornik J. Amer. Chem. Soc. 1975,97 1614. 41 C. B. Reese J. C. M.Stewart J. H. van Boom,H. P. M. de Leeuw J. Nagel and J. F. M. de Rooy J.C.S. Perkin I 1975 934. 42 T. Sowa and S. Ouchi Bull. Chem. SOC.Japan 1975,48 2084. 43 T. Hata K. Furusawa and M. Sekine J.C.S. Chem. Comm. 1975 196. 403 Biological Chemistry -Part (iv) Nucleic Acids OH R (19) R = H or OH (18) B = protected base R = Ph Me or MeOCH ribosomal preparation from E. coli it is possible to synthesize gram quantities of the 'magic spot' guanosine polyphosphates ppGpp and pp~Gpp.~~ A chemical synthesis of the corresponding deoxyribonucleotides dppGpp and dpppGpp has been described.45 Cyclic UMP.-The only 3',5'-cyclic phosphate of the four common ribonucleosides not hitherto detected in natural sources has now been isolated from rat liver extracts and microbial culture It is not yet established whether cUMP is an authentic cell constituent rather than an artifact of the isolation procedure and if so whether it is responsible for any pronounced physiological effects.The observation that cUMP like CAMP and cGMP delays the onset of growth of leukaemia cells in culture whereas cCMP initiates their proliferation could imply that in common with the cyclic purine nucleotides cUMP participates in regulation of the cell cycle. Physical Studies.-Temperature-jump relaxation measurements have been used to investigate the tautomerism between the N(7)H and N(9)H forms of adenine.47 A detailed kinetic analysis shows that the interconversion between the forms is catalysed by protons hydroxide ions and the adenine anion.At 20 "C in water the ratio of the tautomers is ca. 4 1in favour of the N(9)H form. A comprehensive study of substituent effects on the I3Cchemical shifts of the bridgehead carbon atoms (C-4 and C-5) in purines and 7-deazapurines has provided a set of parameters which can be used to estimate the tautomeric populations of purine derivative^.^^ The method is applicable to prototropic tautomerism involving N-7 and N-9 of the imidazole moiety as well as lactam-lactim and thione-thiol tautomerism in the pyrimidine portion of the purine ring. Proton spin-lattice relaxation times have been to define the conformational dynamics of NAD' in solution; the nicotinamide moiety interconverts rapidly between the syn and anti conformers with respect to the adjacent ribose ring.The conformations of NMN'' and five 3',5'-cyclic nu~leotides~' have been investigated by analysis of lanthanide-ion-induced pseudocontact shift and broadening data. Conformational studies based on 'H n.m.r. have also been 44 M. Cashel Analyt. Biochem. 1974 57 100. 45 E. Hamel E. P. Heimer and A. L. Nussbaum Biochemistry 1975,14,5055. 46 A. Bloch Biochem. Biophys. Res. Comm. 1975,64 210; J. Ishiyama ibid. 1975,65 286. 47 M. Dreyfus G. Dodin 0.Bensaude and J. E. Dubois J. Amer. Chem. SOC.,1975,97,2369. 48 M.-T. Chenon R. J. Pugmire D. M. Grant R. P. Panzica and L. B. Townsend J. Amer. Chem. Soc. 1975,97,4627,4636. 49 A. P. Zens T. J. Williams J. C. Wisowaty R. R. Fisher R. B. Dunlap T. A. Bryson and P. D. Ellis J.Amer. Chem. SOC.,1975,97 2850. 50 B. Birdsall N. J. M. Birdsall J. Feeney and J. Thornton J. Amer. Chem. Soc. 1975,97 2845. 51 M. Kainosho and K. Ajisaka J. Amer. Chem. SOC.,1975,97 6839. 404 R.J. H.Davies reported for all the common 2’-and 3‘-ribonucleotide~,~~ and the 5’-phosphates of 8-aza-adenosine and 8-azag~anosine.~~ Analysis by i.r. shows that base-pairing between uracil and adenine derivatives in chloroform solution is virtually unaffected by the presence of added water. The lack of competition by water molecules for the hydrogen-bonding sites involved in base-pairing is explained by the formation of double hydrogen-bonds by the uracil carbonyl groups. This result suggests that hydrogen-bonding interactions may make a greater energetic contribution to the stability of double-helical nucleic acids than previously supposed.Chemical ionization mass spectra have been recorded for a number of bases and nucleosides; this technique allows their basicities in the gas phase to be e~timated.~’ Of the many crystal structures of nucleic acid components and their analogues published this year the following deserve special mention. The structure of 5’-CDP is the first to be reported for a nucleoside diphosphate. The same group have also determined the structure of the nucleotide coenzyme cytidine-5’-diphosphocholine which is an important intermediate in the metabolism of phospholipid^.^^ Deoxyuridine-5’-phosphate adopts an unusual conformation5’ not previously encountered in the X-ray crystallography of nucleotides the sugar ring shows Cl’-exo puckering and the disposition of the phosphate about the C4’-C5‘ bond is trans -gauche.3 Oligonucleotides and Polynucleotides The RNA ligase isolated from E. coli infected with bacteriophage T4should prove a valuable tool in oligoribonucleotide This enzyme catalyses the forma- tion of a 3‘-5’ internucleotide bond between an oligoribonucleotide ’with a free 3’-hydroxy-group and another oligoribonucleotide bearing a 5’-phosphate. The dodecanucleotide (Ap),Cp(Up),U was synthesized almost quantitatively from the hexamers (Ap),C and p(Up),U by this method. The enzyme does not appear to show any base specificity and oligomers as short as trimers can be joined. An improved preparation of 2’(3’)-O-isovaleryl ribonucleoside 5’-diphosphates has been pub- li~hed.~~ Polynucleotide phosphorylase catalyses the addition of these blocked diphosphates to the 3‘-hydroxy-groups of oligonucleotide primers.In this way a number of tetranucleoside triphosphates incorporating 3’-terminal-modified nuc- leosides such as 1,A@-ethenoadenosine have been prepared. The insoluble polymer poly(3,5-diethylstyrene) sulphonyl chloride and the N-methylpyridinium salt of dichlorophosphoric acid have been evaluated as novel reagents for internucleotide bond synthesis.60 A computer program DINASYN has been developed61 which will 52 D. B.Davies and S. S. Danyluk Biochemistry 1975,14,543. 53 C.-H. Lee F. E. Evans andR. H. Sarma J. Biol. Chem. 1975,250 1290. 54 A. D’Albis M. P.Wickens and W. B. Gratzer Biopolymers 1975 14 1423. 55 M.S.Wilson and J. A. McCloskey J. Amer. Chem. Soc.,1975,97,3436. 56 M.A.Viswamitra T. P. Seshadri M. L. Post and 0.Kennard Nature 1975,258,497. 5’ M.A.Viswamitra T. P.Seshadri and M. L. Post Nature 1975,258,542. 58 G. C. Walker 0.C. Uhlenbeck E. Bedows and R. I. Gumport Proc.Nat. Acad. Sci. U.S.A.,1975,72 122. 59 G. C. Walker and 0.C. Uhlenbeck Biochemistry 1975,14,817. 6o M.Rubinstein and A. Patchornik Tetrahedron 1975 31 1517 2107. G. J. Powers R. L. Jones G. A. Randall M. H. Caruthers J. H. van de Sande and H. G. Khorana J. Amer. Chem. Soc.,1975,97 875. Biological Chemistry -Part (iu)Nucleic Acids 405 generate optimal synthetic routes utilizing both chemical and enzymatic steps for the preparation of single- and double-stranded polydeoxyribonucleotidesof defined sequence.Its application to the tRNAA'" gene synthesized by Khorana produced another reaction pathway estimated to require only half as much effort-a saving of about 10 man years! Six oligodeoxyribonucleotides which collectively constitute the duplex sequence of the lactose operator of E.coli have been chemically synthe- sized by improved versions of the phosphodiester and phosphotriester approaches.62 A 31-base-pair duplex DNA which contains the lactose operator sequence has been independently ~ynthesized.~~ An artificial DNA duplex consisting of 45 base pairs and designed to code for a modified S-peptide of ribonuclease A has been con- ~tructed.~~ A simple and sensitive method6' for sequencing the oligonucleotides obtained by digestion of 32P-labelled RNA with pancreatic ribonuclease has been described.Esterification of the terminal phosphate groups of polynucleotides with sorbitol allows them to be separated by chromatography on supports containing the dihyd- roxyboryl group.66 5-Mercuriuridine and 5-mercurideoxyuridine have been incorporated into enzymatically synthesized polynu~leotides.~~ The mercurated polymers are selec- tively retained on columns of sulphydryl agarose and may then be used as probes in low-temperature hydridization studies. Poly(8-bromoadenylic acid) is the first example of a polynucleotide with all its bases in the syn conformation.68 In contrast to poly(A) it assumes a very stable helical secondary structure (presumed double- stranded) at neutral pH and does not complex with poly(U).The 8-amino-group of poly(8-aminoguanylic acid) participates in hydrogen-bonding to give an unusually base-paired secondary structure at ne~trality.~' At pH 10-1 1a different hydrogen- bonding pattern not involving the 8-amino-group is observed. Poly(C) and poly(5- bromocytidylic acid) form 1:1 complexes7o with the homopolyribonucleotide derived from formycin B. The poly(A).poly(U).poly(I) triplex7' is the first triple- stranded complex to be characterized which incorporates three fundamentally different heterocyclic bases. The complex of poly(2'-O-methylcytidylic acid) with oligo(dG) is a specific template primer for viral reverse transcripta~e.~~ The kinetics and thermodynamics of the helix-coil transition of three self- complementary oligonucleotides have been analysed in great detail dATGCAT73 and rAAGCUU74 were investigated by n.m.r.spectroscopy rA7U775 by calorimetry and other spectroscopic techniques. At high concentrations and low temperatures 62 K. Itakura N. Katagiri S. A. Narang C. P. Bahl K. J. Marians and R. Wu J. Biol. Chem. 1975,250 4592. 63 M.H. Caruthers D. V. Goeddel and D. G. Yansura Fed. Roc.,1975,34 554,Abstract 1891. 64 C. L. Harvey K. Olson A. de Czekala and A. L. Nussbaum Nucleic Acids Res. 1975,2 2007. 65 G.Volckaert and W. Fiers Analyt. Biochem. 1975,62 573. 66 N.W.Y.Ho Fed. Roc. 1975,34,607 Abstract 2199. 67 R.M. K.Dale and D. C. Ward Biochemistry 1975,14,2458. 68 F. B.Howard J. Frazier and H. T. Miles J. Biol. Chem. 1974 249 2987; ibid. 1975,250 3951. 69 M. Hattori J. Frazier and H. T. Miles Biochemistry 1975,14 5033. 70 P. F. Torrence E. De Clercq J. A. Waters and B. Witkop Biochem. Biophys. Res. Comm. 1975,62 658. 71 E.De Clercq P. F. Torrence P. De Somer and B. Witkop J. Biol. Chem. 1975,250,2521. 72 G. F. Gerard P. M. Loewenstein M. Green and F. Rottman Nature 1975 256 140. 73 D. J. Patel and C. W. Hilbers Biochemistry 1975,14,2651,2656; D.J. Patel ibd p. 3984; D. J. Patel and A. E. Tonelli ibid. p. 3990. 74 P. N. Borer L. S. Kan and P. 0.P. Ts'o Biochemistry 1975,14,4847,4864. 406 R.J. H. Davies in aqueous solution complementary dideoxynucleotides associate by ba~e-pairing.~~ The stability of the minature double helices so formed depends on base sequence as well as base composition.4 Intercalation New insight into the mechanism of intercalation has been gained from physical studies of the complexes formed between dinucleoside phosphates and intercalating agents. The structure determinati~n~~ of the crystalline complex of ethidium with 5-iodouridylyl(3’-5’)adenosine is particularly important in this respect because it allows for the first time the direct visualization of intercalative binding by a drug to a fragment of a nucleic acid double helix. The basic structural unit in the crystal comprises a Watson-Crick base-paired dimer to which two ethidium ions are bound. One of these is positioned between the base pairs of the miniature double helix while the other stacks externally on top of them.Besides providing a wealth of structural information regarding intercalative interactions the structure allows the unwinding of the double helix upon intercalation to be estimated. Assuming that the results for the model system can be extended to DNA and RNA an unwinding angle of -29” is predicted for ethidium intercalation into DNA in excellent agreement with the currently accepted value. Investigations of ethidium :dinucleoside phosphate com- plexes in solution by spectroscopic method^,^' indicate a marked preference for ethidium binding to pyrimidine(3’-5’)purine sequences rather than the isomeric purine(3’-5’)pyrimidine sequences. Thus CpG interacts with ethidium to give a miniature double helix bearing an intercalated ethidium ion whose spectroscopic properties closely resemble those of nucleic acid intercalation complexes.The ethidium is instrumental in bringing about association of the two CpG molecules as they do not base-pair under these conditions in its absence. The sequence isomer GpC does not form a complex of comparable stability. Support for this sequence preference arises from work on the crystallization of the complexes ethidium complexes of pyrimidine(3’-5’)purine dinucleoside phosphates crystallize well but repeated attempts to crystallize complexes of the reverse sequences have been U~SUCC~SS~U~.~~ Stacking forces between the nucleotide bases and the planar interca- lated ring are believed to dictate the sequence specificity. The crystal structure of a complex of 9-aminoacridine with ApU has also been determined79 but is less relevant to intercalation in RNA and DNA.In this case the base-pairing between the ApU molecules is of the Hoogsteen rather than the Watson-Crick type and the complex has an open non-double-helical structure. The base pairs are stacked parallel to each other with 9-aminoacridine sandwiched in between them. High- quality X-ray diffraction patterns have been obtained of polycrystalline fibres containing the terpyridylplatinum(I1) complex cation (20)bound to DNA by interca- lation.” These strongly support the neighbour-exclusion model of intercalation in which intercalation occurs only at alternate interbase-pair sites. 75 K. J. Breslauer J. M. Sturtevant and I.Tinoco jun. J. Mol. Biol. 1975,99 549. 76 M. A. Young and T. R. Krugh Biochemistry 1975,14,4841. 77 C.-C. Tsai S. C. Jain and H. M. Sobell Proc. Nut. Acad. Sci. U.S.A. 1975,72,628. 78 T. R.Krugh and C. G. Reinhardt J. Mol. Biol. 1975 97 133;T. R. Krugh F. N. Wittlin and S. P. Cramer Biopolymers 1975 14 197. 79 N. C. Seeman R. 0.Day and A. Rich Nature 1975,253,324. 80 P.J. Bond R. Langridge K. W. Jennette and S.J. Lippard Proc.Nut. Acud. Sci. U.S.A.,1975,72,4825. Biological Chemistry -Part (iv)Nucleic Acids Dimers of acridines have been prepared" in which the aromatic rings are connected by alkylamine bridges. When the bridge exceeds a critical length both acridine moieties can intercalate into the same DNA molecule. Differential dialysis measurements8* have been used to assess the effects of heteroatoms and ring substituents on the intercalative properties of a series of proflavine and acridine orange analogues (21).Specificity for G.C base pairs increases as the absorbance maximum of the chromophore moves to longer wavelenths; ring substituents affect both the sequence and base specificity of intercalation. The interaction between nucleic acids and compounds of limited water solubility can be investigated using a newly developed solvent -parti tion method .83 1' (21) X = C or N Y = N 0,or S R=HorMe 5 tRNA Further analyses of the crystal structures of the mono~linic~~ and orthorhombic forms" of yeast tRNAPhe have revealed an extensive network of tertiary hydrogen- bonding interactions.The more detailed picture emerges from the model of the monoclinic form based on an electron-density map at 2.5 A resolution. Besides confirming the main features of the structure discussed in last year's Report,86 the new modelg4 reveals the nature of the interactions between the D loop and T$C loop which could not be interpreted unambiguously at 3 A resolution. Two invariant G residues G18 and G19 of the D loop form a tightly knit cluster of conserved bases with residues 54 to 58 (T$CGm'A) stabilized by a complex array of hydrogen bonds and stacking interactions. For example G18 is hydrogen-bonded to both the base and ribose of $55 and to ribose 58 while 0-2' of ribose 18and 0-1'of ribose 19 are hydrogen-bonded to the amino-group of G57 which is intercalated between G18 and G19.Such hydrogen bonds between the bases and the ribose-phosphate backbone are common throughout the molecule. The pyrimidine always found at position 11in tRNA is hydrogen-bonded to the 2'-OH of ribose 9 and the invariant base A21 does not form a base triplet with the reversed Hoogsteen base pair U8-Al4 but is hydrogen-bonded to riboses 8 and 48. Hydrogen-bonding between some adjacent ribose moieties also occurs. As an added bonus the stereochemistry of the J. B. Le Pecq M. Le Bret J. Barbet and B. Roques Proc. Nat. Acad. Sci. U.S.A. 1975,72 2915. 82 W. Miiller and D. M. Crothers European J. Biochem. 1975,54,267;W. Miiller H. Biinemann and N. Dattagupta ibid. p. 279. 83 M. J. Waring L. P. G. Wakelin and J. S.Lee Biochim. Biophys. Acta 1975,407 200. x4 J. E. Ladner A. Jack J. D. Robertus R. S. Brown D. Rhodes B. F. C. Clark and A. Klug Proc. Nat. Acad. Sci. U.S.A. 1975,72 4414; Nucleic Acids Res. 1975 2 1629. 85 G. J. Quigley A. H. J. Wang N. C. Seeman F. L. Suddath A. Rich J. L. Sussman and S. H. Kim Proc. Nat. Acad. Sci. U.S.A. 1975,72 4866. 86 R. J. H. Davies Ann. Reports (B) 1974,71 383. 408 R.J. H.Davies 'wobble' base pair G.U has been established. Many other interesting and important structural features are apparent besides those selected for mention here. A very similar pattern of tertiary interactions is seen in the 3 A structure of the orthorhom- bic form of the tRNA.85 The minor discrepancies which exist between the two structures may reflect differences in molecular packing as well as in interpretation.Chemical modification studiess7 are consistent with tRNAPhe having the same conformation in solution as in the crystalline forms. It also appears most likely that other tRNA species adopt a very similar tertiary structure. The n.m.r. spectra of unfractionated tRNA preparations" indicate that common tertiary structure base pairs occur in most yeast and E.coZi tRNA species. Six tertiary base-pair reso-nance~~~ identified in the 360 MHz 'H n.m.r. spectrum of E.coli tRNA""' have been correlated with tertiary base pairs in yeast tRNAPhe. The primary sites of amino-acylation of the tRNAs of E.coZi have been deter- mined using tRNA species terminated with either 2'-deoxyadenosine or 3'-deoxyadeno~ine.~~ The aminoacyl-tRNA synthetase enzymes fall into three classes with regard to their specificity those which attach the amino-acid to the 2'-hydroxyl of the 3'-terminal adenosine those which attach it to the 3'-hydroxyl and those which can amino-acylate both the 2'- and 3'-hydroxy-groups.The enzymes from yeast" have the same specificity as their E.coZi counterparts showing that it has been preserved in the evolution from prokaryotes to eukaryotes. Similar conclusions concerning the specificity of amino-acylation have been drawn by Fraser and Rich9' from experiments with tRNAs terminated by 2'-amino-2'-deoxyadenosineor 3'-amino-3'-deoxyadenosine; some of their assignments however do not agree with those of Cramer and co-worker~.~~ More than a dozen new tRNA sequences have been published this year.For the first time a tRNA tRNAp& from yeast has been sequenced utilizing post-labelling techniq~es.~~ Application of the same method has proved that the sequences of tRNAPhe from human placenta and calf liver are identi~al.'~ An automated degrada- tion pr~cedure,~~ based on the amine-catalysed elimination of the terminal nuc- leoside from periodate-oxidized RNA has been used to sequence 20 residues from the 3'-end of E.coZi tRNAG'". This established the presence of two components which differ by having either A or G at position 66. It is considered unlikely that this sequence variation would have been detected by more conventional sequencing methods. The discoveryg6 that Holley's original sequence for yeast tRNA*'" incor- porates a superfluous GC doublet will necessitate the revision of many textbooks.The highly modified nucleoside Q which is present in the first position of the x7 D. Rhodes J. Mol. Biol. 1975 94 449. HR P. H. Bolton and D. R. Kearns Nature 1975,255 347. My B. R. Reid and G. T. Robillard Nature 1975 257 287. M. Sprinzl and F. Cramer Proc. Nut. Acad. Sci. U.S.A.,1975,72 3049. 91 F. Cramer H. Faulhammer F. von der Haar M. Sprinzl and H. Sternbach F.E.B.S. Letters 1975,56 212. 92 T. H. Fraser and A. Rich Proc. Nar. Acad. Sci. U.S.A. 1975 72 3044. 93 K. Randerath L. S. Y. Chia R. C. Gupta E. Randerath E. R. Hawkins C. K. Brum and S. H. Chang Biochem. Biophys. Res. Comm. 1975,63 157. 94 B. A. Roe M. P. J. S. Anandaraj L.S. Y. Chia E. Randerath R. C. Gupta and K. Randerath Biochem. Biophys. Res. Comm. 1975,66 1097. y5 M. Uziel and A. J. Weinburger Nucleic Acids Res. 1975 2 469. 96 J. R. Penswick R. Martin and G. Dirheimer F.E.B.S. Letters 1975,50 28. Biological Chemistry -Part (iu) Nucleic Acids anticodon of certain E.coli tRNA species has been identified9' as a cyclopentenediol derivative of 7-deazaguanosine (22). 5-Carbamoyl-methyluridine (23) is the first modified nucleoside isolated from tRNA to contain a primary amide group.98 ,OH kibosyl H,*I'\N I Ribosy1 "3)!'. (22) Mnity chromatography on resin-bound tRNA may be used for the specific isolation of precursor molecules for tRNAs possessing the complementary antico- don.99 Precursor molecules for E.coli tRNA7 have been synthesized in vitro by promoter-dependent transcription of DNA fragments containing the tRNA gene.loo 6 RNA Isolation and Sequencing.-The straightforward caesium-salt density gradients so widely used in the isopycnic analysis of DNA are not suitable for work with RNA. New density-gradient media have been developed which permit the simultaneous separation and isolation of both types of nucleic acid. These are based on met- rizamide,"' sodium iothalamate,"* potassium iodide,lo3 and a mixture of caesium chloride and guanidinium chloride. lo4 A new chemical procedure for radioactively labelling the 5'-ends of RNA molecules involves reaction of the [32P]di-imidazolate of orthophosphate with the cetyltrimethylammonium salt of RNA in dimethylfor- mamide.lo5A method for the rapid separation and quantitative recovery of oligonuc- leotides containing 5'-triphosphate end-groups'% should find application in se- quence work on mRNA.T2 ribonuclease cleaves RNA molecules whose C and U residues have been modified with a methoxyamine-bisulphite mixture at purine residues only. lo7 The sequencing of tracts containing up to 100 ribonucleotides is becoming commonplace. A continuous sequence of over 1750 nucleotides from the 5'-end of bacteriophage MS2 RNA (about half the entire molecule) is now established y7 H. Kasai Z. Ohashi F. Harada S. Nishimura N. J. Oppenheimer P. F. Crain J. G. Liehr D. L. von Minden and J. A. McCloskey Biochemistry 1975,144198. 98 D. B. Dunn and M.D. M. Trigg Biochem. SOC. Trans. 1975,3,656. yy G. Vogeli H. Grosjean and D. Soil Proc. Nut. Acad. Sci. U.S.A. 1975,72,4790. loo H. Kiipper,R. Contreras,A. Landy and H. G. Khorana Proc. Nut. Acad. Sci. U.S.A.,1975,72,4754. lol D. Rickwood and B. D. Birnie F.E.B.S. Letters 1975,50 102. lo2 P.Serwer J. Mol. BioL 1975,92 433. Io3 H.Wolf Analyt. Biochem. 1975,68 505. Io4 V.Enea and N.Zinder Science 1975,190,584. loS E.Rapaport and P. C. Zamecnik Proc. Nut. Acud. Sci. U.S.A.,1975,72 314. Io6 K.Grohmann L. H. Smith and R. L.Sinsheimer Biochemistry 1975,14 1951. Io7 A.M. Mazo and L. L. Kisselev F.E.B.S. Letters 1975,59 177. 410 R.J. H. Davies together with a 3'-terminal sequence of 361 nucleotides.lo' Intricate multiple- hairpin-loop secondary structures are proposed for both regions.The 3'-teyminal7 1 nucleotides of tobacco mosaic virus RNA and an internal tract of 232 nucleotides which is encapsidated by protein have been ~equenced.''~ The nuclear U-2 RNA from Novikoff hepatoma cells is very highly modified and contains 11I,9 residues and 11methylated nucleosides in the 5'-terminal 64 nucleotides."' Apart from two I,9 residues no modified bases are found in the rest of the molecule (nucleotides 65 to 196). The 4.5s RNA from E.coZi whose function is unknown contains 107 nuc- leotides which can be arranged into a very stable secondary structure."' QB-replicase has been shown to synthesize self-replicating RNA species whose produc- tion is directed by templates generated de novo during the lag phase of the reaction."* The structure of the synthetic RNA is adapted to the reaction condi- tions.Thus RNAs which are resistant to nuclease attack or which replicate only in the presence of acridine orange may be obtained. One such RNA termed 'micro- variant RNA' is 114 nucleotides long.'13 Sequences comprising ca. 95% of the 16s ribosomal RNA cf E.coZi have now been determined1l4 and encompass a total of 1520 nucleotides. Certain oligonuc- leotide sequences which are highly conserved in the 16s RNA of 27 different prokaryote~"~ are concentrated in the 3'-half of the molecule. Of the 21 proteins present in the 3OSdbosomal subunit six can associate independently with 16s RNA. The specific binding sites of five of these proteins (S4 S7 S8 S15 and S20) have been located within the 16s RNA and they range in size from 40 to 500 nuc-leotides.' l6 mRNA.-While the occurrence of the poly(A) sequences at the 3'-termini of many mRNAs remains unexplained it has been discovered that the mRNAs from a wide variety of cellular and viral sources have a distinctive structure at their 5'-ends.The 5'-terminal sequences generally have the form m7G5'@pp5'(N,p) of2Np. . . and such mRNAs are said to be blocked or 'capped'. The 'capping' nucleoside 7- methylguanosine is joined by a (5'-5') triphosphate linkage to the 5'-terminal nucleotide of the mRNA. Two distinct types of capped mRNA are encountered type I in which the 5'-terminal nucleotide alone is 2'-O-methylated and type I1 in which the adjacent nucleotide is 2'-O-methylated as well.Both types are found in lo8 W. Fiers R. Contreras F. Duerinck G. Haegmean J. Merregaert W. Min Jou A. Raeymakers G. Volckaert M. Ysebaert J. Van de Kerckhove F. Nolf and M. Van Montagu Nature 1975,256,273;A. Vandenberghe W.Min Jou and W. Fiers Proc. Nut. Acad. Sci. U.S.A. 1975 72 2559. Io9 H. Guilley G. Jonard and L. Hirth Proc. Nut. Acad. Sci. U.S.A.,1975,72,864;H.Guilley G. Jonard K. E. Richards and L. Hirth European J. Biochem. 1975 54 145. H. Shibata,T.S. Ro-Choi R. Reddy Y. C. Choi D. Henning and H. Busch J. Biol. Chem. 1975,250 3909;T.S.Ro-Choi Y. C. Choi D. Henning J. McCloskey and H. Busch ibid. p. 3921. ll1 B. E.Griffin J. Biol. Chem. 1975 250 5426. 11* M. Sumper and R. Luce Proc. Nut. Acad. Sci. U.S.A. 1975 72 162.113 D.R. Mills F. R. Kramer C. Dobkin T. Nishihara and S. Spiegelman Roc. Nut. Acad. Sci. U.S.A. 1975,72,4252. l l4 C. Ehresmann P. Stiegler G. A. Mackie R. A. Zimmerman,J. P. Ebel and P. Fellner Nucleic Acids Res. 1975,2 265. l5 C. R. Woese G. E. Fox L. Zablen T. Uchida L. Bonen K. Pechman B. J. Lewis and D.Stahl Nature 1975,254,83. 116 R. A. Zimmerman,G. A. Mackie A. Muto R. A. Garrett E. Ungewickell C. Ehresmann P. Stiegler J. P. Ebel and P. Fellner Nucleic Acids Res. 1975,2,279;E.Ungewickell R.Garratt C. Ehresmann P. Stiegler and P. Fellner European J. Biochem. 1975 51 165. Biological Chemistry -Part (iv)Nucleic Acids 411 the polyadenylated mRNA from mouse myeloma cells117 and in duck globin mRNA."' In HeLa cells the major type I mRNA has the novel nucleoside Nd,02'-dimethyladenosinelinked to 7-methylguanosine.'19 A substantial portion of the HnRNA of mouse L cells contains type I 5'-terminal sequences but both type I and type I1 sequences are found in the mRNA.12' The capped 5'-end-group uf tobacco mosaic virus RNA m7GS'pppS'Gp. . . is unusual in that 2'-O-methylated nucleotides are absent. 12' The functional significance of capping is not fully under- stood but it is required for the translation of reovirus and vesicular stomatitis virus mRNAs in uitro.122 Enzymes have been solubilized from vaccinia virus cores whose concerted action converts the viral mRNA and even poly(A) into type I capped structures.' 23 A fragment of tryptophan operon mRNA from E.coli has been isolated which contains the junction between the trp A and trp B cistrons.Sequence analysis shows that the UGA termination signal for the B protein is out of phase with the AUG initiation codon for the A protein. Remarkably the final A of the termination triplet is the initial A of the initiation don.'^^ The mRNAs for the a-and P-globin chains may be separated electrophoretically. Nucleotide sequences in the P-globin mRNA which are not normally translated match amino-acid sequences found in two abnormal human haemoglobins which arise by frameshift mutation^.'^^ Highly purified polyadenylated mRNAs can now be rapidly isolated from eukaryotic cells by a simple and gentle one-step procedure.'26 Protein Synthesis.-The direct involvement of the 3'-terminus of 16s ribosomal RNA in the initiation of protein synthesis which is suggested by its sequence,127 has now been confirmed128 by the isolation of a complex between a 30-nucleotide mRNA fragment and a tract of 50 nucleotides from the 3'-end of 16s RNA.The complex is held together by seven base pairs between the 16s RNA fragment and a polypurine stretch common to initiator regions in E.coli and bacteriophage mRNAs. That a similar phenomenon occurs in eukaryotic systems is suggested by the isolation of a 1:l complex of mRNA and 18s ribosomal RNA from rabbit reticulocyte polyribosomes.129 Somewhat paradoxically extensive complementarity is also found between the 5'-end of 16s RNA and protein initiation sites of bacteriophage RNA. A possible role for the 5'-termirial sequence of 16SRNA in the recognition of initiation sequences has been propo~ed.'~' It has been shown that ir protein 117 S.Corry and J. M. Adams J. Mol. Biol. 1975,99 519. 11* R. P. Perry and K. Scherrer F.E.B.S. Letters 1975 57 73. 119 C.-M. Wei A. Gershowitz and B. Moss Nature 1975,257,251. lZo R.P. Perry D. E. Kelly K. H. Friderici and F. Rottman Cell 1975 4 387; ibid. 1975 6 13. lZ1 J. Keith and H. Fraenkel-Conrat F.E.B.S. Letters 1975,57,31;D. Zimmern Nucleic Acids Res. 1975 2 1189. 122 S. Muthukrishnan G. W. Both Y. Furuichi and A. J. Shatkin Nature 1975,255,33; Cell 1975,6,185; G. W. Both A. K. Bannerjee and A. J. Shatkin Proc. Nut. Acud. Sci. U.S.A. 1975,72 1189. lz3 M. J. Ensinger S. A. Martin E. Paoletti and B. Moss Proc. Nut. Acad. Sci. U.S.A.1975,72 2525. 124 T. Platt and C. Yanofsky Proc. Nut. Acud. Sci. U.S.A. 1975 72 2399. B. G. Forget C. A. Marotta S. M. Weissman and M. Cohen-Solal Proc. Nut. Acud. Sci. U.S.A. 1975 72 3614. A. Krystosek M. L. Cawthon and D. Kabat J. Biol. Chem. 1975,250 6077. lz7 J. Shine and L. Dalgarno Nature 1975,254 34. Iz8 J. A. Steitz and K. Jakes Proc. Nut. Acad. Sci. U.S.A.,1975 72 4734. 12y D. Kabat J. Biol. Chem. 1975 250 6085. I3O P. H. van Knippenburg Nucleic Acids Res. 1975 2 79. 412 R. J.H.Davies synthesis binding of fMet-tRNA to the small ribosomal subunit must occur before mRNA can be bound and phased c0rrect1y.l~~ A linked cell-free transcription- translation system has been developed'32 which can direct the synthesis of authentic viral polypeptides from SV40 DNA.7 DNA Apart from the characterization of the subunit structure of chromatin discussed earlier the main developments in DNA chemistry this year concern sequence studies and the properties of closed circular DNA. Sequence Studies.-The use of restriction endonucleases in the sequence analysis and restructuring of DNA molecules has been reviewed.'33 While many of their applications are becoming standard techniques new possibilities for their exploita- tion continue to arise. The EcoRI restriction endonuclease and methylase enzymes are capable of cleaving or methylating the duplex formed by the self-complementary octanucleotide dpTGAATTCA whose internal six base pairs constitute their com- mon recognition site. 134 Such cleavage of short double-helical regions formed by self-complementary sequences may explain'35 why some restriction endonucleases cleave the single-stranded viral DNAs from bacteriophages fl 4x174 and M13.Alteration of pH and ionic reduces the specificity of the EcoRI endonuc-lease so that it will cleave duplex regions formed by the self-complementary tetranucleotide sequence dAA?T. DNA which is protected by methylation from digestion at the normal recognition sites may now be cleaved at other sites which incorporate this simpler nucleotide sequence. The DNA from eukaryotic cells contains polypyrimidine sequences which are typically 25-200 nucleotides in length. 137 In D.melunoguster DNA where their length may exceed 1000 nucleotides the polypyrimidine tracts are localized in a cryptic satellite species from heterochromatin whose sequence is largely made up from the repeating unit d(CTCIT)n.138 A simple procedure for transferring elec- trophoretically separated DNA fragments from agarose gels to cellulose filters allows the efficient detection of specific sequences in the fragments by hybridization techniques.139 An ingenious new method for the rapid determination of DNA sequences by primed synthesis with DNA polymerase14o depends on the combined use of DNA G. Jay and R. Kaempfer J. Biol. Chem. 1975,250 5742. 132 B. E.Roberts M. Gorecki R. C. Mulligan K. J. Danna S.Rozenblatt and A. Rich Roc. Nut. Acud. Sci. U.S.A.,1975 72 1922. 133 D. Nathans and H. 0.Smith Ann. Rev. Biochem. 1975,44 273. 134 P.J. Greene M. S. Poonian A. L. Nussbaum L. Tobias D. E. Garfin H. W. Boyer and H. M. Goodman J. Mol. Biol. 1975 99 237. 135 R. W. Blakesley and R. D. Wells Nature 1975 257 421; K. Horiuchi and N. D. Zinder Roc. Nat. Acad. Sci. U.S.A.,1975 72 2555. 136 B. Polisky P. Greene D. E. Garfin B. J. McCarthy H. M. Goodman and H. W. Boyer Proc. Nar. Acad. Sci. U.S.A.,1975,72 3310. 137 H. C. Birnboim and N. A. Straus Cunad.J. Biochem. 1975,53,640;S.T. Case and R. F. Baker J. Mol. Biol. 1975,98 69. 138 H. C. Birnboim N. A. Straus and R. R. Sederoff Biochemistry 1975,14,1643;H. C. Birnboim and R. Sederoff Cell 1975,5 173; R. Sederoff L. Lowenstein and H. C. Birnboim ibid. p. 183. 13y E. M. Southern J. Mol. Biol. 1975,98 503. F. Sanger and A. R. Coulson J.Mol. Biol. 1975,94 441. Biological Chemistry -Part (iu) Nucleic Acids 413 polymerase I from E.coZi and DNA polymerase from phage T4 under conditions of different limiting nucleoside triphosphates and concurrent fractionation of the products according to size by polyacrylamide gel electrophoresis. Although at its present stage of development the results cannot be regarded as entirely reliable and may sometimes require independent confirmation in favourable circumstances it should permit the sequencing of 50 nucleotides in a few days. .The effectiveness of this method has been demonstrated by its application to the sequence analysis of a ribosome-protected tract of 5 1 nucleotides from 4x174 DNA whose primary structure had been previously established both by the ribosubstitution technique using primed DNA polymerase I repair ~ynthesis'~~ and by direct DNA sequencing methods.14* It has also been employed,'43 in conjuction with the sequence determi- nation of RNA transcripts to deduce a DNA sequence of 195 nucleotides coding for the N-terminal67 amino-acids of the gene G protein of phage 4x174.In this case the primers for the DNA polymerase were obtained from restriction endonuclease cleavage fragments . A great deal of interest has centred on the nature of promoters i.e. the sites at which RNA polymerase interacts with double-stranded DNA to initiate transcrip- tion. Such sites are included in the sequences of the entire E.coli lac control region (122nucleotide~)~~~ which and the rightward operator of phage A (79 nu~leotides)~~' have appeared this year.A shorter sequence of 33 nucleotides from the major leftward operator of phage h is also known.'46 The various approaches used in these studies provide excellent examples of the power and scope of modern sequencing techniques. When E.coli RNA polymerase binds to DNA it protects a fragment of about 40 base pairs from nuclease digestion. One such fragment from phage fd DNA,147 and two from phage T7 DNA,148 have been sequenced. These fragments are isolated as stable pre-initiation complexes with RNA polymerase and code for the first 20 or so bases of mRNA. A comparison of all known promoter-region sequences has led Pribno~'~~ to formulate a model for promoter structure and function. In essence this involves a recognition sequence presently unknown located about 35 base pairs before the mRNA initiation point and a conserved A.T-rich seven-base-pair polymerase binding site separated by five or six base pairs from the initiation point.The initiation point itself is defined by the requirement that the first nucleotide in the mRNA transcript be A or G. Closed Circular DNA.-Covalently closed circular molecules of the same DNA which differ by just a single superhelical turn can be resolved by agarose gel electrophoresis. This system affords a convenient assay for the purification of a DNA-relaxing enzyme (or nicking-closing enzyme) which removes both positive and 141 J. E. Donelson B. G. Barrell H. L. Weith H. Kossel and H. Schott European J.Biochem. 1975,58 383. 142 B. G. Barrell H. L. Weith J. E. Donelson and H. D. Robertson J. Mol. Biol. 1975 92 377. 143 G. M. Air E. H. Blackburn F. Sanger and A. R. Coulson J. Mol. Biol. 1975,96,703. 144 R. C. Dickson J. Abelson W. M. Barnes and W. S. Reznikoff Science 1975 187 27. 145 V. Pirotta Nature 1975,254,114; T. Maniatis A. Jeffrey and D. G. Kleid Roc. Nut. Acud. Sci. U.S.A. 1975,72,1184. 146 T. Maniatis M. Ptashne B. G. Barrell and J. Donelson Nature 1975,250,394;J. E. Dahlbergand F. R. Blattner Nucleic Acids Res. 1975 2 1441. 147 H. Schaller C. Gray and K. Herrman Proc. Naf. Acad. Sci. U.S.A.,1975 72 737; K. Sugimoto T. Okamoto H. Sugisaki and M. Takanami Nurure 1975,253,410. 14x D. Pribnow J. Mol. Biol. 1975,99 419. 414 R.J.H.Davies negative superhelical turns from closed circular DNA in a stepwise manner.149 Its action ultimately converts native closed circular DNAs into a series of DNA molecules differing by an integral number of superhelical turns which are distri- buted in Boltzmann fashion about a superhelical density of zero i.e.perfectly relaxed closed circles. 15* Polynucleotide ligase acting on nicked circular DNA generates an identical distribution of closed circular DNAS.'~~,'~' This happens because ther- mally induced torsional fluctuations of the DNA helix give rise to an equilibrium distribution of molecules differing slightly in their number of duplex turns. Upon ligation this configurational equilibrium is frozen and a population of covalently closed molecules is produced having different numbers of superhelical turns.Analysis of the products formed by the action of the DNA-relaxing enzyme on closed circular DNA in the presence of increasing amounts of ethidium has confirmed that intercalation by an ethidium ion unwinds the helix by 26-28'. This value is also indicated by experiments involving electron microscopic examination of supercoiled DNA molecules annealed with short fragments of DNA which are complementary to one of the By using chromatography on hydroxyapatite in the presence of ethidium bromide supercoiled DNAs may be isolated on a preparative scale without the need for an ultracentrifugation 14y W. Keller Proc. Nut. Acad. Sci. U.S.A. 1975,72 2550. lS0 D. E. Pulleyblank M. Shure D. Tang J.Vinograd and H.-P. Vosberg Proc. Nar. Acad. Sci. U.S.A. 1975,72,4280. '5' R. E. Depew and J. C. Wang Proc. Nat. Acad. Sci. U.S.A.,1975 72 4275. 152 W. Keller Proc. Nut. Acad. Sci. U.S.A. 1975,72 4876. 153 L. F. Liu and J. C. Wang Biochim. Biophys. Acta 1975,395 405. W. Pakroppa W. Goebel and W. Miiller Analyt. Biochem. 1975,67 372.

ISSN:0069-3030    

DOI:10.1039/OC9757200396

出版商:RSC    

年代:1975

数据来源: RSC

摘要:

14 Biological Chemistry Part (v) Biosynthesis By R.B. BOAR Department of Chemistry Chelsea College London SW3 6LX and D. A. WIDDOWSON Department of Chemistry Imperial College London SW7 2AY 1 Introduction This year's literature on biosynthesis has been dominated by applications of "C n.m.r. spectroscopy. Experiments using precursors in which two adjacent atoms are labelled with carbon- 13 have proved to be particularly informative. The conditions under which carbon-13 studies are most likely to be successful were defined last year,' and have since been further reviewed.2 The decline in more classical investiga- tions employing radiochemically-labelled precursors is exemplified by the area of alkaloid biosynthesis which no longer warrants a separate section.2 Terpenoids The incorporations of [14C]carbon dioxide and [14C]acetate into a representative range of monoterpenes have been ~tudied.~ Comprehensive degradations have established that not only does the half of the molecule derived from isopentenyl pyrophosphate contain the majority of the radioactivity but that labelling within this unit itself is uneven. Thus a sample of (+)-isothujone (1) isolated from l7zujaplicata after administration of [2-14C]acetate was labelled as shown. Such results are understandable if one accepts the concept of compartmentalized pools of particular intermediates. R. B. Boar and D. A. Widdowson Ann. Reports (B) 1974,71,455. T.J. Simpson Chem.Soc. Rev. 1975,4497; G.A. McInnes and J. L. C. Wright Accounts Chem.Res.1975,8,313;R.E.London V. H. Kollman and N. A. Matwiyoff J.Amer. am.Soc. 1975,97,3565. 3 D. V. Banthorpe 0.Ekundayo J. Mann and K.W. Turnbull Phytochernistry 1975,14,707. 415 R. B. Boarand D.A. Widdowson The additional carbon atoms of the homosesquiterpenoid insect juvenile hor- mones (3) and (4) are supplied by propionate units. [l-*4C]Propionate gave JH I (3) in which the radioactivity was shown to be equally divided between C-7 and C- 11.4 In an independent study a mixture of [Me-14C]methionine and [5-3H]homomevalonic acid (2) was administered to an in vitro culture from Manduca sexta.’ The derived homosesquiterpenoid JH I1 (4) and the unmodified sesquiter- pene JH 111 (5)were each labelled with carbon-14 in the methoxy.-group but only the former was labelled with tritium (at C-9).R‘ R2 (3) R’= R2 = Me (4) R’= Me; R2 = H (5) R’ = R2 = H In contrast it is well established that the additional carbon atom(s) present at C-24 of many triterpenoids originate from the S-methyl group of methionine. The exact mechanism of the alkylation varies from one system to an~ther.~” Feeding experi- ments with [Me-2H3]methionine and [2-I4C 4R -3H] mevalonic acid (MVA) sup-port the biosynthesis of the sitosterol side-chain in the higher plant Hordeurn vulgare by the pathway shown in Scheme 1. Scheme 1 Work defining the occurrence of stereospecific 173-hydride shifts in the biosyn- thesis of certain classes of sesquiterpenes has been When initial cyclization of the farnesol precursor involves bond formation bztween C- 1and C- 11 M.G. Peter and K. H. Dahm Helv. Chim. Acta 1975,58,1037. R. C. Jennings K. J. Judy and D. A. Schooley J.C.S. Chem. Comm. 1975,21. E. I. Mercer and W. B. Harries Phytochemistry 1975,14,439; E. I. Mercer and S. M. Russell ibid. pp. 445 451. J. R. Lenton L. J. Goad and T. W. Goodwin Phytochemistry 1975,14 1523. D. Arigoni Pure Appl. Chem. 1975,41 219. 9 F. Dorn P. Bernasconi and D. Arigoni Chimia (Switz.) 1975 29 24. lo J. R. Hanson and R. Nyfeler J.C.S. Chem. Comm. 1975 824. l1 A. Corbella P. Gariboldi G. Jommi and M. Sisti J.C.S. Chem. Comm. 1975 288. Biological Chemistry -Part (v) Biosynthesis there follows migration of a pro-S-hydrogen from C-1 to C-10 whereas bonding between C- 1and C-10 results in a pro -R-hydrogen migrating from C-1to C-11.8-11 The isomeric hydrocarbons (-)-longifolene (6)and (-)-sativene (7)are thus biosyn- thesized in Helminthosporium species according to Scheme 2.**' A similar rationale applies to the biosynthesis of culmorin (8)" and dendrobine (9).8*11 Scheme 2 The labelling patterns of the fungal metabolite fusicoccin (10) when biosyn- thesized from [l-13C]acetate (@) and [2-13C]acetate (A)are fully consistent with an unexceptional formation via geranylgeranyl pyrophosphate.l2 Precursors doubly labelled with carbon-13 at adjacent sites can provide rapid solutions to otherwise complicated problems. Feeding experiments using [1,2-13CJacetate and [2-14C,4R -3H]MVArequire that the biosynthesis of the diterpene l2 K.D. Barrow R. B. Jones P. W. Pemberton and L. Phillips J.C.S.Perkin 1 1975 1405. R. B.Boar and D.A. Widdowson OAc Ho -A 0H Ho\ /Q A' A ACH,OMe (10) antibiotic aphidicolin (11) proceeds according to Scheme 3.13The usual observation that it is the 4a-methyl group of 4,4-dimethyl-di- and -tri-terpenoids which is derived from C-2 of MVA is confirmed by this13 and elated'^*'^ studies. J" 1 T+> H &:T\oHk' HO** + f' OH (1 1) Scheme 3 l3 M. R. Adams and J. D. Bu'Lock J.C.S. Chem. Comm. 1975,389. l4 J. Polonsky G. Lukacs N. Cagnoli-Bellavita and P. Ceccherelli TetruhedronLetters 1975,481. S. Seo Y. Tomita and K. Tori J.C.S. Chern. Comrn. 1975 270 954. Biological Chemistry -Part (v)Biosynthesis 419 The use of [1,2-13C2]acetate as a precursor permits an unambiguous definition of the rearrangement leading from (3s)-squalene 2,3-epoxide to triterpenoids of the a-amyrin (urs-12-ene) group [for example (l2)]." The absence in the 'I3Cn.m.r.spectrum of coupling between carbon atoms 20 and 21 indicates that this bond has not survived intact from acetate and therefore rules out the possible intermediacy of the carbonium ion (13) (Scheme 4). +l CH,-CO; -+ HO H t Scheme 4 H The chirally labelled MVA (14) has been used to show that in the rearrangement leading from (3s)-squalene 2,3-epoxide to lanosterol the 1,2-shift of a methyl group from C-14 to C-13 proceeds with retention of configuration.16 l6 K. H.Clifford and G.T.Phillips J.C.S.Chem. Comm. 1975,419. R. B.Boar and D.A. Widdowson 3 Polyketides The use of acetate that is doubly labelled with 13Chas become dominant in this area of biosynthesis. In addition to the vicinal coupling which shows the incorporation of intact acetate units long-range (1,3)coupling has been observed in the product of a 1,2-alkyl shift.17 The method promises to be useful in the general area of 1,2-alkyl migrations. Terrein (15) a metabolite of Aspergillus terreus is now known to be derived from 3,4-dihydro-6,8-dihydroxy-3-methylisocoumarin(16). The proposed pathway including the novel extrusion of one carbon atom from the aromatic ring is given in Scheme 5.'* A pyrone metabolite (17) of Aspergillus melleus has been studied by using ['3C-Jacetate.Simpson suggested" a tropolone intermediate with a contraction of the side-chain as the method of generation of the unusual juxtaposition of two H02C Scheme 6 T. J. Simpson and J. S. E. Holker Tetrahedron Letters 1975,4693. 1s R. A. Will R. H. Carter and J. Staunton J.C.S. Gem. Comm. 1975,380. Biological Chemistry -Part (v) Biosynthesis 42 1 acetate methyl carbons (Scheme 6). In a subsequent collaboration with Holker” a simpler process (Scheme 7) was proposed. From the [13C2]acetate feeding they used the 1,3-coupling of 6.2 Hz between C-1 and C-7 in the metabolite to show the origin I Scheme 7 of these carbons to be as indicated. This result rules out the earlier Simpson hypothesis although a modified form would fit the later results.The necessity for formate involvement (Scheme 6) was not established. A similar juxtaposition of two acetate methyl carbons is found in the aflatoxins. [13C]Acetate feedings to Aspergillus flavus and A.parasiticus confirmed2’ the earlier results using 14C-labelled compounds and the double-labelled work21 gave the pattern shown for aflatoxin B1(18). A related metabolite sterigmatocystin (19) has previously been shown22 to be an efficient precursor of aflatoxin B1 (18) in A. parasiticus and a reassignment of the 13C spectrum of (19) has led23 to a new hypothesis for aflatoxin B1 biosynthesis (Scheme 8). The earlier hypothesis24 is no longer compatible with the newly established acetate-labelling pattern. The new scheme is based on a suggestion by Thomas2’ and encompasses the related metabo- lites averufin (20) and versicolorin A (21).The six-carbon side-chain would seem superfluous however for the aflatoxin route beyond (22) and a four-carbon analogue could be The labelling pattern in the furanoid system could be explained by a process related to the pyrone (17) pathway. l9 T. J. Simpson Tetrahedron Letters 1975 175. 2o D. P. H. Hsieh J. N. Sieber C. A. Reece D. L. Fitzell S. L. Yang J. I. Dalezios G.N. LaMar D. L. Budd and E. Motell Tetrahedron,1975,31,661. 21 P. Steyn R. Vleggaar and P. L. Wessels J.C.S. Chem. Comm. 1975 193. 22 M. T. Lin and D. P. H. Hsieh J. Amer. Chem. Soc. 1973,95 1668. 23 K. G. R. Pachler P. Steyn R. Vleggaar and P. L. Wessels J.C.S. Chem. Comm. 1975 355. 24 M.Bollaz G. Buchi and G. Milne J. Amer. Chem. SOC.,1970 92 1035. 25 R. Thomas personal communication to M. 0.Moss in ‘Phytochemical Ecology’ ed. J. B. Harborne Academic Press London 1972 p. 140. z6 (a)J. G. Heathcote M. F. Dutton an3 J. R. Hibbert Chem. andZnd. 1973,1021; (b)J. S. E. Holker and L. J. Mulheirn J.C.S. Chem. Comm. 1968 1576. R. B. Boar and D.A. Widdowson 0 -H,C-C02-J 0 0 (22) 1 0 0 0 (18) Scheme 8 The polyketide antibiotics continue to attract attention. The aglycone of the macrolide tylosin (23) is derived27 from acetate propionate and butyrate (2-ethylmalonate) as indicated. The structure of piericidin A has been revised to (24).28 The biosynthesis in Streptomyces mobaraensis had been shown to be from four acetate units and five propionate This was confirmed by the I3C-feedings.The isolation of erythromycin E (25) from Streptomyces erythreus and its forma- tion from erythromycin A (25) throws further light on the later stages of biosynthesis of this class of complex macrolide~.~~ 27 S. Omura A. Nakagawa H. Takeshima J. Miyazawa C. Kitao F. Piriou and G. Lukacs Tetrahedron Letters 1975,4503. S. Yoshida S. Shiraishi K. Fujita and N. Takahashi Tetrahedron Letters 1975 1863. 29 Y. Kimura N. Takahashi and S. Tamura Agric. and Bid. Chern. (Japan) 1969,33 1507. 30 J. R. Martin R. S. Egan A. W. Goldstein and P. Collum Tetrahedron 1975,31 1985. Biological Chemistry -Part (v)Biosynthesis mycinose I m ycaminose I mycarose (23) \ acetate; A Propionate; butyrate NMe, 1 Me H0.-12 HO..Me :e$o,J$Me . 0 Me 0 Me .VOH Me Me Me -Me 4 Shikimic Acid Metabolites Some interesting new routes to modified shikimic acid metabolites have appeared. Thus echinatin (27) a retro-chalcone is formed in Glycyrrhiza echinata by an apparent reversal of the substitution patterns of the aryl rings. Feeding experi- ment~~’ (Scheme 9) showed a 1,3-transposition of the unsaturated ketone function after formation of the ‘normal’ intermediate (28). 31 T. Saitoh S. Shibata and U. Sankawa Tetrahedron Letters 1975,4463. R. B. Boar and D.A. Widdowson (27) Scheme 9 Eucomin (29) one of a group of 3-benzylchroman-4-ones from Eucornis and Scilla spp. is formed by an oxidative cyclization of a methoxy-group onto the chalcone system.Scheme 10 shows a suggested pathway.32 The sequence of the OH (29; R = Me) Scheme 10 biosynthesis of the pterocarpan demethylhomopterocarpin (30) from an isoflavone in red clover (Trifoliurn pratense) has been determined to be as shown in Scheme 11-33.34 5 Nitrogen Metabolites The preparation from Catharanthus rmew of cell-free systems which can for example convert tryptamine and secologanin into alkaloids of the Corynanthe group with considerable efficiency promises to revolutionize the study of the 32 P. W. Dewick Phytochemistry 1975,14 983. 33 P. W. Dewick Phytochemistry 1975 14 979. 34 P. W. Dewick J.C.S. Chem. Comm. 1975,656. Biological Chemistry -Part (u) Biosynthesis J J Scheme 11 biosynthesis of indole alkaloids.35 Not only are more indicative incorporations thus obtained but the way is opened to studies using carbon-13-labelled precursors.Full details of Battersby's work on the biosynthesis of tetrahydroprotoberberine-type alkaloids have been published.36 The ambiguity in the origin of the cytochalasan D (31) has been resolved by experiments using d~ubly-'~C-labelled acetate.38 The C- 18 methyl group is derived from methionine and C-18 C-19 are derived from acetate [see (31)]. The A0 CH,-CO,-Ay$yOH H CH,-methionine (31) related macrolide cytochalasan B (phomin) (32) is formed39 by oxygen insertion into a carbocyclic precursor (33)' in a Phoma sp. (S 298) (Scheme 12). After the determination of the chiral origins of the methyl groups in penicillin G (34) attention has been turned to the nuclear hydrogen atoms.The a-hydrogens of both D-and L-valine are lost in the formation of penicillin G in a commercial high-yielding strain of Penicilliurn chrysogen~m.~~ Both isomers are equally effective precursors. D-Valine is therefore not directly incorporated and the chirality at C-3 in penicillin G is generated at a later stage. 35 A. 1. Scott and S.-L. Lee J. Amer. Chem. Soc. 1975,97 6906. 36 A. R. Battersby J. Staunton,H. R. Wiltshire B. J. Bircher and C. Fuganti J.C.S. Perkin I 1975 1162 and references there cited. 37 R. B. Boar and D. A. Widdowson Ann. Reports (B),1974,71,464. 38 J. C. Vederas W. Graf L. David and Ch.Tamm Helv.Chim. Acfu 1975,58 1886. 39 J.-L. Robert and Ch. Tamm Helv. Chim. Acfa 1975,58 2501. 40 B. W. Bycroft C. M. Wels K. Corbett A. P. Maloney and D. A. Lowe J.C.S. Chern.Cornrn. 1975,923. R.B.Boar and D.A. Widdowson -+ 0 (33) (32) Scheme 12 (34) Two groups have shown independently that the hydrogen at C-5 is derived largely (60-70%) from the 3-pro-R hydrogen of cy~teine.~',~~ The formation of the N-C-5 bond occurs therefore with overall retention of configuration. Porphyrin and corrin biogenesis continue to attract considerable interest. Akhtar has by feeding chiral a! -ketoglutarate to a haemolysed erythrocyte prepara- tion that the decarboxylation of the acetic acid side-chains in haem biosynthesis occurs with overall retention of configuration (Scheme 13).Uroporphyrinogen 111 Coproporphyrinogen 111 Scheme 13 Chemical degradation of the haem gave acetic acid from the methyl groups attached to rings c and D that had S-chirality. In an analogous series of experiments using Euglena gracilis Battersby has showna that the vinyl side-chains of protoporphyrin IX (35) are generated by an apparent antiperiplanar elimination of H' and CO from the propionic acid groups (Scheme 14). The stereospecifically labelled porphobilinogen (36) with (S,S)-41 D. J. Morecornbe and D. W. Young J.C.S. Chem. Comm. 1975 198. 42 D. J. Aberhart L. J. Lin and J. Y.-R. Chu J.C.S. Perkin f 1975 2517. 43 G. F. Barnard and M. Akhtar J.C.S. Chem. Comm. 1975,494. A. R. Battersby E. McDonald H. K. W.Wurziger and K. J. James J.C.S. Chem. Comm. 1975,493. 427 Biological Chemistry -Part (0) Biosynthesis (36) CO,H C0,H Scheme 14 chirality in the side-chain gave the monodeuteriated vinyl group as shown in (35). The (R,R)-isomer gave the converse 1,2-dideuteriovinyl function. By means of a specifically 14C-labelled uroporphyrinogen I11 (37) feeding Bat- tersby has also demonstrated4’ that the pro-S-methyl group in ring cof the cobyrinic acid derivative is derived from the acetic acid side-chain of (37) in Propionibacterium shermanii (Scheme 15). This transformation is the first unambiguous proof of the intermediacy of (37) in corrin biosynthesis. C0,H C0,Me CO,H C0,H Me0,C C0,Me (37) (38) Scheme 15 As a result of new studies on the origin of the angular methyl group in ring A of vitamin BIZ,Scott has proposed46 a new theory for corrin biogenesis.This methyl group is not derived from porphobilinogen and the meso-carbon of the uropor- phyrinogen I11 (37) precursor but from the C pool. 45 A. R. Battersby,M. Ihara E. McDonald F. Satoh and D. C. Williams J.C.S. Chem. Comm. 1975,436. 46 A. I. Scott C. A. Townsend K. Okada R. J. Cushley and P. J. Whitman J. Amer. Chem. Soc. 1974,% 8069.

ISSN:0069-3030    

DOI:10.1039/OC9757200415

出版商:RSC    

年代:1975

数据来源: RSC

摘要:

Errata Volume 71B,1974 Page 21 The title of the book in reference 35 should read “Advances in Molecular Relaxation Processes” and the publisher is Elsevier (Amsterdam). Page 44 Owing to a printing error line 8 of the main text appears as a repeat of line 4. Line 8 should read “. . .separation. The terse term ‘LC’ (liquid chromatography) is often encountered in. . .”. 428

ISSN:0069-3030    

DOI:10.1039/OC9757200428

出版商:RSC    

年代:1975

数据来源: RSC

摘要:

Aalbersberg W. G. L. 123 Aarssen A. M. 398 Abbott F. 21 Abbott S. J. 391 Abels B. N. 237 Abelson J. 413 Abenhaim D. 146 Aberhart D. J. 426 Abiko S. 183 Abraham M. H. 48 Abramova Z. A. 181 Abramovitch A. 144 222 Abramovitch R. A. 108 110 269,270,272 Acharya S. P. 236 Acher A. 366 Acheson R.M. 15 271 Acquadro M. A. 3 15,359 Adams C. D. 259 Adams D. R.,197 Adams J. M. 41 1 Adams M. R. 418 Adcock W. 216 Adeleke B. B. 98 Adinolfi M. 372 Adler V. E. 348 Adrian F. J. 100 Agami C. 372 Agapiou A. 126 Agranat I. 239 Aharon-Shalom E. 239 Ahlbrecht H. 138 Ahlgren G. 41 294 Ahrens W. 239 Ainsworth S. 392 Air G. M. 413 Ajisaka K. 403 Akabori S. 226 Akasaka K.20 Akashi K. 323 Akermark B. 41 195,294 Akhmedov V. M. 124 Akhtar M. 16,426 Akiyama S. 247 248 Aksenov V. S. 189 Akutagawa S. 141 Albanbauer J. 256 Albeck M..78 Albert A. 401 Author Index Alder R. W. 244 Alegrii A. E. 101 Alexander D. 234 Alexandre C. 342 Ali S. M. 336 Allan Z. J. 234 AIICaume M. 202 Allgeier H. 231 Allinger N. L. 41 212 294 Almlof J. 43,44 207 Alper H. 132,250,309 Alrichs R. 37 40 AI-Sader B. H.,117 188,307 Alscher A. 246 Alston P. V. 63 197 Altland H. W. 146,228 Altman J. 335 Altman L. J. 171 Altmann J. A. 48 116 Alverhne G. 250 Alzerreca A. 240 Amaro A. 113 Amaro A. H. 105 Ambles A. 375 Ames D. E. 266 Amick D. R. 263 Ammon H.L. 240,242 Anadaraj M. P. J. S. 408 Anapolle K. E. 67 Anastassiou A. G. 281 Andersen J. R. 267 Anderson A. B. 38 Anderson C. D. 279 Anderson D. J. 53 283 Anderson J. E. 198 Anderson P. J. 393 Anderson P. S. 257 Anderson R. C. 200 Anderson R. J. 183 193,311 356 Anderson W. F. 392 Anderson W. G. 209 Ando I. 52 Ando W. 116 146 Andrews G. D. 56 Andrews L. 113 Andrist A. H. 45 Androse J. D. 47 Aneja R. 372 429 Aneshansley D. 353 Anet F. A. L. 12 13 27 120 221,293,294,295 Anh N. T. 209 Annarelli D. C. 147 Anoiske E. O. 390 Ansari H. R. 321 Anthon E. 348 Anthony M.T. 124 Aoki K. 23 Aoki T. 324 Aoyama T. 107 Apeloig Y. 74 Appel R.,317 Appleby C.A. 379 Apisimon J. W. 19 Araki S. 240 Archibald J. I. C. 126 Arigoni D. 57 199 416 Armour M. A, 108 Armstrong V. W.. 326 Arn H. 348,349,355 Arnett E. M. 80 Arnold D. R. 169 Arnstein H. R. V. 378 Arnstein R. E. 378 Arsenyiadis S. 250 Arthur A. P. 348 Asao T. 244 Asche R. 7 Ashby E. C. 137 Ashfaq A. 226 Asleson G. L. 14 Asmus P.. 50 299 Ast T. 8 Atherton N. M. 103 Atkins P. W. 98 Atkins T. J. 140 Atkinson R.S. 63 110 182 189,251 Atsuta S.. 132 Atusmi K. 16 200 Au A. 65 276 Aue D. H. 60,255 Auerbach J. 61 312 Aumann R.,280,303 Avaca A. L. 163 Averbeck H. 280 Ayling G. M. 30 Ayres €3. E. 266 Ayres D. C. 272 335 Ayscough P.B. 98 Ayyar K. S. 197 Azerbaev I. N.. 181 Baardman F. 189 Baba S. 145 Babiak K. A. 176 Babin V. N. 14 Babler J. H. 362 Babsch H. 265 Bach R. D. 188 189 Baciocchi E. 223 Back T. G.,191 315 Backes J. 67 Bacon E. .7 Bacon 0.G. 348 Bacquet C. 139 Backvall J.-E. 41 195 294 Baer T. A. 193 Baes M.,238 Bagatur’yants A. A. 43 Bagnell L. 146 Bahl C. P. 405 Baier H. 149 277 278 Baigrie B. D. 236 Bailey P. M. 124 Bailey R. J. 117 Bailey R. T. 35 Bailey W. F.. 11 Baird W. C. 142 Baizer M. M. 160 Bajorek J. J. S. 61 312 Balsar J. D. 309 Bakayev V. V. 397 Baker F. C. 16 Baker R. 133 195,351 353 Baker M. W. 102 Baker R. F. 412 Bakke A. 350 Baklien S.277 Bal K. 367 Balanson. R. D. 322 Balchunis R. J. 399 Baldwin J. E. 56 65 332 Baldwin J. P. 396 Baldwin S. W. 345 Ballard D. G. H. 119 Balle T. 229 Bally T. 1 15 Baltimore D. 398 Baluzov W. M. 194 Bampfield H. A. 60 191 Bando K. 127 Bhhidai B. 370 Bank S. 237 Bannerjee A. K. 411 Bannerman C. G. F. 118 Banthorpe D. V. 415 Barager H. J. 114 Baran J. S. 182 316 Barbet J. 407 Barcelo J. 110 278 Barclay L. R. C. 88 Barcza S. 148 Bard A. J. 158 Bard R. B. 281 Bares J. E. 79 Barfield M.,13 52,215 290 Barile G. C. 177 Barili P. L. 194 Barkovich A. J. 123 Barlow M.G. 218 Barltrop J. A. 167 220 257 273 Barnard E. A. 393 Barnard G. F. 426 Barnes K.K. 153 Barnes W. M. 4 13 Barnett J. W. 223 Barnier J. P. 330 Barone G. 372 Barreiro E. 186 Barrell B. G. 413 Barron R.,8 Barrow K. D. 16,417 Bartell L. S. 40 Barthelat M. 41 Bartholmei P. 114 Bartlett A. J. 185 Bartlett R. J. 49 Bartmess J. E. 79 80 Bartnik R. 108 Bartoletti I. 127 Barton D. H. R. 191 213 228,309,315,323,325,340 366,368,371,372,376,377 Barton T. J. 116 147 Bashall A. P. 320 Basselier J. J. 362 Basu N. K. 368 Basus. V. J. 120,221 Batchelor J. G. 11 Bates D. J. 394 Bates G. S. 338 Bates R. B. 290 352 Bathurst E. T. J. 374 Battersby A. R. 17,425 426 427 Battiste D. R. 210 Battiste M. A. 300 Bauer D. P. 330 Bauer K. 119 Bauer S.H. 217 Bauld. N. L. 69 Baum J. W. 193 Baum K. 2 12 Baxter G. J. 113 258,286 Bayer E. 129 Bayne D. W. 271 Beak P.. 215 Beard C. D. 115,212 Beasley G. H. 364 Beatson R. P. 78 Beauchamp. J. L. 4 7. 8 Beavers W. A. 290 Author Index Bechgaard K. 267 Beck A. K. 319 Becker D. 176 Becker E. D. 10 Becker H. J. 145 Becker J. Y. 152 157 Becker K. B. 292 312 Beckey H. D. 3,8 Beckhaus H. 370 Beckley R. S. 301 302 Beckmann W. 181 Bedoukian R. H. 363 Bedows E. 404 Bee L. K. 187 289 Beeby J. 187 289 Beeby P. J. 281 Beetz T. 185 Beevor P. S. 348 356 Begley. M.J. 334 Begley R. F. 33 Behan J. M.,192 315 Behling J. R. 164 Beierbeck H. 19 Belcher R. S. 30 Belgaonkar V.H. 272 Bell W. J. 208 Bellamy A. J. 160 Bellamy F. 270 Bellamy L. J. 32 Bellas T. E. 349. 353 Bellucci G. 194 Benassi R. 215 Benedek-Vamos M. 274 Benedetti E. 121 Ben-Ishai D. 335 Benkovic P. A. 393 Benkovic S. J. 393 Bennett M. A. 120 Bensaude O. 403 Bensel N. 336 Benson S. W. 39 94 Bentley F. F. 33 Bentley T. W. 7 Bentvelzen P. 398 Benz J. 66 306 Berestova S. S. 14 Berg A. 18 Berg P. 398 Berger H. O. 277 Berger J. G.. 259 Berger S. 19 Berghauser J. 388 Bergman R. G. 73 85 106 217 Bergstrom G. 351 355 393 Berkowitz E. M.,397 Berlan J. 189 Berler Z. 335 Bernadon C. 146 Bernardi F. 43,45,50,64,372 Bernardi L. 340 Bernasconi P.416 Berndt A. 239. 256 Bernstein H. J.. 32 Author Index Beroza M. 347,348,350,355 Berridge J. C.,168 220 Berrier C. 375 Bershas J. P. 312 Berson J. A. 54,61 197. 303 Bertelo C. A. 133 Berthod H. 49 Berthou J. 390 Bertran J.. 44 Bertrand M.,60 191 Bertrand R.,11 Bestmann H.J. 356,358,359 Betkouski M. F. 297 Bevan. P. 133 Beyer R. L. 33 Beynon J. H. 3,5 8 Bhanu S. 182 316 Bhargava A. K. 393 Bhatnagar S. P. 197 Bianchi M. 121 Bianchi R. 246 Bichan D. J. 61 Bickelhaupt F. 142 149 217 218,278,300,304 Bicker R. 298 Bidan G. 362 Bieber L. 252 Bied-Charreton C. 14 Biehl E. R.,107 Bieniek D. 220 Bienvenue-Goetz E. 194 Bieri G. 51 Bierl B.A. 348 350 Biggs I. D. 235 Bigley W. S. 35 1 Bilkis I. I. 225 Biller S. A. 196 Billeter M. A. 398 Bindra J. S. 346 Bindra R.,346 Binger P. 124 143 220 243 Bingham. E. M.. 189 Bingharn R. C. 38,216 Binkley E. S. 341 Binkley J. S. 40 Biollaz M. 368 Birch A. J. 16 268 Bircher B. J. 425 Birdsall B. 13,403 Birdsall N. J.M. 13 403 Birmingham L. 34 Birnbaum D. 176 Birnbaum E. R. 21 Birnboirn H. C. 412 Birnie B. D. 409 Birnstock F. 37 Birshtein T. M. 41 Bischof P. K. 44 Biserte G. 390 Bister K. 19 Black D. St. C. 53,284 Black J. A. 393 Blackburn E. H. 413 Blackbum T. F.. 133 317 Blackburne I. D. 284 Blackham A. U. 153 Blackman G. L. 228 Blacksberg I. R.,290 Blackwell J.35 Blake C. C. F. 392 Blake M. R. 125 Blakesley R.W. 412 Blanco A. 226 Blankenhoern G. 378 Blaser H. U. 125 Blatchly J. M. 229 Blattner F. R.,41 3 Bleha T. 39 Blinn R. C. 30 Bloch A. 403 Block A. McB. 101 102 Block F. 30 Blomberg C. 142 Bloodworth A. J. 143 Blount J. F. 202 366 Blum J. 291 Blurn M. S. 350 35 1,353 Blum Z. 157 Blume E. 261 Blume G. 237 Blumenthal T. 8 Boar R. B. 376,415.425 Bobbitt J. M. 158 Boche G. 66,306 Bocian D. F. 294 Bock M. G. 320 Boden R. M.,310 Bodewitz H. W. H. J. 142 Boeckman R. K. 332 Boekelheide V. 170,230,23 1 247,282 Bonnemann H.,+123 Bottcher A. E. 186 Bogachev Yu. S. 14 Bogdanowicz M. J. 343 Boggiz L.M. 41 Bogs R. A. 302 Bohlmann F. 7 Boireau G.. 146 Bojanovski M.,391 Boldt P. 114 237 Boll P. M. 200 Bollaz M.,421 Bolonsky J. 16 Bolton P. H.,408 Bomse D. S. 198 315 Bond F. T. 315 Bond P. J. 406 Bonds W. D. jun. 129 Bonen L. 410 Bonet G. 253 Boniewin W. 47 Bonjouklian R. 276 338 Boop J. L. 301 Booth H. 12 Booth R. J. 212 Boppre M. 349,355 Borch R. F.,61 43 1 Borchers F. 5 Borden J. H. 350 Borden W.T. 42 50 53 55 240 Borders C. L. 389 Bordwell F. G. 79 80 Borer P.N. 405 Boriack C. J. 349 355 Borisvo E. V. 43 Bornatsch W. 155,237 Borovsky J. 113 171 172 Bose A. K. 13 320 372 Boseley P. G. 396 Bosyakov Yu. G. 181 Both G.W. 41 1 Bott K. 194 Botteghi C. 121 Boublik M. 22 Boue S. 170 Bouis P. A. 203 Bourson J. 260 Boutigue M.-H.b 372 Boutin N. E. 372 Bovill M. J. 294 Bowen D. V. 8 Bowers. L. 245 Bowie J. H. 8 Bowler D. J. 293 Boxler D. 334 Boyer H. W. 412 Boyer J. S. 379 Boyle P. F. 130 Bracher G. 133 Bracke J. W. 355 Bradbury E. M. 22.396 Bradbury J. H. 22 Bradshaw J. S. 283 Bradshaw J. W. S. 351 Brady L. E. 6 Brady R.F. jun. 31 Braekman J. C. 353 Braitsch D. M. 134 227 Brand J. M. 353 355 Brandsma L. 186 Brauchaud B.. 345 Braurnan J. I. 56 Braun A. 254 Breheret E. 173 Bremser W. 246 Brenan J. 236 Brennan M. M. 348 Brenner G. S. 206 Brenner S. 290 398 Brenner.W. 185 Breslauer K. J. 406 Breslow R.,81 106 171 174 241,291,371 Breuninger M. 219 Breunlich G. 33 Breuss B. 43 Brevet A. 390 391 392 Brew K. 378 Bridges A. J. 203 Brill W. J. 379 381 Briscese S. M. J. 225 Britten-Kelly M. R.,191 315 Broadhurst M. D. 364 Brodie T. D. 235 Brodrick A. 257 Brook P. R.,60 191 Brookliart M. 66 306 Brooks C. J. W. 16 Brooks D. J. 386 Brouwer A. C. 21 1 Brown C. A. 184,210 Brown C. W. 30 Brown D. 87 Brown H. C. 136 143 145 182,183,195,267,320,346 Brown J. M. 18.66 246 281 Brown R.D. 228 Brown R. F.C. 113 258 286 Brown R.S. 407 Brown. W. V.. 353 Browne L. E. 355 Brownlee R.G. 349 Broxton T. J. 226 Broyde S.B. 25 Brubaker C. H. jun. 129 Brucker G. E. 79 Brum C. K. 408 Brummel R. N. 189 Brunelle D. J.. 341 Brunsvold W. R. 131 Bruza K. J. 332 Bryant T. N. 392 Bryce-Smith D.. 168,220 Bryson T. A. 20 337,403 Buc H. C. 386 Buc-Caron M. -H. 386 Buchan R. 229 Buchanan B. G. 9 Buchanan G. W. 12 Buchardt O. 275 Buck H. M. 41,93 Buck V. 246 Budd D. L. 17,421 Buchi G. 289 345,421 Bunemann H. 407 Burstinghaus R. 138 332 Bulachinski A. B. 262 Bulen W. A. 379 Bull J. R. 369 Bu'Lock J. D. 418 Buncel E. 225 235 Bungaard T. 23 Bunton C. A. 85 226 234 Burdett K. A. 67 172 Burger K. 254 256 262 Burger U. 58 Burgi H. B. 27 Burgoyne L. A. 396 Burgstahler A. W. 208 360 Burfitt I.13 52 Burk P. L. 125 Burke S. D. 138 Burke S. S. 250 Burkholder W. E. 349 Burns A. 379 Burns R. C. 379 Burns R. H. 378 Burri P. 106 Burris R.H. 381 Bursey M. M. 3,44 Bursey J. T. 3 Burton D. J. 118 285 Busby S. J. W. 385 386 Busch H. 410 Busch M. A. 126 Buser H. R.,348 355 Busetta B. 202 Bush R.D. 146 Buswell R. 301 Bushweller C. H. 209 Busman S. C. 234 Butin K. P. 143 Butler A. R.,226 Butler R. N. 284 Buttlaire D. H.. 389 Bycroft B. W. 204 206 335 425 Byrd L. R.,152 Byrne J. W. 125 Byrne K. J. 348 350 355 Bystrov V. F. 13 Zacace F. 223 Zadicot P. 185 Zadogan J. I. G. 107 108 110 118,222,236,262 Zael J. J. 35 Zagniant D. 283 Zagniant P.283 Zagnoli-Bellavita,N. 16,418 "alas R. 148 Zalderon J. 354 Zalb V. 208 253 Caluwe P. 269 Xzaferri G. 47 Zambon A. R.,372 Campbell A. C. 375 Campbell B. S. 266 Campbell 1. D. 19 23 rampbell R.0.169 Cantacuzene J. 12 Cantrell T. S. 168 220 Caple R.,75 183 Capman M. L. 189 Capozzi G. 253 Carbo R.,41 Carby T. J. 386 Carde R.,348 Carde R.N. 110 Carde R. T. 348 Cardenas J. M. 392 Carder R. 167 273 Cardin D. J. 125 Carey F. A. 11 3 Cargill R.L. 363 Carlsen P. H. J. 58 114 178 249 Author Index Carlson B. A. 272 Carlson D. A. 350 Carlson G. L. 41 Carlson G. M. 385 Carlson R.G. 61 292 Carlson R.M. 138,332 336 Carmody M. A. 315 359 Carmody M.J. 67,307 Carnahan E. 265 Carpenter B. K. 54 303 Carr D. D. 125 CarriC R. 268 Carroll F. L. 16 Carroll S. E. 110 Carroll T. X.,199 Carruthers R. J. 246 Carsky P. 52 Carter B. J. 225 Carter R.E. 216 Carter R. H. 420 Carty T. J. 385 Caruthers M. H. 404,405 Cary P. D. 22 Casalone G. 246 Case R.S. 241 Case S. T. 412 Casey C. P. 131 Casey M. L. 16 Cashel M. 403 Caspi E. 200 Cass K. H. 387 Cassar L. 181 Cassatt W. A. 33 Castagnino E. 370 Castiello F. A 235 Catalan J. 43 Caubtre P. 107 128 258 Cault D. 18 Cava M. P. 263,267,283 Cavill G. W. K. 351 Cawkill E. 259 Cawthon M. L. 41 1 Ceccherelli P. 16,418 Cech D. 399 Cedheim L. 151 157 Cella J.A. 323 Cerfontain H. 224 Cessac J. 69 Cessna A. J. 15 234 Chaabouni R.,141,250 Chachaty C. 12 Chaimovich H. 226 Challand S. R.,110 Challis B. C. 205 244 Chalvet O. 225 Chambers R.D. 167,273 Chambon P. 396 Chan J. A. 200 Chan K. C. 117 Chan S. H. 30 Chan S. I. 22 Chan T. C. 148 Chan T. H. 267,287,360 Ehandrasekaran E. S. 129 Author Index Chandrasekhar B. P. 376 Chandy M. J. 73 Chang A. C. Y. 398 Chang C. C. 107 Chang E. 360 Chang H. M. 51 171 Chang S. H. 408 Chang S. M.,193 Chanot J. J. 128 Chao L. C. 336 Chapman A. J. 128 Chapman B. E. 390 Chapman 0.L. 12 107,293 347 Chaput G. 201 Chatt J. 131 273 382 Chauhan M. S. 13 Chavdarian C.G. 332 Chawla. 0.P. 102 Chayet L. 226 Chen C.-N. 22 Chen G.-S. J. 272 Chen M.C. 35,36 Chen S. L. 209 Chihevert R. 205 Cheney J. 233 Cheng A. K. 295 Cheng J. -D. 234 Cheng-fan J. 250 Chenier J. H. B. 90 212 Chenon M. T. 14,403 Cherton J. -C. 275 Cheunat T. 268 Cheung A. S. 51 Chevli D. M.,372 Chia L. S. Y. 408 Chiong K. N. 158 Chiou B. L. 144 Chisholm M. D. 348 Chishti N. H. 326 Chiu K. -W., 144 Chodkiewicz W. 189 Choe F. W. 231 Choi Y.C. 410 Chong A. O. 209 Chong B. 353 Chong H. L. 11 7 Choo K. Y. 98 Chou S. 174 Chou T. -C. 292 Choy E. M.,201 Christensen,B. W. 212 Christensen,H. 139 Christensen,J. J. 283 Christensen K. A. 11 Christensen L.F. 400 Christiansen,P. A. 47 Christie M.,65 Christl M.,55 117 Christoffersen R. E. 40 51 245 Christy M. E. 257 Chu C. Y. 341 Chu J. Y.-R.,426 Chum K. 13 Chung C. 196 Chung S. -K., 301 Chyng -Yam Shiue 400 Cinquini M.,344 Claesson A. 187 Clardy J. 244 Clark B. A. J. 259 Clark B. F. C. 407 Clark F. R. S. 134 227 Clark G. 131 227 Clark H. C. 132 Clark N. G. 259 Clark R. A. 307 Clarke J. A. 266 Clarke J. K. A. 185 Clarke R. 321 Clarke,T. C.,73,106,241,291 Claus P. 250 Clauss G. 244 Clegwidden W. R. 388 Clem T. R. 10 Clementi S. 49 223 Clifford K. H. 419 Clifft B. E. 98 Cline B. L. 401 Clish A. M. 277 Closs G. L. 64,233 Clubnall A.C. 354 Clunie I. J. G. 16 Coates R. M. 327 Cochran D. W. 11 Cocivera M.,23 Cockerill A. F. 79 Coffett J. A. 355 Coffey P. 37 Coggins J. R. 386 Cohen A. H. 64,233 Cohen E. 175 Cohen J. F. 362 Cohen P. 385 Cohen S. N. 398 Cohen T. 143 228 Cohen Z. 319 Cohen-Solal M.,41 1 Cohn M.,387,388,389,391 Cole R. A. 348 Coleman M. M.,31 Coles B. F. 181 Colle K. S. 217 Collignon N. 140 Collins J. F. 320 Collrnan J. P. 120 Collum P. 422 Colbn M.,102 Colussi A. J. 93 Combertiati J. R. 30 Combrisson S. 12 Combs L. L. 38 Comi R. 320 Comisarow M. B. 30 Commercon A. 317,360 Comnick R. W. 297 Conecpcibn R. 101 102 Conger J. L. 235 Conia J. M. 53 292 330 Conlin R.T. 114 Connert J. 354 Conrad W. E. 334 Conred M.E. 41 Constantinides,D. 343 Contreras R. 409,410 Cook J. 236 Cook K. A. 378 Cook M. J. 223 Cook P.D. 401 Cook R. B. 32 Cook R. J. 7 Cooke M. 120 Cooke M.P. 131,322 Cooks R. G. 3,5,8 Cookson R. C. 133,197 Coombs M. M. 369 Cooney K.E. 367 Cooper J. W. 88 Cooper N. J. 126 Cooper R. 352 Coppens P.,24 Corbally R. P. 167 273 Corbella A. 416 Corbett K. 425 Corbin V. L. 183 193,357 Corcoran R. J.; 174 371 Corderman R. R.,4 Corey E. J. 61 134,136 138 148 193,201,269,290,309 319,320,322,323,326,328 329,331,339,345,372 Corey J. Y. 148 279 Cornelisse J. 220 Cornforth F. J. 79 Cornforth J. W. 200 Corosine M.41 Corrie J. E. T. 202 Corry S. 41 1 Costa L. M. 226 Cotterrell G. 183 Couch D. A. 23 Coulson A. R. 412 413 Coulson D. R. 227 Courot P. 316 Cousse H. 293 Coutrot P. 336 Couturier R.,209 Covey W. D. 225 Cowherd F. G. 345 Cowles C. R. 209 Cox B. G. 82 Cox G. R. 183 Cox M. T. 61,312 Cox R. A. 235 Coxon J. M.,374 CrabbC P. 186 Crabtree R. H. 120 Craig J. C. 115 Craig J. T. 238 Crain P. F. 409 Cram D. J. 204,231,282,323 Cramer F. 21 408 Cramer R. 227 Cramer S. P.,406 Crampton M. R. 226 Crandall J. K. 251 Crane-Robinson C. 22 Crasnier F. 4 1,293 Crass G. 320 Cravador A. 353 Crawley L. C. 25 1 Crerner D. 246,293 Cresp T. M. 247 Crewe R.M.,355 Crick F. H. C. 396 Criegee R. 191 Cristol S. J. 172 Croisy A. 170 Crornbie L. 189 Crossland I. 141 Crothers D. M. 407 Crow W. D. 116,269 Crowe H. R. 98 Crowley K. J. 6 Crozier R. F. 53 284 Crumrine D. S. 11 8 Csizmadia I. G. 44,45,48,116 Cullen D. L. 25 Cullen W. R. 128 Culvenor C. C. J. 349 Curphey T. J. 161 Current S. 61 197 Curtin D. Y.,28 Curtis W. D. 204 282 Cushley R.J. 11 427 Cussler E. L. 201 Cuthbert R. A. 350 Cvetkovic D. 50 Cyr N. 13 Czczodrowska B. 47 Czuba W. 271 Dabbagh A. M.,218 Dahlberg J. E. 413 Dahlqvist U. 393 Dahm K. H..416 Daigle D. J. 277 D’Albis A. 404 Dale J. A. 371 Dale R. M.K. 399,405 Dalezios J. I.17,421 Dalgarno L. 41 1 Dalling D. K. 11 Dallos J. L. 10 Daloze D. 353 Dalton D. R. 75 194 Dalton. J. C. 175 Damasevitz C. A. 124 Damico J. N. 8 Damji S. W. H. 23 Damon R. E. 200 D’Amore M.B. 56 Dana G. 165 Dang T. -P.,121 d’Angelo J. 138 330 Daniels. C. K. 322 Danna K.J. 412 Dantrevant M.,390 Danyluk S. S. 404 Darnall D. W. 21 Das K. G. 8 Dassanayake N. L. 223 Daterman G. E. 208,347,360 Dattagupta N. 407 Dauben W. G. 49 53 292 312,321,344,364 Davalian D. 221 289 Daves G. D. 208 David L. 425 Davidson A. H. 191,311,346 Davidson N.,397 Davies A. P.,372 Davies B. 146,224 Davies D. B. 404 Davies G. D. 360 Davies R. J. H. 407 Davis D. G. 14 Davis J.115 Davis J. H. 2 17 Davis L. C. 379 381 Davis L. P. 2 10 Davis R. C. 391,392 Davis R. W. 397 Davis V. C. 53 284 Dawes C. C. 325,377 Dawson D. A. 10 Day A. C. 50 57 167 220 257,273 Day M. J. 340 368 Day R. O. 27 125 172,406 Day,-V.W. 125 172 Dea P.,401 Dean D. 251 Dean F. M.,340 De Bernardo S. 204 De Boer C. D. 175 de Boer T. 110 Debuire B. 390 De Buyck L. 250 De Buys Scott 16 de Castiglione R. 340 Decker J. A. jun. 30 De Clercq E.,405 Declerq J. P. 25 Decoret C. 225 Decorzant R. 341 de Czekala A. 405 De Fay N. 61,239 De Fontaine D. L. 18 Degen I. A. 29 34 Degner D. 165 de Graaf S. A. G. 14 De Graff B. A. 110 259 de Groot F. G. 398 DeHaan F.P. 225 de Haas N.. 97 Dehm D. 176,264,276 Dehmlow E. V. 15 Author Index Dehmlow S. S. 15 de Jong F. 282 De Jongh D. C. 7,263 De Kimpe N. 250 De las Heras F. G. 401 Delbaere L. T. J. 240 293 Del Bene J. E. 41,42 de Leeuw H. P. M. 402 Delhaye M.,32 Dell’Erba C. 226 De Lorenzo R.A. 94 Dc Luca H. F. 367 De Lucchi O. 253 de Mayo P. 174 de Meijere A. 50 299 Demoulin A. 249 Denney D. B. 266 Denney D. Z. 266 Dennis N. 62 270 Dennis R. W. 92 Den Tonkelaar W. A. M.,30 Depew R. E. 414 Depezay J. C. 138,330 De Puy C. H. 285 Derocque J.-L.,6 de Rooy J. F. M. 402 Derrick P. J. 5 7 Desbene P.-L. 275 Deshayes H. 3 10 Deshpande R.R. 168,270 Desimoni G. 283 Deslauriers R.,18 Deslongchamps P.49 205 De Somer P.,405 Desvages G. 390,391 De Titta G. T. 25 Devadoss M.,108 Devaquet A. 68 de Villardi G. C. 198 Dewar M.J. S. 38,42,44,46 48 53 55 56 64 105,216 218,240,241,283,295,305 Dewick P. W. 424 de Wit G. 15 de Wolf W. H. 217,218,300 304 Dexheimer E. M.,147 Deyrup J. A. 297 D’Hinterland L. D. 293 Dial J. L. 225 Diamantis A. A. 131 273 Di Blasi R. 13 Dichman K. S. 26 Dickinson R. A. 197 229 Dickson R. C. 413 Dieck H. A. 181 Dill J. D. 45 227 Dillon J. 209 377 Dilworth J. R. 131 Dirnroth K. 88 222 277 Dingwall J. 261 Dinizo S. E. 324 Dinkel R. 67 Di Nunno L. 358 Author Index Dirheimer G. 408 Dirke G.W. 364 Dixon D. A. 75 Dixon H. B. F. 378 Djerassi C. 9 Doali J. O. 31 Dobbs A. J. 98 Dobkin C. 410 Dobson A. 123 Dobson C. M. 23 Doddrell D. 13 52 Dodin G. 403 Doering W. von E. 54,304 Doerjer G. 181 Dotz K. H. 236 Dolbier W. R. 67 117 188 305,307 Dolenko A. J. 235 Doll R. J. 345 Dollat J. M. 186 Dolling U. H. 64 233 Dollish F. R. 33 Donaubauer A. 277 Donelson J. 413 Donelson J. E. 413 Donndelinger P. 322 Doolittle R. E. 348 Dopper J. H. 218 291 Dorlars A. 284 Dorn F. 416 Dorn H. C. 10 Dorofeenko G. N. 275 Doss S. H. 31 Dotsevi G. 204 Doty P. 397 Dougherty R. C. 53 Douglas K. L. 240 Douglas K. T. 78 Douglas W. M. 129 Doupeux H. 161 Douse C.S. 31 Dowd P. 199 Doyle M. J. 124 125 Doyle M. P. 147 234 Dradi E. 10 Dreux M. 316 Drewes S. E. 3 Dreyfus,M. 403 Driessler F. 37 Dromey R. G. 9 Drouin J. 292 Drucker G. E. 80 Drummond P. C. 348 Dubois J. E. 80,126,194,403 Dubois R.A. 86,320 Dubord C. 390 Duck M. W. 11 Duerinck F. 410 Durr H. 221 Duffaut,N. 148 Duffield,R.M.,351 Duke B. J. 37 40 Duke C. B.. 51 Dumont W. 252,321 Dunbar B. I. 340 Dunbar R. C. 6 Duncan W. G. 229 Dunitz J. D. 27 Dunkerton L. V. 106 Dunlap R. B. 20,403 Dunn D. B. 409 Dunne K.,203 Dunning T. H. jun. 40 Dunogues J. 148 Duret C. 23 Durst H. D. 228 340 Durst T. 12 256 Duschek Ch. 194 Dutasta J. P.,13 Dutta N.C. 47 Dutton M. F. 421 Du Vernet R. B. 247 Dvortdk P.,202 D'yakonov I. A. 189 Dyall L. K. 109 Dykstra C. E. 108 Dymerski P.P. 4 Eadon G. 7 Eady C. R. 119 Eady R. R. 378,380 Eakin R. T. 15 Earls D. W. 79 Easterby J. S. 394 Eastwood F. W. 113 Ebel J. P.,410 Eberson L. 151 157 Echegoyen L. 101 Echols R. E. 18 Eck C. R.,200 Eckert-MaksiC,M.,39 Edgar J. A. 349 Edlund U. 19 20 21 Edmonds J. W.,25 Egan R. S. 201,422 Eger K. 257 Eggar K. W. 194 Eggerding D. 178 230 Eggers S. H. 350 Eggersdorfer M. 256 Eggiman W.. 227 Eguchi S. 190 260 Eguchi Y.,366,367 Ehresmann C. 410 Eilers J. E. 37.40 Eisch J. J. 64 124 145 146 279 Eisenmann E. 307 Eisenstein O.209 Eisner M.,353 Eisner T.,349 353,354 Eisner. U. 268 Eiter K. 347 Ekman P. 393 Ekundayo O. 415 El-Bayoumi M.A. 52 Elder D. L. 352 Elgin S. C. R.. 396 Elguero J. 284 Eliel E. L. 11 272 Elliott A. J. 98 Elliott R. L. 10 Ellis.'P. D. 20,403 Ellison R. H. 338 Ellison W. R. 394 Elphimoff-Felkin I. 285 Elson 1. H. 93 Elzinga M. 387 Embree D. J. 6 Emoto S. 200 Enders D. 210,322 Endo T. 270 Enea V. 409 Engel J. D. 400 Engel P. S. 176 177 English T. H. 98 Engstrom L. 393 Ensinger M.J. 41 1 Epiotis N. D. 43,50 64 372 Epling G. A. 156 320 Epps L. A. 274 Erhard K. 391,392 Erhardt W. D. 240 Eriksen J. 178 Erinholf N. 8 Ermakov V. I. 158 Ermishkina S.A. 224 Emst R. R. 17,22 23 Ertl H. 241,288 Essenbries H. 354 Estrabaud E. 273 Etz E. S. 33 Etzold G. 399 Evans A. G. 102 Evans A. J. 61 Evans C. A. 93,221 Evans D. A. 16,66,326,347 352,377 Evans D. F. 198 201 Evans F. E. 404 Evans J. C. 102 Evans P.R. 392 Evans S.L. 351 Evans T. J. 259 Evens G. 269 Eventoff W. 387 Everett G. W. 14 Everett J. W. 187 289 Ewig C. S. 37 Ewing G. D. 243,244 Faber S. 367 Fakhrai M.,225 Fales H. M. 8 Farges C. 202 Farid S. 175 Farina J. S. 342 Farneth W. E. 56 Farnham W. B. 301,302 Farnum D. G. 307 Farona M. F. 125 Farrugia L. 126 Fateley W. G. 33 Fattoum A. 390 391 Faulhammer H. 408 Faurme R.390 Faure R. 51 Featherman S. I. 284 Fedin E. I. 14 Fedot’ev B. V. 148 Fedorynski M. 112 Fedot’eva I. B. 148 Feeney J. 11 13 23 403 Feeney R. E. 378 Feld W. A, 261 Felkin H. 120 124 191 194 Fellner P. 410 Fendler E. J. 84 Fendler J. H. 84 Fenselau C. 8 Ferguson D. C. 3 18 Ferguson J. 397 Fermaandjian S. 13 Fernandex-Alonso J. I. 43,44 Ferrer-Correia A. J. 8 Fessenden R. W. 99 102 Fetizon M. 371 Fiato R. A. 300 Fiaud J. -C. 344 Fichter K. C. 146 Field D. J. 67 Field F. H. 8 Field M. 13 Fields T. R. 113 172 Fiers W. 405 410 Fieser L. F. 346 Fieser M. 346 Figeys H. P. 38 Filler R. 231 Finch J. T. 397 Findlay R. H. 40 114 Fink C. G. 155 Finkelstein M.151 Finocchiaro P. 47 Fioshin M. Ya. 158 Fischer A. 223 224 Fischer E. H. 385 Fischer H. 94 97 Fish L. J. 353 Fishbein R. 393 Fisher R. P. 182 313 Fisher R. R. 20,403 Fitjer L. 190 Fitzell D. L. 17,421 Fitzwater S. 40 Flarnini A. 41 Flapper W. M. J. 294 Fleming I. 107 148 191,236 325,346 Fletcher B. S. 352 Fletcher D. J. C. 353 Fletcher J. L. 24 Fletterick R. J. 394 Fleury G. 34 Fliszar S. 11 Flitzsch W. 281 Floor J. 369 Florio S. 358 Flowerday R. F. 15 Flowers M. C. 56 Flowers W. T. 218 Floyd D. M.,342 Floyd J. C. 229 Flygare W. H. 215 Foag W. 256 Fog T. R.. 67 Fokin A. V. 283 Fokina T. V. 43 Folkes M. J. 35 Fonken G. J.64 Forbes C. P. 323 Ford M. E. 139 253 314 Ford W. T. 228 Forget B. G. 41 1 Forrey A W. 385 ForsCn S. 13.43 207 Forsyth D. A. 10. 44 Foster D. 134 Foster M. S. 4 Fosty R. 145 Foti A. E. 47 Fouda H. G. 348 Fourrey J. L. 273 Fox D. P. 115 Fox G. E. 410 Fox J. J. 401 Fraenkel G. 205 Fraenkel-Conrat H. 411 Frank-Neumann M.,130,243 Frank C. W. 14 Franklin T. 156 Frappier F. 374 Fraser G. V. 35 Fraser T. H. 408 Frasnelli H. 370 Fratev F. 52 Frazer-Reid B. 200 Frazier J. 405 Freed J. H. 98 Freedman H. H. 86,320 Freeman D. 366 Freeman F. 53 191 Freeman R. 19 Freeman S. K. 33 Freitag W. 12 Frey A. 245 Frey H. M. 56 112 118,241 Friderici K. H.41 1 Fridovich I. 378 Friedman A. E. 148 Friedman L. 172 Friedrich L. E. 300 Frigerio A. 3 Fritz H. 219 254 Fritz H. -G. 291 Froborg J. 155 Frostl W. 61 271 312 Author Index Fromageot P. 13 Fromm H. J. 394 Frones J. A, 41 Frushour B. G. 35 Fry A. J. 162 Fuentis R. 19 Fuerniss S. J. 172 Fuganti C. 425 Fujikura Y. 127 Fujita K. 170 422 Fujita S. 231 Fujiwara H. 268 Fukami H. 350 Fukuil H. 349 Fukui K. 43,44 53 Fukui T. 386 Fukushima Y. 260 Fukuzawa A. 201,338 Fukuzumi K. 122 Funarnizu M. 244 Funk R. L. 123 Furuhata K. 201 Furuichi Y.,41 1 Furukawa J. 128 Furukawa S. 138 Furukawa Y.,399 Furusawa K. 402 Furuta T. 107 Fusco R. 258 261 271 Fyfe C.A. 23 Fyles T. M.. 88 222 Gachon P.,201 202 Gadet A. 390 Gadian D. G. 385 Gait S. F. 62 Gajewski J. J. 190 Gakis N. 179 Gal P. 171 Gallagher R. E. 398 Gallaher K. L. 217 Galle J. E. 64 124 279 Gallenkamp B. 219 Gallo R. C. 398 Gallucci R. R. 116 147 Games D. E. 355 Games G. 33 Gammill R. B. 337 Ganem B. 323 Garanti L. 261 271 279 Garcia B. A. 193 Gardner J. N. 141 Gardner K. H. 35 Garfin D. E. 412 Gariboldi P.,416 Garin F. 186 Garito A. F.. 267 Garnett J. L. 123 Garratt D. G. 253 Garratt P. J. 185 187 221 257,289 Garrett R. A. 410 Author Index 437 Garst J. F. 237 Garza 0.T. 117,307 Gaskell A. J. 40 Gaspar P. P.,114 Gassman P.G. 140 153 263 Gaston L. K. 348 Gatti G. 10 Gaudenner A. 14 Gault F. G. 186 Gaunitz S. 7 Gavazzo E. 26 Gavezzotti A. 48,64 Gavina F. 87 240 288 Gavrilov Ya. D. 13 Gawley R. E. 345 Gay B. W. jun. 30 Gayoso. J. 38 Geerlings P. 38 Gehret J. C. E. 308 Gell K. 53 Gemmer R. V. 61 197 Genies M. 154 Gentry C. R. 348 Georghiou P. E. 338 Gerace M. J. 241 288 Gerad G. F. 405 Gerchman L. L. 138 Gerlach H. 202 Germain A. 203 Gerrnain G. 25 Germond J. E. 396 Gershowitz A.. 41 1 Gesson J.-P. 373 Ghosez L. 249,287 Giacomello P. 223 Giacometti G. 94 Gibbons. C. 129 Gibbons E. G. 141 Gibson D. H. 285 Gibson M.S. 272 Gielen M. 145 Gierke T. D. 215 Gifkins K.B. 244 Giglio E. 26 Gil G. 60 191 Gilbert A. 168 220 Gilbert J. C. 158 189 Gilchrist T. L. 15 203 260 26 1,274 Gilgen,P.,250 Gill D. 36 Gill R. J. 348 Gillespie D. H. 398 Gillespie D. W. 110 259 Gillespie S. J. jun. 236 Gilman L. B. 94 Gilman R. R. 40 Gimarc G. M. 40 Gimbarzevsky B. P. 256 Givens R. S. 174 Glaser R. 121 Glaspie P. S. 217 Gleispach H. 377 Gleiter R. 47 Gloor J. 64 Glyde E. 225 Goad L. J. 416 Goasdoue C. 242 Goddard W. A. tert. 39 46 210,212 Goebel W. 414 Goeddel D. V. 405 Goeke G. L. 125 Goff D. L. 176 Goggin P. L. 35 Gogte V. N. 279 Goh S. H. 117 Gokel G. W. 204,282 Gold A. M.,386 Gold P. 7 Goldberg I.B. 98 Golden R. 216 Goldstein A. W. 422 GoliE L. 274 Golik U. 279 Golino C. M. 146 Golob A. M.,66 Gornbos J. 202 Gomez J. E. 21 Gompper R. 184,241,289 Gonzalez-Diaz P. 38 Gonzenbach H. U. 65,305 Goodin R. D. 158 Goodman H. M. 412 Goodwin T. W. 416 Goosen A. 230 Gordon M. S. 52 Gore J. 60 191 Gore W. E. 350 355 Gorecki M. 412 Gorenstein D. G. 227 Gorissen H. 249 Gorlov Y. I. 41 Gorsane M.,238 Gosney I. 118 Goto G. 356 Gotthardt H. 190 Gottlieb D. 16 Gould K. J. 144 182 313 Gould R. O. 262 Gouord P. 23 Gozonka Z. 18 Grace D. S. B. 108 Graf E. 283 Graf W. 425 Graffeuil M.,49 Graham W. D. 58 Grakauskus V. 212 Granier F 126 Grant,D. M.11,13,14,18,19 21,215,403 Grant J. L. 227 Grant M. E. 19 Grantham G. D. 215 Gras J.-L. 60 191 Gratzer W. B. 404 Gravel D. 7 Graves D. J. 385 386 Gray C. 413 Gray R. 231 Gray R. T. 200,283 Green C. L. 347 Green M. 116 146,240 405 Green M. L. H. 124 126 Green M. M. 7 Green R. D. 15,234 Green R. J. S. 229 Greenberg R. S. 44 Cireene P. J. 412 Greenhill. J. V. 200 Greenhouse R. 269 Greenlee W. S. 125 Gregor I. K. 123 Greig C. C. 224 Greijdanus B. 218,291 Grellier P. L. 48 Gresson P. 185 Greving B. 280 Gribble G. W. 318 Grider R. O. 177 Grieco P. A. 138 143 321 334,337 Griesbaum K. 336 Griffin A. C. 46 56 Griffin B. E. 410 Griffin I. M.,143 Griffin T.S.. 283 Griffith D. W. T. 217 Griffith J. D. 396 Griffith R. C. 13 Griffiths D. V. 12 Griffiths J. R. 386 Griffiths P. R. 30 Griller D. 88,93,95 222 Grimaldi J. J. 23 Grimbert D. 68 Grimm K. G. 326,377 Grimmer V. M. 41 Grimshaw J. 162 Grob K. 351 Grohrnann K. 105 113,409 Grohmann K. G. 215 Gronowitz S. 264 Grosclaude,J. P.,65 305 Grosjean H. 409 Gross S. 21 Gross-Bellard,M. 396 Grossman W. L. 32 Groth P. 277 Grovenstein E. jun. 226 Groves J. T. 298 Grubbs R. H. 125,129 Gruber G. W. 106 Gruber L. 317 Gruenfeld M. 30 Griitze J. 230 Griitzmacher,H.-F. 7 Grunewald G. L. 51 245 Grunstein M. 398 Grutzner J. B. 23 Gryaznov V. M. 185 Grzejszczak S. 344 Gschwend H.W. 139 Guanti G. 226 Gubanov V.A. 41 Giinther H. 10.246 Guggenberger. L. J. 126,259 Guilley H.,410 Guillou A. 390 Gulbis J. 14 Gumport R. I. 404 Gund P. H. 45 50 299 Gund T. M.,50,299 Gupta R. C. 408 Gupta S. K. 136 183 267 Gurevitz E. 352 Gusel’nikov L. E. 146 Gust D. 47 Gutfreund H. 378 Gutman I. 39 50 235 Guttmann H. 67 Guziec F. S. 191 315 Haas C. K. 143 Haber S. B. 65 Haberkamp T. J. 118 Hackman R.H. 354 Haddon,R. C. 40,42,216,296 Hadfield K. L. 379 Haegmean G. 410 Hafner K. 244 Hagedorn A. A. 307 Hagen J. P. 65 Hagihara N. 128 133 134 135 181,229 Hagler A. T. 26 Hahnfeld J. L. 118 285 Haines R. J. 119 191 Halbritter K.209 273 Halevi E. A. 50 Hall D. R.,348 356 Hall H. T. 131 227 Haller. G. 119 Halton B. 238 Hamaguchi H. 324 Hamana M. 270 Hamaoka T. 145 Hamdan A. 139 Hamel E. 403 Hamer N. K. 178 Hamilton G. A. 196 Hamilton J. B. 172 Hammond G. S. 190,293 Hamon D. P. G. 291 Han K. K. 390 Hanack M. 72,73 Hanafusa T. 224 Hancock K. G. 177 Hand E. S. 81 Hanes R.M.,128 Hanifin J. W.. 175 Hannan W. 123 Hansen C. E. M.,98 Hansen H.-J. 67 250 Hansen L. D. 283 Hansen P. E. 13 18 270 Hanson J. R. 16,416 Hanst P. L. 30 Hanzawa Y. 219,372 Hanzlik R. P. 202 Hara H. 231 Harada F. 409 Harada N. 201,209 Harbison K. G. 106 227 Harding J. R. 167 273 Harding L. B. 39 212 Hardy R.W. F. 378 Harel M.,24 Harel Z. 176 Hargrove R. J. 186 317 Harries W. B. 416 Harrington K.J. 113 286 Harris C. J. 274 Harris R. K. 13 Harrison A. G. 6 Harrison C. R. 144 182 196 313 Harrison,J. F. 40 108 Harrison S. 346 Harrison T. T. 346 Hart A. J. 10 Hart D. J. 321 344 Hart D. W. 133 317 Hartman J. S.. 13 Hartman S. E. 175 Hartshorn M. P. 374 Harvey A. B. 33 Harvey C. L. 405 Harvey M. 378 Harvey T. M. 8 Hase T. 326 Hasegawa K. 264 Haselbach E. 115 Hashimoto H. 118 142,285 Hashimoto K. 229 Hashimoto T. 324 Haslinger E. 202 Hass B. S. 25 Hasselmann D. 306 Hassner A. 53,250,283,320 327,402 Haszeldine R.N. 218 Hata K. 231 Hata T. 402 Hatano H.20 Hatch C. E. 110 254 Hatcher A. S.. 138 332 Hattori M. 405 Hatz M.,358 Haubenstock H. 318 Haugh M. J. 75 194 Hauptmann H. 25 187 Hauser S. 44 Havelka U. D. 378 Havinga E. 220 Hawkins E. R.,408 Hayashi H. 242 Hayashi T. 51 122 Hayles W. J. 307 Author Index Haynes R.K. 371 Hayward R. J. 260 Heath G. A. 131 273 Heath R.R.,348 Heathcock C. H. 332,341 Heathcote J. G. 421 Heck F. R. 181 Heck R.F. 127 Hedgecock H. C. 195,319 Hedin P. A. 353 Hedrick J. L. 385 Hedstrand D. M. 234 Hegarty A. F. 81 206 Hege H..G.,273 Hegediis B.. 202 Hehre W. J. 38 40 45 55 216 Heibl C. 263 Heidrich D. 41 Heil B. 121 129 Heilbronner,E. 51 Heimbach P.185 Heimer E. P. 403 Heimgartner. H. 67 179 Heimgartner M. 250 Heinmann U. 55 Heinrich S. 149 278 Heinzelmann W. 250 Hekman M. 241 Helenbrand D. F. 16 Helgee B. 151 157 Helgeson R.,23 1 Helquist P. 134 193 290 Helwig G. S. 60 Henderson T. R. 400 Hendra P. J. 34. 35 Hendricks D. E. 348 Hendricks L. 3 Hendrickson J. B. 323,345 Hendry L. B. 348,354,359 Henneburg D. 196 Henning D. 410 Henrici-Olive C. 124 Henrick C. A, 183 193 311 356,357 Henricks P. M. 21 Henriquez R. 222 Henry P. M. 119 Henry-Basch E. 146 Henson R.D. 353 Henzel K. A. 245 297 Herberich G. E. 145 Heremans K. A. H. 381 Herkes F. E. 203 Hermans J. jun. 26 Herndon W. C. 39 235 Herriott A.W. 86,. 343 Herrman K. 413 Hers H.-G. 386 Herschbach D. R. 75 Herzog W. 47 Hesbian-Frisque A. M. 249 287 Hesse M.,3 Author Index Hesse R. H. 340 366 368 Hesson D. 65 Heumann A. 11 Hewett C. L. 375 Hewish D. R. 396 Hexem J. G. 21 Heyde E. 391 Heyn A. S. 70 Heyne T. R. 118,286 Hibbert J. R. 421 Hiberty P. C. 48 Hibino K.,324 Hickey M.J. 212 Hicks K. 353 Hidai M.,127 Hidalgo H. 101 Higgins R. J. 244 Higgs M.D. 352 Hikita T.;127 Hilbers C. W. 405 Hill A. S. 348 Hill J. H. M. 67 Hill R. L. 378 Hillard R. L. tert. 123 185 22 1 Hillenbrand D. F. 295 Hinchcliff A. 39 Hindenlang D. M.,359 Hine J. 210 Hine. K. E. 129 Hingerty B.25 Hino K.-I. 122 Hino T. 237 Hinshaw W. S. 22 Hirai H. 173 Hirai T. 242 Hirama M. 242 Hiramoto H. 128 Hirano S. 23 1 Hirano T.. 44 Hiroi K. 333 368 Hirotsu T. 238 Hirsekorn F. J. 120 125 221 Hirshfield F. L. 24 Hirt B. 396 Hirth L. 410 Hitchcock P. B. 27 Hitzel E.. 120 Hixson S. S. 113 171 172 Hiyama T. 112 231 Hjelmeland L. M. 4 I Ho C. T. 114 Ho N. W. Y.. 405 Ho T.-L. 213 358 Hoa K. 123 Hobrock B. W. 3 Hochmann P. 52 Hocks L. 119 Hodge P. 196,317 Hodgson P. K. G. 191,346 Hoffelner H. 157 Hoffman C. W. 148 Hoffman D. H. 323 Hoffman,M. K. 5 Hoffman V. L. 108 Hoffman W. H. 320,372 Hoffmann H. M.R. 208 Hoffmann R. 54,595 Hoffmann R. W.,’67 115 Hogarth M.J. 48 Hogberg H. E. 231 Hogenkamp H. P. C. 19 Hogness D. S. 398 Hohener A. 17 Hohner G. 232 Hoinowski A. M. 206 Holcomb W. D. 269 Holker. J. S. E. 16 420 421 Hollenstein R. 61 Holloman M. 38 Holmes J. D. 169 Holmes J. L. 4 7 Holmes S. A. 216 Holubka J. W. 189 Honda K. 112 Honda M.,219,372 Hong. P.-K. 301 Honig B. 52 Honjo M. 399 Hooper D. L. 34 Hoornaert C. 287 Hoornaert G. 183 Hootele C. 353 Hopf H. 187 Hoppe B. 274 Hoppe I. 261 Horiuchi K. 412 Hornback J. M. 174 Homer L. 165 Horton W. J. 18 Howell D. C. 376 Hossain A. M. M. 272 Hosteny R. P. 40 Houghton E. 351 Houk K. N. 53,63 House H. O. 140 208,341 House W. V. 22 Houseman D.398 Howard F. B. 405 Howard J. A. 90 212 Howarth 0.W. 23 Howe W. J. 345 Howell J. A. S. 14 Howells D. 191 346 Howse P.E. 351 353 Hoyle C. E.. 59 169 Hoz S. 78 Hsieh D. P. H. 17,421 Hsu C. J. 22 Hsu C. Y. 127 Hsu Lee L. F. 200 Hu H.-Y. 386 Huang C.-Y. 379 Huang F.-C. 200 Huang S. J. 158 Hubbard. D. R. 392 Huchi M. 185 Hunkowska E. 271 Hudrlik P. F. 192 3 11 Hudson B. S. 33 Hudson R. F. 95,209 Hunig S. 138 Hughes D. R. 30 Hughes P. R. 350 355 Hughes R. P. 240 Hughes W. B. 119 Huhn M.,202 Huisgen R. 59,287 Huler E. 26 Hull R. 272 Humbert H. 188 304 Humble E. 393 Hummel H. E. 348 Hunkler D. 219 Hunt D. F. 8 Hunt E 17 Hunter D.H. 69 Hunter W. 240 Hunziker E. 224 Hush N. S. 51 Hutchings M. G. 137 195 Hutchins R. O. 320 Hutchinson D. A. 98 Hutchinson S. A. 16 Hutmacher H. -M., 29 1 Hutzenbuhler D. A. 113 Huwyler S. 351 Huxley T. H. 385 Hvistendahl G. 7 8 Hyde A. J. 35 Iacobelli J. A. 366 Ibel K. 396 Ibrahim B. 62 270 Iddon B. 110 Igami M. 324 Igier C. 11 1 278 Ignatiadou-Ragoussis V. 371 Ihara M. 427 Ikeda M.,52,255,259 Ikeda T. 52 Ikekawa N. 342,367 Ikenberry D. 215 Ilan Y. 168 Ilenda C. S. 172 Illuminati G. 223 Imahashi Y. 117 Imai H. 122 Imaizumi S. 11 Imamura A. 44 Imoto T. 20 Imuta M.,258 Inagaki S. 44 53 Inagaki Y. 263 Inamoto N. 263 266 Ingold K.U. 88 93 95 222 Inokuchi H. 26 Inone Y. 113 172 Ipaktschi J. 176 Ippen J. 246 440 Iriuchijima S. 335 Isaacson A. D. 207 Isenour T. L. 8 Ishaaya I. 352 Ishibashi H. 255 Ishibe N. 229 Ishiboti H. 12 Ishida S. 260 Ishii F. 276 Ishii S. 350 Ishii Y. 128 Ishikawa H. 141 Ishikawa M. 366 367 Ishikawa N. 134 Ishimoto S. 342 Ishiyama J. 11,403 Ishizaki K. 224 Isobe M. 331 Isaacson A. D. 43 Itakura K. 405 Ito I. 279 Ito K. 326 Ito S. 242 286 363 Ito Y. 117 Itoh K. 128 Itoh M. 144 182 183 193 316,330 Itzel H. 97 Ivanov V. T. 206 Ivanovin G. 401 Ivaroska C. 39 Iwaki S. 359 Iwama A. 232 Iwamura H. 57 117 172 Iwata S. 42 Iyoda M.247 248 Izatt R. M. 283 Izumi T. 269 Izumiya N. 21 Izydore R. A. 252 Jablonski C. R. 132 Jack A. 407 Jackson A. H. 355 Jackson B. 250 Jackson G. E. 118 Jackson W. R. 342 Jacob R. A. 138 Jacobs P. 64 Jacobsen C. S. 267 Jacobson S. E. 128 Jacobson U. 245 280 308 Jacobus J. 21 Jacquesy J.-C. 373,374 Jacquesy R. 372 373 374 375 Jacquignon P. 170 Jackel K.-P. 72 Jaenicke O. 119 Jaffe H. H. 51 Jain S. C. 27,406 Jakes K. 41 1 Jakobsen H. J. 23 James B. R. 121 James K. J. 426 James M. N. G. 240,293 James T. L. 391 Jamieson W. D. 6 Janssens F. 183 Jantzen R. 12 Janzen E. G. 93,221 Jaouen G. 131 Japenga J. 306 Jardine I. 8 Jarre W. 220 Jarreau F.-X. 374 Jay G. 412 Jeanmaire D. L. 33 Jeffery. E. A. 146 Jefford C. W. 58,308 Jeffrey A. 413 Jeltes R. 30 Jeminet G. 201 Jen J. S. 199 Jencks W. P. 81,83 378 Jenkins -4.D. 128 Jenkins P. A. 189 Jennette K. W. 406 Jennings K. R. 8 Jennings R. C. 416 Jensen H. H. 42 Jespers J. 61 239 Jessup P. J. 239 Jesthi P. K. 145 325 Jetuah F. K. 376 Jochem M.,6 Jonsson B. 43 207 Joh T. 128 Johansen H. 52 Johnson A. W. 347 Johnson B. F. G. 129 Johnson C. D. 217,223 Johnson C. R. 139,253 Johnson D. E. 59 Johnson K. W. 98 Johnson L. N. 384 Johnson P. E. 391 Johnson P. Y. 110 254 Johnson T. 172 Johnston D. E. 80 Johnstone R. A. W. 7 192 315 Joho R.H.398 Joiner C. M. 196 206 Jolly P. W. 119 Joly G. 373 Jommi G. 416 Jonard G. 410 Jones C. R. 22 Jones C. W. 379 Jones D. N. 130 Jones D. W. 67,229 Jones Sir E. R. H. 371 Jones G. 110,315,359 Jones G. H. 204,282 Jones J. G. 393 Jones J. R. 79 Author Index Jones M.,112 114 116 146 147 Jones R. B. 11 16,417 Jones R. L. 404 Jones R. R. 106,246,247,281 Jones R.W. 138,332 Jones S. P. 205 Jones W. M. 113 Jongsma C. 278 Jonkers F. L. 193 31 1 Jordan F. 42 Jorgensen W. L. 50 Joseph-Nathan P. 10 Jouin P. 273 Joyce M. A. 11 2 Jubault M. 165 Juckett D. A. 237 Judy K.J. 416 Jugovich J. 359 Juhasz A. A. 31 Juillard J. 201 Junk G. A. 7 Jurs P. C. 8 Just G.194 Justice J. B. 8 Jutz C. 238 Jutzi P. 137,283 Kaae R. S. 348 Kaba R. A. 88 Kabalka G. W. 195 309 319 Kabat D. 411 Kacprowin A. 112 Kadoi S. 128 Kadunce W. M. 140 Kaempfer R. 412 Kafka W. A, 355 Kagabu S. 219,254,287 Kagan H. 238 Kagan H. B. 121 Kagel R. O. 32 Kagi D. A. 197 Kai M. 263 Kainosho M. 403 Kaiser C. 219 254 Kaiser K. H. 64 Kajikawa A. 342 Kakis F. J. 371 Kakuta S. 366 Kalechits I. V. 43 Kalicky P. 371 Kalinowski H. O. 15 320 Kallen R. G. 82 KP16y K. 202 Kalvoda J. 368 Kam T. S. 11 7 Kamata S. 201 338 339 Kamigata N. 112 249 Kamiyama Y.,147,352 Kammula S. 116 146 Kamogawa A. 386 Kan L. S. 405 Kan S. K. 23 Author Index Kandasamy D.320 Kane F. J. 391 Kane J. 265 Kaneko C. 366,367 Kang K.. 199 Kaplan L. 323 Kapoor M. 393 Karady S. 206 Karakhanov E. A. 268 Kariv E. 164 Karl R. R. jun. 21 7 Karle I. L. 25 Karlsson R. 353 Karlstrom G. 43 207 Karpinski Z. 185 Karplus. M. 47 50,52 Karplus S. 21 Karten M. J. 140 Kasahara A. 269 Kasai H. 409 Kasang G. 355 Kashdan D. S. 343 Kashitani T. 248 Kassab. R.,390 Kastrup R. V. 22 Kata H. 43 Katagiri K. 359 Katagiri N. 405 Katagiri T. 195 268 Katalinic J. P. 67 Kates M. R. 47 Kato H. 38 106 Kato M. 235 Kato Y. 367 Katritzky A. R. 49 62 179 223,269,270,275,284 Katz J.-J. 136 195 Katz T. J. 125 168 218 Katzenellenbogen J.A. 360 Kau L. S. 21 KauEif V. 274 Kauffmann T. 280 Kawahara A. 263 Kawakami J. H. 195 Kawamura T. 38 Kawano Y. 287,330 Kawase T. 267 Kawashima T. 231 232 237 Kawauchi H. 301 Kayama Y. 289 Kayane Y. 238 Kazakos A, 372 Kearns D. R. 22,408 Keay R. E. 196 Kebarle P. 80 Keck G. E. 59 177 Kedes L. H. 398 Keefer L. K. 210 Keeley D. E. 363 Keinan E. 319 Keith J. 41 1 Keller A. 35 Keller R. 219 252 Keller W. 414 Kellerer H. 149 Kelley D. E. 41 1 Kelley J. A. 323 Kellner R. 31 Kellogg R. M. 185 Kelly T. R. 287 Kelso P. A. 190 Kernball C. 186 Kemp W. 32 Kenchan E. F. 323 Kende A. S. 134 227 285 343 Kenkare U. K. 394 Kennard O.404 Kennedy G. J. 112 Kent A. B. 385 Kent J. E. 217 Kenyon G. L. 389 Keough T. 8 Kerek F. 209 Kerekes I. 186 Kergomard A. 202 Kern J. 343 Kershaw M. J. 218 Kessler H. 15 298 Keul H. 336 Kevan L. 188 Khan A. N. 116.269 Khan S. A. 282 Khorana H. G. 404,409 Khosrovi M. 225 Khuong-Huu Q. 109 Kido H. 348 Kieboom A. P. G. 15 Kiefer G. W. 383 Kiefer W. 33 Kiemar N. G. 21 Kiers C. Th. 185 Kiji J. 128 Kikugawa Y. 319 Kilgour J. A. 116 147 Kilkan R. C. G. 24 Kim B. 169 Kim B. T. 129 Kim C. U.,338 Kim C. W. 298 Kim J. J. 35 Kim K. H. 250 Kim M. -G.,312 Kim S. H. 407 Kimura A. 247 Kimura Y. 422 Kinashi H. 202 King D. L. 75 King J.F. 78 Kingsbury C. A, 209 Kingston D. G. I. 3 Kinoshita M. 141 Kinoshita T. 85 Kinson P. L. 238 Kirby G. W. 202 Kirino Y. 102 Kirk D. N. 368 374,377 Kirmse W. 86 441 Kirschner S. 48 55 56 64 218,241,305 Kiselev V. D. 237 Kiso Y. 140 147 231,282 Kispert L. D. 240 Kisselev L. L. 409 Kistenmacher T. J. 274 Kitagawa Y. 193 315 Kitahara K. 402 Kitahara Y. 117 243 244. 289 Kitai M. 144 315 357 Kitamura T. 128 Kitao C. 200 422 Kitaura K. 42 Kitazawa M. 158 Kitching W. 13,245 297 Kituchi K. 319 Kizer K. L. 31 Kjaer A. 212 Klabunde K. J. 119 Klayman D. L. 283 Kleid D. G. 413 Klein J. 196 Klein R. S. 401 Klessinger M. 50 299 Klopper D. 37,47 Kloosterziel H.67 Klopman G. 196,206 Klug A. 396,407 Klumpp G. W. 306 Klun J. A. 347 Klyne W. 377 Knaggs J. A. 372 Knauer B. 355 Kneen G. 67,229 Kneidl F. 149 Knifton J. F. 120 322 Knight D. W. 334 338 Knights E. F. 195 Knolle J. 159 364 Knothe L. 219 Knowles J. R. 113 391 Knowles W. S. 121 Knox S. D. 130 KO E. C. F. 72 234 Kobayashi M. 342 Kobayashi T. 242 Kobayashi Y. 219 372 Kobler H. 340 Kochansky J. 348 Kochetkova N. S. 14 Kochi J. K. 223 Kodama M. 363 Kodama S.-I. 140 231 282 Koeberg-Telder A. 224 Kohler H.-J. 37 41 47 Koelbe H. 217 Koenig J. L. 30 31 35 Koenig T. 41 206 229 Koenuma M. 202 Koerner von Gustorf E. A. 119 Kossel H. 413 442 Author Index Koster R.143 Kogure T. 122 Kohno Y.,26 Koizurni N.,367 Kojima M. 263 Kolbl H. 171 Kolc J. 107 Kolchin I. M. 191 Kollman P. 45 Kollman V.H. 16,17. 19,415 Kollmar H. W., 42 240 295 Kolomiets A. F. 283 Kolpack R.L. 30 Kolshorn H.,11 Komin J. B. 251 Komoroski R.A. 18 Kondo K. 144,261,318,328 338,358 Kondo T. 352 Kondrikov N.B. 158 Konig J. 280 Konno M. 26 Konno T., 205 Konyushenko V. P. 194 Kool M.,306 Koopman H. 210 Koosha K. 189 Kopp L. D. 11 Kopple K. D. 13 Kornberg R.D. 396,397 Korte F.,211,220 Korth M. 52 Korver O. 198 Koneniowski S. H. 359 Kosarych Z. 359 Kosley R. W. 317 Kossanyi J. 362 Kossemehl G. 265 Koster J.F. 392 Koster S. K.,230 Kostikov R. R.,189 Kotani E. 158 Kottwitz. J. 286 Kouwenhoven A. P. 189 KovaEeviC K. 39 Kovacic P. 206,322 Kowanko N.,16 Kozerski L. 27 Kozikowski A. P.,328 Kozmutza K. 52 Kraatz U.,211 Krajca K. E. 113 Kramer D. M. 22 Kramer F. R.,410 Krane J. 13 Kraniz A. 274 Krapcho A. P. 343 Kratzl K. 310 Krauch C. H. 175 Krause J. G. 341 Krauss D. 354 Krausz P.,126 hwayk H. 186 Krebs E. G. 385 Krech R. H. 97 Kreeger R.L. 116 147 Kreissl F. R.,19 Krenmayr P.,3,4 Kretschmer G. 254 Krief A. 192 252 321 Krimm S. 35 Krishna B. 37 Krishnamurthy S. 320 Kristof w., 55 Kristoff J. S. 125 Kroger C.-F. 225 Krogh-Jespersen K.178 Krolikiewicz K. 402 Kroll L. C. 129 Kropp P. J. 113 172 Krow G. R.,64 Krugh T. R.,406 Kruglaya 0.A. 148 Kruijt J. K. 392 Kruszewski J. 39,216 Krygowski T.M.,39,43,216 Krystosek A. 411 Kucherov V.F. 75 183 Kiipper H. 409 Kuhn J. 52 Kulbe K. D.,391 Kulic J. 225 Kulikov A. V.,380 Kulkarni P.S. 8 Kumada M.,122 140 147 231,282 Kumadaki I. 219,372 Kumadaki S. 270 Kumanotani J. 294 Kumar A. 22 Kunieda N.,141 Kurabayashi M.,268 Kuroda S. 244 Kuroda Y. 231 Kurozumi S. 333,342 368 Kushida K. 23 Kutney J. P. 269 Kutz A. A. 108 Kutzelnigg W. 37 40 Kuwano H. 201 Kuzuya M. 107 Kwak S. 153 Kwan C. Y. 391 Kwart L. D. 124 194 Kwiatkowski J.S. 47 284 Kwon S. 259 Kyburz R. 243 Kysel O. 43 Labarre J.-F. 41.49,293 L'AbM G., 283 Labinger J. A. 133 Labovitz J. N.,193,357 Ladner J. E. 407 Laemmle J.T. 137 Lagow R.J. 138,196 Laguerre M. 148 Laidler D. A. 204 282 Laird T. 185 Lakshmikantham M. V. 267 283 Lal B. 320,372 Lalloz L. 107 258 La H.-Y. 367 La Mar G. N. 17,421 Lambert G. 98 Lambert J. B. 282 284 Lamprecht W.,391 Landberg B. E. 257 Landheer 1. J. 217 218 300 304 Landick R. C. 344 Landini D. 87 Landor P. D. 187 Landor S. R. 187 Landy A. 409 Lane C. F. 136 Langler R. F. lY8 Langley P.A. 350 Langridge R. 25,406 Langs D. A. 25 Lanier G. N.,350 Lappert M.F. 49 125 Large G. L. 340 Larock R.C. 142 183 Larsen J. W., 203 Larson W. D. 120 221 Larsson-Raznikiewin M. 3Y1 Latajka Z. 41 Lathan W. A. 43 Laub R.J. 182,313 Lauer G. 240 Lauer R. F. 372 Laurent A. 108 141 250 Lauterbur P. C. 22 Lavie D. 352 Lawesson S. O. 271 Lawson A. J. 95 Lawson J. A. 170,247 Layloff T. 161 Lazzeretti P. 215 Leandri G. 226 Lebediv V. L. 43 Le Bret 407 Lechner M. 117 Le Clair-Lanteigne P. 7 Leclerc G. 371 Led J. J. 18 21 Lederberg J. 9 Ledlie D. B. 245 Lee C. C. 15 72 234 Lee C. -H. 404 Lee D. P. 3 17 Lee G. A. 276 Lee G. R. 204,335 Lee J. S. 407 Lee L. 20 Lee S. J. 343 Lee S.-L.,425 Lee T.B. K. 206 Author Index Leete E. 16 Lefohn.A. S. 30 Leforestier C. 49 Legris C. 336 Lehmkuhl H. 196 Lehn J. -M. 283 Leibovici C. 49 Leichter L. M. 343 Leigh,G. J. 119 131. 191,273 Leighton P. 187 Lemal D. M. 241,288 le Noble W. J. 63 Lenton J. R. 416 Lentz C. M. 341 Leonard J. E. 293 Leonard N. J. 400 Le Pecq J. B. 407 Le Perchec P. 53 330 Lerch K. 385 Le Roux J.-P. 242 P75 Lester R. 348 356 Leung H. W. 225 Leung K. H. 345 Lever 0.W. 332 Levin C. C. 47 Levin. G. 39 Levin R. H. 208 Levine R. 107 140 Levinson A. R. 349 Levinson H. Z. 349 365 Levisalles J. 372 Levsen K. 5 Levy A. B. 143 182 Levy E. C. 352 Levy.G.C. 18 19 20,21 Lew G. 14.5 Lewis B. J. 410 Lewis C. P. 66 306 Lewis D.215 Lewis E. S. 217 Lewis F. D. 59 169 Lewis G. E. 106 Lewis J. 129 Ley B. E. 47 Ley K. 262 283 Ley S. V. 2 13 Leyendecker F. 292 Li H. J.. 22 Li W. K. 10.5 Li. W. S. 210 Liang G. 14 216 Libbey L. M. 350 Liberles A. 37 40 Libert V. 238 Libman J. 169 Lichtenthaler F. W. 402 Liebeskind L. S. 134,227,343 Liehr. J. G. 409 Lienhard G. E. 80 Lifson S. 26 Likhtenshtein G. I. 380 Liler M. 205 Lim. D. C. K. 263 Lirnacher H.. 349 Lin D. C. K. 7 Lin H. C. 203 Lin L. J. 426 Lin M. T. 42 1 Lin P. -H. 6 Linda P. 284 Lindner H. J. 238 244 Lindoy L. F. 283 Lindqvist L. 173 Lindsay D. 88 95 Lines R. 157 Linstrumelle G. 139 186,3 17 Liotta C. L.49 Lipari N. O. 51 Lipina E. S. 245 Lipkowitz L. B. 364 Lippard S. J. 406 Lipshutz B. H. 324 340 Lipsztajn M. 43 Lischka H. 37 40 Li-Shang Shih. 266 Litton J. F. 5 Liu K. 177 Liu K. T. 195 Liu L. F. 414 Liu R. S. H. 169 Liu S. 371 Livingston D. C. 399 Ljones T. 383 Lloyd H. A. 353 Lloyd R. V. 212 Lo D. H. 38,216 LO S.-F. 238 Locke M. J. 229 268 Lobering H.-G. 238 Loehr T. M. 36 Loew G. H. 41 Loew L. M. 296 Loewenstein P. M. 405 Logemann E. 181 Logue M. W. 202 Lornbardo L. 243 London R.E. 16 17 19,415 Long D. R.. 259 Long M. A. 123 Longworth S. W. 266 Lonsky W. 310 Loomis G. L. 313 Loozen H. J. J. 236 Lopez L. 208 253 Lorch H. W. 157 Lord R.C. 35 Losman D. 353 Losing F. P. 242. 295 Loubinoux B. 128 Loudon G. M. 341 Lourens R. 149 Loutfy R. O. 112 Louw R. 203 Low M. J. D. 30 Lowe D. A.. 425 Lowe D. G. 381 Lowenstein L.. 4 12 Lowers R. H.,98 Lown J. W. 257 Lozach N. 276 Lucchini V. 44 253 Luce R. 410 Luche J. L. 186 Lueck J. D. 394 Liickhoff M.,277 Luippold D. A. 52 Lukan G. 16,200,418,422 Lukas J. H. 189 Luknitskii F. I. 209 Lukovkin G. M. 14 Lumrna W. C. 206 Lunazzi L. 88 Lund A. 44 Lund H. 160 16 Lundell G. F. 25 Luria M. 39 Lutz H. 173 Lyerla J. R. 19 Lyons J. E. 120 33 Lynch. P. F. 30 Lythgoe B. 67 193 3 1 1 329 Ma K. W. 298 Ma M. C. 349 McAninch T. W. 345 McBride J.A. H. 273 McCallum R. J. 79 McCandlish C. S. 317 McCarthy B. J. 412 McCleland C. W. 230 McClelland R. A. 205 McCloskey J. A.,404,409,410 McCollurn G. J. 79 McCornbie S. W. 309 372 MacConnell J. G. 347 353 McCorrnack M. T. 206 McCullagh L. 67 McCulloch C. S.. 290 McCullough J. J. 67 230 MacDonald C. G. 7 McDonald E. 17,426 427 McDonald G. G. 388 McDonald R. N.. 244 McDonald R. S. 29 McDonald W. S. 60 191 McFadden D. L. 97 Macfarlane N. 392 McGhie J. F. 376 McGinnis J. 125 McGlinchey M. J. 119 191 242 McGrath J. P. 323 McGregor W. H. 18 MachaEek V. 226 Machida Y. 3 19 322 372 Maciel G. E. 10 McInnes A. G. 16 415 Mclver J. W. 50 Mack M. P.228 Mackay A. L. 26 McKelvey J. 45 444 AuthorIndex Mackie G. A, 410 Mckillop. A. 146 228 McKinney R. 40 McKinnon D. 13 McLafferty F. W.. 4 7 McLauchlan K. A. 98 Maclean D. I. 97 McLean S. 252 McLennan D. J. 76 McLick J. 146 McMahon T. B. 80 McManus S. P. 2 1 1 McMeeking J. 124 220 McMullen G. L. 113 258 McMurry J. E. 315 326 343 McNelis E.. 126 MacNicol D. D. 13 216 Macnicol P. K. 379 McNutt R.W. 287 Macomber R. S. 297 330 McOmie J. F. W. 229 266 McPhail. A. T. 353 McQueen J. E. jun. 26 McVey J. K. 169 McVicker E. M.. 13 McWilliam H. M. 110 Madhavarao M. 192,315 Madhavora M. 125 Madigan D. M. 67 172 Madsen H. 348 Madsen N. B. 384 Maeda M.263 Markl G. 149 277 278 Maggiora G. M. 40 Magnus P. D. 213 228 325 371,377 Magnusson G. 155 Mah T. 107,236 Mahan B. H. 50 Mahany P. G. 348,356 Maia A. M. 87 Maidment M. S. 375 Maier G. 240 241 Maier W. F. 46 203 Maitlis P. M. 124 Majetich G. 143 Maki T. 319 Maki. Y. 107 Mako\za M. 112 Maksii. Z. B. 39 Malcolm A. D. B. 386 Malherbe R. 61 197 Malinski E. 223 Mallon C. B. 58 112 Maloney A. P. 425 Malpass J. R.,63 110 189 25 1 Mametsuka H. 52 Manas A.-R. B. 138,342 Mancini G. J. 305 Mandai T. 159 Mandolini L. 223 Manecke G. 265 Mangano F.,42 Mangini A. 45 Mango F. D. 119 191 Mangoni L. 372 Mangum M. G. 115 Manhas M. S. 320 372 Mani E. 349 Maniatis T.4 13 Manimaran T. 276 Manion M. L. 117 Maniwa K. 335 Mann B. E. 124 Mann C. K. 153 Mann J. 415 Manz F. 254 March J. L. 365 March P. G. 45 Marchand A. P. 2Y2 Marchese F. T. 41 Marchese L. 253 Marcuzzi F. 184 Mareda J. 308 Margolin Z. 79 80 Marguretha P. 155 Marians K. J.. 405 Maricich T. J. 108 Marino J. P. 342 344 Marioni F. 194 Mark H. 30 Mark H. B. jun. 30 Markley J. L. 22 Marko L. 121 Markovetz A. J. 355 Marotta C. A. 41 1 Marrero R. 335 Marsden K. M. 390 Marshall A. G. 30 Marshall D. R. 77 Marshall H. 336 Marshall J. A. 338 Martens H. 183 Martigny P. 155 Martin E. 399 Martin G. J. 11 242 Martin H. D. 241 287 307 Martin J.R. 201 422 Martin M. 41 Martin M. L. 11 Martin R. 63 408 Martin R. H. 61 238 239 Martin S. A. 41 1 Martina D. 130 243 Martinelli M. 242 Marton M. T. 359 Marumo S. S. 359 Maruyama S. 224 Marx F. 387 Marzilli L. G. 274 Marzin C. 284 Masamune S. 73 201 240 293,338,339 Maslakiewicz J. R. 273 Maslen E. N. 24 Masmanides C. A, 51 Mason R.,27 Massiot G. 23 Massuda D. 148 287 Mastre T. A. 193 Masuya H. 356 Masuyama Y. 267 Mateescu G. D. 10 Matheson T. W. 14 Mathis F. 41 Mathis R. 41 Matsuda I. 128 Matsugashita S. 255 Matsugo S. 258 Matsui K.. 110 170 260 352 Matsui M. 356 Matsuki Y. 363 Matsumoto H. 122 147 Matsumoto K. 237 Matsumoto M.338 Matsumoto N. 294 Matsumoto Y. 118 142 285 Matsumura F. 349 Matsumura Y. 152 324 Matsuura T. 258 Matteoli. U. 121 Mattes K. C. 347 Matteson D. S. 143 145 325 Matthes D. 277 Matthews C. R.,20 Matthews W. S. 79 80 Mattson J. S. 30 Matusch R. 202 Matuszewski B. 174 Matveeva N. B. 224 Matwiyoff N. A. 15 16 17. 19.415 Maurin R.,60 191 Maverick E. 27 May I. R. 350 May K. D.. 292 Mayall B. I. 290 Mayeda E. 152 Mayer M. S. 355 Mayo D. W. 33 Mayr H. 59 287 Mays M. J. 14 Mazo A. M. 409 Mazur S. 164 Mazur Y. 185 319 366 Mazza F. 26 Mazzocchi P. H. 303 Meakins G. D. 371 Medlik-Balan A. 196 Meguro H. 205 Meier H. 21 1 252 Meier W. 34 Meijer J.186 Meinwald J. 106 349 353 354,355 Meisters A. 146 Melamud E. 391 Mellon F. A. 7 Melloni G. 184 Melnickov A. A. 397 Melvin L. S.. 339 346 Author Index Mende U. 310 Mendenhall G. D. 95 Mensch S. 184,241 289 Menzies I. D. 371 Mercer E. I. 416 Merkel C. 181 Merlin J. C. 32 Merlini. L. 283 Mermet-Bouvier R. 13 Merregaert J. 410 Merrill R. E. 144,222 Mertz P. 270 Metcalfe D. A, 67 Meth-Cohn O. 260 266 Metzner W. 175 Meyer A. 131 Meyer,.A. Y. 2 12 Meyer E. F. 25 Meyer H. 52 Meyer H.J. 352 Meyer L. U. 50,299 Meyer R. 370 Meyer R. B. jun. 400,401 Meyers A. I. 139 253 268 314,333 Mian A. M. 401 Michejda C. J. 297 Michel M. -A. 155 161 162 Michelot D..139 186 317 Middleton R. 167 273 Midland M. M. 139 143 182 Midorikawa H. 107 Miertus S. 43 Miethchen R. 225 Migita T. 116 146 Mihelich E. D. 139 Mikhailov B. M. 143 Mikolajczyk M. 344 Mildvan A. S. 391 Miles H. T. 405 Milevskaya I. S. 41 Miller B. 233 Miller C. H. 337 Miller J. G. 237 Miller J. M.,392 Miller J. P. 400 Miller J. R. 354 Miller,L.L. 152,153,157 164 Miller M. J. 341 Miller R. D. 299 Miller R. S. 28 Miller R. W. 350 353 Millington D. S. 355 Mills D. R. 410 Milne G. 42 1 Milne G. W. A. 8 Milner J. R. 329 Milner-White E. J. 388 Minabe M. 228 Minematsu H. 229 Miners J. 0..371 Min Jou W. 410 Minks A. K. 348 Minter D. E..64 Mishra P. C. 52 Mishra S. P..207 212 Mislow K. 47 Mison P. 152 Misumi S. 231 232 237 Mitchard D. A. 189 Mitchell E. R. 348 365 Mitchell P. J. 216 Mitchell R. H. 230 246 Mitscher L. A. 201 Mitschkev A. 280 Mitsudo T. 131 324 325 Mittal R. S. D. 200 Miura I. 352 Miura S. 342 Miwa. T. 106 Miyake N. 147 Miyakoshi S. 243 Miyamoto R. 243 Miyano S. 118 142 285 Miyashita M. 337 Miyashita O. 399 Miyaura N. 144 182 183 193,316,330 Miyazaki H. 289 Miyazaki T. 52 Miyazawa J. 16 200 422 Miyoshi H. 184 Miyoshi N. 21 Mizuno K. 169,237 Mo O. 43 Mobbs J. 396 Mochalov S. S. 224 Mock W. L. 59 266 Modena G. 44 253 Modro T. A. 223 Modlhammer U. 187 Moestrone T.345 Moffitt H. R. 348 Mok K.-L. 53 Mol J. C. 119 Mole T. 146 Montanari F. 87 344 Monteiro P. M. 226 Monti S. A. 345 Moodie R. B. 223 Moody C. J. 260 Moody R. J. 145,325 Moore B. P. 353 Moore H. W. 229,268 Moore W. M. 35 163 Mooser G. 378 Moradpour A. 238 Moran T. A. 193 31 1 More K. M. 279 More P. 23 Moreau C. 205 Morecombe D. J. 426 Morehead S. R. 352 Morel J.-P. 201 Moreland B. H. 390 Moreland C. G. 16 More O’Ferrall R. A. 78 Moreshead J. 41 Morgan E. D. 8 Morgan L. O. 15 Morgan M. E. 350 Morgan R. P.,5 7 Morgan T. K. jun. 176 Mori E. 338 Mori K. 356 357 359 361 362,364 Morigaki M.,247 Morimoto C. N. 25 Morio K. 73 Morioka M.238 Morisaki M. 342 367 Moritani I. 116,144,309.318 322 Morizur J. P. 362 Morokuma K. 42,43,207 Morren G. 238 Morris D. G. 205 Morrison H. 175 Morrison J. 390 Morrison J. A. 138 196 Morrison J. F. 391 Mortenson L. E. 378 383 Morton A. A. 143 Morton J. R. 93 Morton R. A. 31 Morton T. H. 7 198 315 Mosberg H. I. 20 Moser J. C.,351 Mosher H.S. 187 Mosley J. 384 Moss B. 41 1 Moss M. O. 421 Moss R. A. 58 112 Moss R. H. M. 106 Motell E. 17 421 Moulijn J. A. 119 Mousset G. 161 162 Mouzin G. 293 Moxon P. D. 167,257 Moyes W. 40 Miiller C. 253 Mueller D. D. 16 Muller E. 186 Miiller W. 407,414 Muelly M. 233 Miinck E. 381 Muetterties E. L. 120 125 126,221 Muir D.M. 226 Muirhead H. 392 Mukai K. 241 Mukai T. 173 243 Mukaiyarna T. 141 324 336 Muke B. 280 Mukerjee A. K. 284 Mukherjee-Miiller G. 67 Mulheirn L. J. 421 Muller B. 364 Muller C. 45 240 Mulligan R. C. 412 Mumrna R. O. 348,354 Mumman R. D. 359 446 Author Index Munakata K. 352 Mundy B. P.,364 Munro M. H. G. 16 Munson B. 8 Mura A. J. 143 Murahashi S.-I. 116,122,309 Murai S. 261 264 Muraki M. 324 Murata I. 235 238 243 Murdock T. O. 119 Murray R. K. jun. 176 Murrell J. N. 37 Muschik G. M. 347 Musgrave 0.C. 229 Musierowicz S. 186 Musser J. H. 343 Musso H. 291 Muthukrishnan S. 411 Muto. A. 410 Muus L. T. 98 Muzard G. 362 Myhre D.V. 34 Myhre P. C. 224 Nachod F. C. 108 NU F. 341 Nagai Y.,122 147 Nagakura I. 317,342 Nagashima S. 122 147 Nagel J. 402 Nair V. 250 Nakadaira Y. 147 Nakagawa A. 16,200,422 Nakagawa M. 247,248 Nakai T. 330 Nakajima T. 51 Nakamura N. 240,293 Nakamura Y. 61 Nakanashi H. 132 Nakanishi A. 139 253 Nakanishi K. 209 352 377 Nakanishi N. 139,253 Nakanishi S. 243 Nakatsuji S. 247 Nakatsuka T. 140 231 282 Nakayama J. 107 Nakazawa J. 235 Nakazawa T. 243 Nambudiry M. E. N. 193,3 11 Nametkin N. S. 146 Napierski J. 106 241 291 Narang S. A. 405 Narang S. C. 224 Naruse M. 144 357 Naruto S. 279 Narwid T. A. 364,367 Nasielski-Huikens R. 274 Nathans D. 412 Natod K.,259 Natori Y.122 Natsukawa K. 26 1 Nauman R. V. 210 Nay B. 110 Neel W. W. 353 Neet K. E. 393 Nefedova M. Yu. 30 Negishi A. 358 Negishi,E. 136 143 144 145 195.222 Neidert E. 340 Neidlein R. 273 Neilson G. W. 212 Nelson D. 30 21 2 Nelson G. V. 103 Nelson J. A. 174 Nelson T. R. 309 Neoh S. B. 185,257 Nesbitt B. F. 348 356 Nesmeyanov A. N. 14 Nestle M. O. 130 Neuenschwander M. 243,245 Neumann T. 237 Neumann W. P. 149 Neurath H. 385 Neuwirth J. 355 Newman G. A. 29,34 Newmark R. A. 16 Neywick C. V. 220 Ng Q. Y. 240 Nibbering N. M. M.,7 Nicholas K. M. 130 315 Nichols P. R. 353 Nicol A. J. 271 Nimlaou K. C. 201,319,322 339,372 Niedballa U. 402 Nielsen S.F. 283 Nikiforov A. 202 Nilsson A. 156 222 Nilsson B. 216 Nir Z. 126 Nishida R. 350 Nishiguchi I. 153 154 Nishiguchi T. 122 Nishihara T. 410 Nishimura S. 409 Nishioka A. 52 Nishiwaki T. 283 Nishiyama K. 23 1 Noda L. 387 388 Nbgradi M. 282 Noguchi I. 158 Nokami J. 141 Nolan C. 385 Nolf F. 410 Noll M.,397 Nolte D. J. 350 Nonhebel D. C. 222 Nooter K. 398 Noren G. K. 195 Norman R. 0.C. 134,227 Normant J. F. 139 181 316 317,360 Norris C. L.. 215 Norris D. M. 332,355 Northrop R. C. 322 Norton J. R. 127 336 Noth H. 277 Nour T. A. 208 Nourse J. G. 200 Novoa W. B. 385 Noyori R. 301 Nozaki H. 112 144 193,231 315,357 Nozoe T. 242 Nucci L.242 Nussbaum A. L. 403,405,412 Nutter D. E. jun. 93 Nwe K. T. 267 Nyberg K. 151 157 Nyburg S. C. 26 Nye M. J. 53 Nyfeler R. 416 Nyquist R. A. 32 Oberhansli W. E. 61 250 O’Brien D. H. 10 O’Brien E. J. 30 Ocasio I. 102 O’Connor B. H. 24 Oda M. 117,243,289 O’Dea J. J. 162 Odiot S. 11 O’Dwyer M.F. 217 Oei H. A. 164 Oertel R. P. 34 Oertle K. 202 Ogata I. 121 Ogata Y. 178 Ogawa T.. 190,260 Ogilvy M. M. 66 Ogiwara J.. 116 Ogoshi H. 240 Ogura H. 201 Ogura. K. 138 Ohashi Z. 409 Ohgo Y. 122 Ohi M. 241 Ohkubo. K. 164 Ohloff G.,329 Ohoi F. 286 Ohrui H. 200 Ohsawa A. 219 372 Oine T. 251 276 Ojima I. 122 Ojima J. 247 Ojosipe B. A. 63 Oka M.15,234 Okada K. 144,226,427 Okamoto K. 85 Okamoto T. 413 Okano M. 184 Okawa M. 154 Okawara M. 267,330 Okazaki R. 263 266 Oku A. 237 Oku M.,245,280,308 Olah. G. A. 10 14 44 107 184 186,203,209,211,216 222,227,296 Author Index Olbrysch. O. 196 O’Leary B. 37,40 Olf H. G.. 35 Olin G. R. 223 Olive S. 124 Oliver J. E. 208 360 264 Ollis W. D. 64 185 233 282 Olson J. S. 389 Olson K. 405 Olsson K. 216 222 224 Olson L. 187 Omura S. 16 200,422 O’Neil. J. W. 209 Onions A. 195 Onoe A. 184 Oosterhoff L. J. 68 Oppenheimer. N. J. 409 Oppolzer. W. 61 271 312 Orahovats A. 250 Orchin M. 127 Orf H. W. 345 Orfanides N. 354 Ori. H. 238 Orlov V.V. 158 Orme-Johnson W. H.. 381 Orr G. A. 391 Orville-Thomas W. J. 41 Osborn J. A, 125 Osborne A. D. 4 O’Shea S. F. 37 Oshima K. 193,315 Osicka V. D. 397 O’Sullivan W. J. 390 Otake N. 202 Otieno S. 393 Otomasu H. 259 Otsubo T. 170 230 247 282 Otsuka S. 141 Ott W. 298 Ottenbrite R. M. 63 197 Otter R. 280 Ottersen T. 42 Ouchi S. 402 Oudet P. 396 Oudman D. 218,291 Ovchinnikov Yu. A. 206 Owens R. M.. 86 Owens W. 276 Oyler A. R. 336 Ozawa H. 237 Pac C. 169 237 Pace D. 240 Pachier K. G. R. 16 23,421 Pacifici J. A. 237 Pack G. R. 43 Paddon-Row M. N. 53 116 Padwa A. 58 112 114 176 178,249,25 1,264,276 Paik C. 226 Painter P. C. 31 Pais M. 374 Paiva A.C. M. 18 Pak s.J.. 97 Pakkanen 44,295 Pakroppa W. 414 Palke W. E. 47 Palmer J. L. 153 Palmer M. H. 40 Palmisano G. 342 Pancrazi A. 109 Pandit U. K. 14 Panzica R. P. 14 401 403 Paoletti E. 41 1 Paoloni L. 42,44 Papperman A. B. 277 Paquer D. 141,209 Paquette L. A. 67 218 219 243,244,245,280,29 1,297 301,302,307,308 Parbo H. 98 Pardi L. 242 Park B. K. 340 Parker A. J. 226 Parker F. S. 31 Parker K. A. 141 317 Parker V. D. 156 Parlman R. M. 131 322 Parrilli M.,372 Parrish R. F. 385 Parrott M.J. 93 Parshall G. W. 120 Parsons J. L. 36 Partchamazed I. 225 Partington P. 23 Partridge J. J. 367 Pasteels J. M. 353 Patane J. 188 Patara A. 223 Patchornik A.320 402 404 Patel D. J. 13 22 405 Patel M. K. 368 Patmore D. J. 128 Patoiseau J. -F. 374 Patrick D. W. 196 Patrick T. B. 116 Pattenden G. 334 338 353 Patti A. 42 Paul 1. C. 28 Paulick R. C. 16 Pawlak M.,289 345 Peacock J. W. 350 Pearce A. 148 325 Pearce D. S. 229.268 Pearce G. T. 350 355 Pearman A. J. 382 Pearson A. J. 130 Pearson H. 12 Pearson P. K. 48 108 Peat I. R. 18 Pecher J.. 238 Pechet M.M. 340,366,368 Pechman K. 410 Pedersen C. L. 275 Pedersen E. B.. 271 Pedersen J. B. 54 98 100 Pedersen L. D. 303 Pedersen L. G. 4 1,44 Pedulli G. 43 Peek M. E. 203,274 Peelan F. C. 218 Peeters. J. F. M. 381 Peeters H. 281 Peichardt C. 273 Pekary A. E.22 Pellegata R. 342 Pelter A. 137 144 145 182 183 195,313,316 Peltier D. 165 Pemberton P. W. 16,417 Penkovsky V. V. 41 Pennings J. F. M. 41 93 Pensak D. A. 345 Penswick J. R. 408 Penton J. R. 224 Peppard D. J. 364 Perekalin V. V. 245 Perin F.. 170 Perkins M. J. 91 Perlberger J. C. 65 305 Perlin A. S. 13 Pero F. 226 Perriot P. 181 316 Perry R. P. 41 1 Persoons C. J. 348 Perun T. J. 201 Pesce G. 253 Pesnelle P. 364 Pete J. P. 310 Peter M. G. 4 16 Peter W. 147 Peterlin A. 35 Peters B. A. 393 Peters D. 215 Peters D. G. 163 Petersen H. 265 Peterson D. 192 31 1 Peterson G. 345 Peterson P. E. 211,372 Peticolas W. L. 35 Petronogolo C. 41 Petrov A.A. 181 Petrova J. 316 Petrzilka M.,61 339 Pettit R. 241 Petty M. S. 33 Petty R. L. 355 Phillips G. R. 4 Phillips G. T. 419 Phillips L.. 11 16 216 417 Phillips L. R. 360 Phillips W. V. 177 Photis J. M.,244 Piancatelli G. 370 Pickard F. H. 26 Picker D. 86 343 Pickering M.,40 Pickering M.W. 110 Pickett H. M.,294 Piechucki C. 310 Piekos A. 223 448 Piepenbrock A. 185 Pierre L. J. jun. 30 Piers E. 3 17 342 Pietra F. 242 Pilipovich D. 98 Pilkiewicz F. G. 112 Pimley R. W. 350 Pinchin R. 206 Pine S. H. 233 Pinnavaia T. J. 129 Pinnick H. W. 327 Pinsky B. 82 Pipano A. 40 Piriou F. 16 200 422 Pirisi F. M. 87 Pirkle W. H. 69 Pirotta V. 413 Pisarskaya T.N. 380 Pitt C. G. 148 Pittman C. U. jun. 124 128 129,240 Pitts W. 200 Pitzele B. S. 182 316 Pizey J. S. 346 Plank D. A. 229 Platt T. 41 1 Plenchette A. 356 Pletcher D. 151 Pocker A. 385 Podgornova N. N. 245 Pohl H. H. 277 Polisky B. 412 Pollack R. M. 202 Polonsky J. 418 Pomerantz M. 295 Ponticello G. S. 257 Poole C. F. 8 Poonian M. S.,412 Pople J. A. 38 40 45 216 293 Popp F. D. 283 Poppi R. G. 348,356 Poppinger D. 40 Popplestone R. J. 195 Poradowska H. 271 Portella C. 310 Porter A. P. 228 Posner G. H. 122 313 318 34 1 Post M. L. 404 Postgate J. R. 378 Potts K. T. 108 265 Potworvorski J. A. 26 Poulin J. -C. 121 Pouliquen J.168 218 Poulsen 0.K.. 18 Pourcelot G. 185 Powers G. J. 404 Powers S. G.. 390 Prabhu K. V. 177 Pradel L. A. 390 391 392 Praeger D. 13 Prager R. H. 106 Prange T. 12 Pratt A. C. 173 Preckel M. 343 Pregosin P. S. 133 Prestegard J. H. 11 Prestien J. 10 Preston K. F. 93 Pribnow D. 413 Price J. A. 282 Price N. C. 386 387 389 Prinzbach H. 219,.252 254 265 Pritt J. R. 76 Pritzkow W. 194 Proehl G. S. 174 Ptashne M. 413 Pugmire R. J. 14,403 Pulay P. 52 Pulleyblank D. E. 414 Pullman A. 49 Pullman B. 284 Putnam T. B. 350 Pyron R. S. 53 Quast H. 252 Quattrone A. J. 19 Quigley G. J. 407 Quijano L. 354 Quinkert G.. 64 Quinney J. C. 228 Quiocho F. A.389 Rabiller C. 242 Radda G. K. 385,386 Rademacher P. 210 Radics L. 317 Radom L. 40,4 1,49 Raduchel B. 310 Radzilkowski P. 43 Raeymakers A. 410 Rai D. K. 52 Rajagopalan M. S. 368 Rakowski M. C.. 120 221 Rall R. B. 181 Ramage R. 326 Ramaiah M. 62 270 Ramakrishnan V. T. 276 Ramel A. H. 393 Ramli M. 200 Rance M. J. 62 Randall G. A. 404 Randall R. F. 393 Randerath E. 408 Randerath K. 408 Randic M. 235 Range P. 358 Rao D. V. 228 Rao V. S. 11 Raoult E. 165 Rapaport E. 409 Rappoport Z. 74 78 Author Index Rasmussen J. K.. 114 Rassat A. 50 57 Rastetter W. H. 279 Ratajczak E. 41 Ratajczak H. 41 Ratajczak H.-J. 86 Rathburn I. M. 67 Ratner M. A.178 Rattle H. W. E. 22 Ratych R. E. 176 Rauleder G. 86 Raulins N. R. 233 Rauscher S. 348 Rautenstrauch V. 195 Ravikumar P. R. 200 Read J. S. 348 Rebek J. 87 240 288 Rebell J. 286 Record K. A. F. 95 Rector D. 148 Reddy R. 4 10 Redfield A. G. 20 Redker. V. D. 394 Reece C. A, 17,421 Reed G. H. 389 Reeder R. A. 283 Rees C. W. 15,626,203,255 260.26 1,274 Reese C. B. 402 Reetz M. T. 46 203 Reeve L. 367 Regen S. L. 87 317 323 Regitz M. 112 21 1 Regnouf F. 390 Reich H. J. 192 319 327 Reichardt C. 209 Reichmanis E. 281 Reid B. R. 408 Reid D. H. 266 Reilly J. 64 Reinehr D. 196 Reinhardt C. G. 406 Reinhoudt D. N. 283 Reiss J. A. 239 Reitano M. 320 Renaud M..390 Renfrow W. B. 108 Rennekamp. M. E. 5 Renner C. A. 168 218 Renner R. 186 Renwick J. A. A. 350 Rirat B. 390 Rkrat C. 390 Resofszki G. 202 Reusch W. 368 Reuss R. H. 327 Reutov 0.A. 143 Rey M. 64,233 Reynolds W. F. 10 Reznikoff W. S. 413 Rhee W. Z. M. 148 Rhoads S. J. 233 Rhodes D. 407,408 Rhodes H. 381 Author Index Ribeiro O. 266 Rice R. 348 Rich A. 27,406,407,408,4 12 Richards B. 52 Richards C. G. 259 Richards F. M. 386 Richards K. E. 410 Richards R. E. 385 Richards R. L. 382 Richardson F. S. 200 Richardson G. 317 Richmond J. M. 244 Rickwood D. 409 Ridley D. D. 318 Riecke E. E. 285 Ried W. 244 Rieke R. D. 142,336 Rietveld G.G. A. 218 Rigaudy J. 1 1 1 278 Riley R. G. 351 360 361 Rimai L. 36 Rindorf G. 267 Rinehart K. L. 16 Rio G. 264 Riordan J. F. 389 390 Rios T.. 354 Ris C. 224 Ritchie C. D. 205 Rithstein S. M. 47 Ritter F. J. 348 351 Rivera-Ortiz J. M.. 381 Riveros J. M. 225 Rizvi. S. Q. A. 275 Rizzardo E. 366 Robbins. J. D. 292 312 Roberge. R. 11 Robert D. U. 372 Robert J. B. 13 Robert J.-L. 425 Roberts B. E. 412 Roberts B. P. 91 92 93 Roberts F. E. 206 Roberts G. C. K. 11 13 Roberts J. D. 13 19. 200 Roberts R. D. 237 Roberts S. M. 336 Robertson H. D. 413 Robertson P. J. 186 Robertus J. D. 407 Robillard. G. T. 408 Robin R. O. 398 Robin Y. 390 Robins R. K.400,401 Robinson M. 376 Robinson M.J. T. 23 Robinson P. J. 21 8 Robinson P.R. 383 Robinson S. D. 123 Roblin J. 189 Robson M.J.. 330 Rocchio J. J. 31 Ro-Choi T. S. 410 Rode B. M. 43 Roe B. A.. 408 Roderer R. 223,224 Riihle G. 377 Roelofs W. L. 348 365 Riimer M. 244 Rogers R. J. 222 Rohmer M. M. 45 Roling P. V. 227 Rolison D. 67 Roller P. 210 Romano L. J. 134 184 Romers C. 294 Rona R. J. 192,311 Ronald R. C. 139 Ronlan A. 156 Rooney J. J. 126 Roos 43,45 207 Roques B. 407 Rosasco G. J. 33 Rose I. A. 378 Rosen. M. H.. 253 Rosenberg S. 81 Rosenberry T. L. 378 Rosenblum M. 125 192 315 Rosenfeld M.N. 228 Rosenfeld S. M. 23 Rosenquist N. R. 107 Rosini G.376 Ross D. K. 18 Ross F. P. 200 355 Ross S. D. 15 1 Rosskamp G. 287 Rossmann M. G. 387 Rosso P. D. 67 172 Roth H. D. 117 Roth H. J. 257 Roth W.-D. 262 Roth W. R. 55 188 304 Rothschild. A. J. 116 147 Rottenberg M. 333 Rottman F. 405 41 1 Rouessac F. 342 Roundhill D. M.,119 Rounds T. C. 294 Rousseau R. J. 401 Roustan C. 390 391 392 Rowan R. 20,21 Rowe K. 137 145 195 Rowland A. T. 372 Rowley A. G. 107 110 222 Roy R. B. 7 Royer J. 225 Rozenblatt S. 412 Rozental JI M. 355 Ruasse M. F. 194 Rubin M. B. 209 Rubin R. J. 85 234 Rubinstein M. 320 402 404 Rubottom G. M. 335 Rudd E. 151 Ruden R. A. 276,338 Rudinsky J. A. 350 Ruchardt C. 69 303 Rummens F.H. A. 15,234 Rumney T. G. 79 Runquist A. W. 122 318 Russ R. L. 322 Russell C. R. 10 Russell J. W. 8 Russell M.E. 4 Russell S. M. 416 Rust M. K. 208 Ruston S. 193 31 1 Rutowski R. 353 Ryan J. F. 8 Rykowski A. 275 Ryono L. S. 134 227 Saari J. C. 385 Sabacky M. J. 121 Sabourault B. 146 Sachdev K. 54,304 Sadd J. S. 261 Saegusa T. 286 Saeki M.,173 Saeki S. 270 Saeva F. D. 223 Safe S. 6 Saidi M. R. 125,192,233,315 Saigo K. 336 Saito I. 258 Saito K. 173 Saito T. 269 359 Saito Y. 26 202 Saitoh T. 423 Sakai M. 73 Sakaki S. 38 Sakamoto H. 367 Sakata Y. 23 1 232 237 Sakurai H. 113,147,169 172 237 Sala A, 271 Sala E.. 13 Salahuddin S.Z. 398 Salajegheh A. 163 Salaneck W. R. 51 Saleh S. A,. 229 Salem L. 49 53 55 179 Salemink C. A. 401 Salmona G. 51 Salomon M.F. 118 286 Salomon R. G. 118 286 Saltiel J. 169 Salzmann T. N. 336 359 Sammes M. P. 269 Sammes P. G. 61,236 312 Samuel C. 273 Samuel C. J. 167 171 Sandefur L. 0..327 Sanderson R. T. 39 San Filippo J. S. 134 184 Sanger F. 412,413 Sankawa U. 423 Sanner T. 386 Sannicolo F. 258 Santine R. E. 23 Santora A. 348 Santry D. P. 37 Santucci S. 242 450 Author Index Sarantakis D. 18 Sarda P. 285 Saris L. E. 263 Sarma R. H. 404 Sarver B. E. 114 Sasaki N. 330 Sasaki. S. 366 367 Sasaki T.. 132 190 260 Satake T. 247 Satani S. 232 Sato S.202 Sato T. 25 231 Sato Y. 107 Satoh F. 427 Satoh J. Y. 372 Satoh S. 23 Sauer W. 241 Saunders J. K. 19 205 Sautiere. P. 22 Savignac P. 3 16 Savitzky G. B. 21 Sawada H. 112 Sawamura M. 367 Saward C. J. 221 289 Sawaya H. S. 3 13 Sayer J. M. 81 82 Sayigh A. A. R. 228 Scaiano J. C. 95 Scandroglio A. 279 Scarponi U. 340 Scartoni V. 194 Scettri A. 370 Schaasberg-Neinhuis 2. R. H. Schaefer H. E.. 108 Schaefer H. F. tert. 48 55 Schafer H. J. 159 364 Schaeffer T. 13 Schaffer O. 277 Schaffner K. 64,65,305 Schaller H. 4 13 Schamp N. 250 Schaub F. 183 Schaublin S. 17 Schaumberg K. 18 Schegolev A. A. 75 183 Schein L. B. 51 Scheinmann F. 182 316 Schell F. M.,11 Schellhammer C.-W.284 Schelling,J. E. 401 Schenck G. 0..175 Schenk C. 224 Schenk H. 25 Schenk W. K. 243 Schemer K. 41 I Schexnayder M. A. 176 177 Schierbeck B. 391 Schiess P. 67 Schildknecht H. 354 Schill G. 181 Schilling B. 69 Schilling P. 186 Schilling,W. 339 Schirmer R. H. 387 Schiwek H. J. 287 Schlegel H. B. 43 45 52 Schlessinger R. H. 200 Schleyer P. von R.,45,50,227 2 99 Schloter K. 239 Schlunegger U. P. 3 Schmalz T. G. 215 Schmand H. L. K. 237 Schmerling L. 225 Schmickler H. 246 Schmid G. H. 253 Schmid H. 61 67 179 233 250 Schmid P. 227 Schmidbaur H. 149 Schmidlin J. 368 Schmidt E. 231 Schmidt E. K. G. 241 Schmidt H 194 Schmidt P. G. 20 22 Schmidt R. R. 283 Schmidt T. 188 304 Schmidt U.108,202 Schmidt W. W. 317 Schmir G. L. 205 Schmitz A. 21 1 Schmitz H. 221 Schmuff N. R. 110,254 Schneider D. 349k 355 Schneider H. 355 Schneider H. J. 12 Schneider H. -W. 219 Schneider M. 299 Schneider M. P. 286 Schneller. S. W. 283 Schnoes H. K. 367 Schnyder J. 333 Schoch W. 228 Schoeller W. W. 64 298 305 Schollkopf U. 261 270 Schoenberg A. 127 Schofeld J. D. 18 Schofield K. 223 Scholl M.-J. 264 Scholl T. 57 199 Scholler D. 310 Scholz M. 41 Schomburg G. 196 Schommer M. 12 Schonbrunn A. 82 Schonholzer P. 250 Schooley D. A. 416 Schott H. 413 Schrader B. 32,34 Schrauzer G. N. 383 Schreiner K. 239 Schrock R. R. 126 Schroder R. 261 Schroeder J. 284 Schubert W. M.225 Schuchardt U.. 124 243 Schuchmann H. -P.,91 Schulte K. W. 240 Schulten H.-R. 3 Schulz G. E. 387 Schurig V. 129 Schuster D. I. 177 178 298 Schuster G. B. 217 Schuster P. 43 Schuttler R. 115 Schutzenhofer D. L. 116 Schwab J. M. 7 Schwartz,J. 127,133,317,336 Schwartz J. A. 13 Schwartz M. 208 Schwartz S. B. 359 Schwartzman S. M. 323 Schwarz A. 149 Schwarz C. 349 Schwarz H. 6 7 8 Schwarz M. 360 Schweig A. 45 52 240 253 Schwesinger R. 219 252 Scott A. I. 301,425 427 Scott J. A. 209 Scouten C. G. 195 Scriven E. F. V. 110,223,262 Scurrell M. S.. 186 Searle J. B. 229 Seaver S. S. 20 Sederoff R. R. 412 Seebach D. 136 138 164 210,319,320,322,331,332 Seeley P. J. 385 Seeman J. I. 177 Seeman N.C. 27,406,407 Segal G. 49,68 Segel I. H. 378 Seiber J. N. 17 348 Seki M. 367 Sekiguchi. A. 116 146 Sekiguchi S. 226 260 Sekine M.,402 Sekiya A. 134 Sekiya M.,326 StmCriva M.,391 Semmelhack M. F. 131 134 227 Semprini E. 41 Senda Y. 11 Seng F. 262 283 Seng Teh J. 22 Seo S. 16 17,418 Septe B. 14 Serguchev Yu. A. 194 Serve M. P.,261 Servis K. L. 13 293 Servis S. 202 Servin R.,151 Serwer P. 409 Seshadri T. P. 404 Sevilla C. L. 385 Seybold G. 184 203 241 261,263,289 Seyferth D. 130 143 147 Shabarov Yu. S. 224 Author Index Shah S. K. 192,319 Shah V. K. 379,381 Shaikh S.. 341 Shalhoub G. 40 108 Shaltiel S. 385 Shamblee D. A. 236 Shamma M. 279 Shannon J.S. 7 Shapet'ko N. N. 14 Shapiro M.,199 Sharma A. 24 Sharma D. K.S. 8 Sharma R. P.,202 Sharma S. C. 37 Sharp J. T. 107,114,236,261 279 Sharpless K. B.,196,209,323 372 Sharrocks D. N. 145 Shatkin A. J. 41 1 Shavitt I. 40 Shaw P. M.,374 Shea K. J. 217 Shearer G. O. 202 Sheats J. E. 106 227 Schechter H. 116 117 147 Shefter E. 27 Shein S. M.,225 Shellhamer D. F. 60 Shenton K. E. 127 336 Shepard K. L. 107 Shepherd J. 353 Sheppherd W. A. 272 Sheridan R. S. 12,293 Shern C. J. 393 Shetlar M. D.,399 Shiba T. 18 Shibasaki. M. 319 372 Shibata H. 410 Shibata M.,52 Shibata S. 20 423 Shibuya I. 268 Shigemitsu Y.,169 Shigemoto K. 12 Shih C. N. 190 Shih-Hsi Chu 400 Shill J. P. 393 Shillady D.D.,63 Shim S. C. 132 Shima T. 356 Shimada E. 336 Shimanouchi T. 34 Shimokawa S. 14 Shimp D. R. 210 Shin S. 127 Shinde M. B. 8 Shine H. J. 234 Shine J. 41 1 Shiner C. S. 319 372 Shinkai I. 110 270 272 Shipley G. G. 27 Shipman L. L. 40 Shirai H. 107 Shiraishi S. 422 Shizuka H. 110 Shoji Y.,242 Shold D. M.,169 Shono T. 152 154 170,324 Shorey H. H. 348 Shriver D. F. 33 Shulte K. W. 52 Shure M.,414 Sidarons L. 156 Siddall J. B. 183 Sidwell R. W. 401 Sieber J. N. 421 Sieber W. 250 Siefert J. H. 41 Siegel M. G. 23 1 282 Siegel T. M. 152 Sieiro C. 38 Sigman D. S. 378 Sih C. J. 200 Sill A. D. 279 Silveira A. 144 Silver D. M.,50 Silver. S. M.,81 Silverstein R. M.,347 348 349,350,351,355,360,361 Silverthorne W.E. 120 Silvestri M.,315 326 Simchen G. 340 Simeone J. B. 350 Simmonds R. G. 259 Simmons H. E. 203,293 Simon L. N. 400,401 Simonet J. 155 160 161 162 Simonetta H. 64 Simonetta M.,48 246 Simonneaux G. 131 Simpson T. J. 16 415 420 42 1 Sinanoglu O. 50 Singer G. 355 Singer M. F.. 398 Singh A. K. 284 Singh T. D.,83 Sin h,.U. P. 265 Sinsteimer it.L. 409 Sisti M.,416 Siverns M.,16 Skell P. S. 119 191 Skorianetz W. 329 Skuballa W. 301 Skuster J. 385 Sledzinski B. 107 Slegeir W. 24 1 Sletzinger M. 206 Sloane H. J. 32 Slobodin Ya. M. 189 Sluis G. J. 203 Slutsky J. 116 147 Small L. E. 80 Smeyers Y. G. 38 Smillie R. D. 327 Smirnov V.S. 185 Smit W. A. 75 183 Smith B. E. 378 Smith C. Z. 151 Smith D. G. 16,222 324 Smith D. H. 9 Smith D.J. 389 Smith D. J. H. 222 324 Smith E.. 390 Smith H. O. 412 Smith 1. C. P. 18 Smith J. R. L. 261 Smith K. 136 137 144 145 183 195,316 Smith L. 148 Smith L. H. 409 Smith L. R. 124 128 Smith P. 94 148 Smith R. A. 118 Smith R. A. G. 113 Smith R. A. J. 138 342 Smith R. G. 208,347,360 Smith S. 27 Smith S. R. 199 Smith T. N. 195 Smith V. F. 205 Smithson L. D. 147 Smolanoff J. 249 353 354 Snatzke G. 209 Sneeden R. P. A. 119 Snell E. E. 378 Snell W. 229 Snider B. B. 174,371 Snoble K. A. J. 228 Snyder E. R. 385 Snyder G. H. 21 Snyder J. P. 69 SO,Y.-H. 157 Sobell H. M.,27,406 Soll D.,409 Soffer R.L. 378 Sogah Y. 204 Sohma J. 14 Sohma K. 184 Soja P. 334 Soling H. 267 Solka B. H. 4 Solka V. N. 13 Solodar J. 121 Solomon M.D. 115 Somanathan R. 255 Sombroek J. 246 Sommer L. H. 146 Sondheimer F. 246 247 281 Sonnet P. E. 208 347 348 350,356,360,364 Sonoda N. 261 264 Sonogashira K. 133 135 181 Sood A, 197 Sood H. R. 279 Sorarrain 0.M. 41 Sorrell T. 274 Sorriso S. 41 Sousa. L. R.. 323 Southern E. M. 412 Sowa T. 402 Sower L. L. 348 355 Sowinski. A. F. 134 184 Spangler D. 40 5 1 245 Spear R. J. 184.21 1,216,296 Spector L. B. 391 Spencer H. K. 267 Spencer T. A. 174 Spessard G. O. 338 Spialter L. 147 Spiegelman S. 410 Spiro T. G. 36 Sprague J.T. 294 Sprinzl M. 2 1,408 Squillacote M. 12 293 Squires T. G. 3 17 Srinivasachar K. 169 220 Srinivasan P. R. 13 Srinivasan R. 170 Srivastava A. K. 37 Srivastava K. C. 273 Staddon B. W. 355 Staemmler V. 37 Stahl. D. 410 Staley R. H. 4 Staley S. W. 70 Stallberg-Stenhagen S. 355 Stalmans W. 386 Stammers D. K. 392 Stang P. J. 1 15 186 317 Stanovnik B. 274 Stanton J. L. 327 370 Stanton R. E. 50 Staral J. S. 216 Starks C. M.,86 Starner 1. J. 174 Starnes W. H. jun. 229 Staron T. 202 Starratt A. N. 368 Staten R. T. 348 Staunton J. 420 425 Stavaux M. 276 Steck W. F. 348 Steenken S. 91 Stefani F. 41 Stein G. 168 Steiner R. P. 69 Steinman D. H. 182 316 Steitz T. A. 392 394 41 1 Stejny J.35 Stellman S. D. 25 Stellwagen E. 387 Steltner A. 78 Stelzer O. 49 Stemke J. E. 315 Stenhagen E. 355 Stepanova I. P.,224 Stephanatou J. S. 282 Stephenson L. M.,61 197 Stephenson R. W.. 62 Steppel R. N. 261 Sterba V. 226 Sterling J. J. 341 Sternbach D. D. 323 Sternbach H. 408 Sternerup H. 151 Steuber F. W. 67 Stevens R. D. 94 Stevenson G. R. 101 102 Stevenson P. E. 209 Stewart A. 126 Stewart J. C. M. 402 Steyn P. S. 16,421 Stick R. V. 376 Stiegler. P. 410 Stilbs P. 13 Stiles P. J. 41 Still I. W. J.. 277 Stilla E. 44 Stille J. K. 127 Stirling C. J. M. 77 Stockis A. 127 Stoddart J. F. 204 282 Stoffel W. 19 Stohrer W.-D. 64 Stojanac N. 197 229 Stojanac Z. 197 229 Stokkink E.350 Stoodley. R. J. 206 284 Stork G. 138 330 331 362 Storr R. C. 62 105 255 Stout D. M. 268 Stout M. G. 401 Strach S. J. 103 Stralow M. 318 Strand G. 320 402 Strandholm J. J. 392 Stransky W. 358 Strathdee R. S. 279 Straus N. A. 412 Strauss H. L. 294 Strauss J. U. G. 342 Strauss M.J. 28 1 Streeter D. G.. 401 Strege P. E. 130 Streith. J. 270 Streitwieser A. jun. 45 Strohmeier W. 120 Strozier R. W. 63 Sturm W. 246 Sturtevant J. M.,406 SU C.-W. 83 Su,H. C.. 348 356 Suau R. 174 Subrahmanyam C. 182.313 Subramanian E. 25 Subramanian L. R. 72 Suda T. 366 367 Suddath F. L. 407 Suenram R. D. 217 Suerra M.;43 Sugano H. 21 Sugdowdz G. 7 Suggs. J. W. 269,309,323,326 sugi Y.,127 Sugihara Y.,235 Sugimoto A.366 367 Sugimoto K. 413 Sugisaki H. 413 Suguira M.,13 Author Index Suhr H. 287 Sukenik C. N. 85 Sullivan G. R. 187 Sumimoto M. 352 Summers D. B. 155 Sumper M.,410 Sundberg R. J. 110,259 Sundby F. 18 Sunthankar S. V. 376 Surridge J. H. 142 Suschitzky H. 110 262 Sussman J. L. 407 Sustmann R. 69 Sustmann S. 69 303 Susuki A. 330 Suter. C.. 364 Suther D. J. 118 Sutherland D. S. 116 Sutherland. I. O. 64. 233 Sutherland J. K. 6 1 3 12 Suwinski J. W. 269 275 Suzuki A. 144 182 183 193 316,367 Suzuki H. 128,224 Suzuki K. 228 326 Suzuki M. 107 138,366 Suzuki Y. 134 Svec H. J. 7 Svendsen A. 200 Swain C. G. 106 222 227 Swain M. L. 272 Swaminathan K. 145 Swenton J.S. 67 172 399 Swierczewski C. 120 124 191 194 Swigar A. A. 350 Swinborne-Sheldrake R. 39 235 Sykes B. D. 20 2 1,23 Symons M. C. R. 207,212 Syrtsova L. A. 380 Tabata M. 144 183 316 Tabb D. L. 31,35 Taber A. M. 43 Tabusa F. 255 259 Tabushi I. 231 Tacconi G. 283 Tachi K. 122 Taddei F. 215 Tadjer. A. 52 Tae-Kyj Ha 41 Taft R. W. 45. 83 Tagami H. 144 193 Taillefer R. J. 205 Tait B. S. 262 Tait J. C. 90 Takabe K. 195,268 Takagi K. 178 Takahashi H. 259 Takahashi K. 242 Takahashi N. 422 Takahashi S. 134 229 Takahashi T. T. 372 Author Index 453 Takamuku S. 113 172 Takanami M. 4 13 Takaoka S. 106 Takaya H. 301 Takaya T. 108,268 Takegami Y. 131,324,325 Takehira Y.187 Takei S. 287 330 Takeshima H. 16 200,422 Takeshita H. 242 Takeshita T. 367 Takeuchi H.. 34 Takeuchi S. 122 Takeuchi Y. 284 Talman E. 35 1 Tam J. C. L. 64 f 12 Tam S. Y.-K., 401 Tamaki Y. 348 Tamao K. 140 145,147,231 Tamaru Y. 138 324,331 Tamburin H. J. 303 Tamm Ch. 425 Tamura M. 238 Tamura S. 422 Tamura Y. 12 255 259 Tan T.-S. 242 Tanaka H. 159 Tanaka J. 195,268 Tanaka K. 43 139,253 Tanaka M. 121 132 Tanaka S. 193,357 Tanaka T. 342 Tanaka Y. 367 Tang D. 4 14 Tang S.-Y. 30 Tanida J. 12 Tanigawa Y. 322 Taniguchi M. 16 Tano K. 383 Tanswell P. 391 Tardivel R. 152 Tarhan H. O. 49,233 Tasumi M. 34 Tatsuoka T. 235 Tausta J. C. 113 Taylor D. S. 110 Taylor E. C. 146 228 Taylor G.A. 274 Taylor G. F. 291 Taylor R.,140 225 245 Taylor R. T. 219 291 Taymaz K.. 66 Tazuke S. 237 Teich N. M. 398 Teitei T. 168 Telang S. G. 376 Teller. S. R. 259 Tengo J. 351 Tennant G. 271 Teratake S. 148 ter Borg A. P. 67 Terem B. 223 Terlouw J. K. 4 Ternai B. 18 Terrosian E. 390 Terwilliger D. T. 8 Tessmer G. W. 385 Tette J. 348 Tezuka T. 243 Thalmann A. 202 Thea S. 226 Theil W. 46 56 Thierry J. 374 Thies R. W. 301 Thijs L. 21 1 338 Thiruvengadam T. K. 276 Thomas C. 15 261 Thomas C. B. 134 146 224 227 Thomas D. 255 Thomas G. J. jun. 33 36 Thomas J. O. 396 Thomas K. M. 27 Thomas M. T. 277 Thomas P. J. 77 Thomas R. 421 Thomas T. D. 199 216 Thommen W.341 Thompson A. C. 353 Thompson D. J. 129 Thompson M. D. 283 Thompson M. J. 224 Thompson,S. T. 387 Thompson T. B. 228 Thompson,W. R. 355 Thomson J. W. 389 Thomson M. L. 263 Thoren S. 155 Thorneley R.N. F. 380 381 Thornton J. 13 403 Thorogood P. B. 114 Thulin B. 231 Thuomas K. 44 Tideswell J. 193 31 1 329 Tidwell T. T. 95 198 Tigler D. 280 Tikhonov A. Ya. 273 Tilak B. D. 279 Timko J. M.,204 228,282 Timmons R. B. 97 Timms P. L. 119 Tinoco I. jun. 406 Tirpack J. G. 228 TiSler M. 274 Tissot P. 155 Titami K. 385 Titz M.,225 Tkachuch R. D. 19 Tobias L. 412 Tobin M. 172 Tobinaga,S. 158 Toda F. 187 241 Toke L. 279 Tohda Y. 133 135 181 Toi H. 309 Tokoyama T. 247 Tolbert L.M.,171 Tomasi J. 4 1 Tomassini T. 200 Tomi K. 324 Tominaga M. 356 Tomioka H. 107 Tomita Y.. 16 17 418 Tomoskozi I. 317 Tonelli A. E.. 405 Tonnis J. A. 322 Tooke P. B. 31 Toome V. 204 Toone T. W. 223 Torchia D. A. 19 Tordeaux M. 12 Torgerson R. L. 351 Tori K. 12 16 17,418 Torii S. 159 Toros S. 121 Torrence P. F. 405 Toru T. 201 339 342 Toshima N. 173 Tosteson D. C. 14 Toube T. P. 6 Touboul E. 165 Toullec J. 80 Toupet L. 268 Tourwe D. 14 Townsend C. A. 57 199,427 Townsend D. E. 169 Townsend L. B. 14,401,403 Toyoda T. 232 Toyo’oka T. 107 Traeger J. C. 242,295 Traitler H. 310 Tran-Dinh S. 13 Traynor S. C. 6 Tremblay J. P.-A,,212 Tremont S. J. 175 Tremper A. 249 Trifunac A.D. 98 Trogg M. D. M. 409 Trinajstic N. 50 235 Trivedi L. D. 161 Trocha-Grimshaw J. 162 Troll T. 160 Tron L. 386 Trost B. M. 115 130 138 238,324,327,33 1,333,336 337,343,346,359,363,368 370 Trueblood K. N. 27 Truesdale,L. K. 196,326,377 Tsai C. C. 27,406 Tschinkel W. R. 353 Ts’o P. 0.P. 21,405 Tsuchihashi G. 138 335 Tsuda T. 286 Tsui F.-P. 8 108 Tsuji T. 12 Tsukanaka M. 112 Tsunetsugu J. 242 Tsushima T. 12 Tu,J.-I. 386 Tucker J. N. 198 Tufariello J. J. 309 Tuinman A. 369 Tukada H. 172 Tumlinson J. H. 348 Tunds P. 344 Tunemoto D. 328 358 Turchi I. J. 46 56 283 Turnball K. W. 415 Turner D. L. 19 Turner G. L. 379 Turner W. V. 69 Turro N. J. 49 53 168 172 217,218 Tursch B.353 Tuszynski G. P. 82 Tuzimura K. 205 Tweddle N. J. 262 Tyrlik S. 309 Ubik. K. 353 Uccella N. 5 Uchida K. 144 Uchida T. 410 Uchida Y. 127 Uebel E. C. 350 Uebelhart P. 250 Ueda M.,144 Ueda T. 279 Uemura S. 184 Ueno Y. 267 Uetrecht J. P. 215 Uhlenbeck 0.O. 404 Uhm S. J.. 142 336 Uijttewaad A. P. 31 1 Ulrich H. 228 Ulrich P. 138 329 Umeda I. 301 Umemoto T. 231 232 237 Umen M. J. 341 Umeyama H. 42 Umpleby J. D. 124 194 Underhill E. W. 348 Undheim K. 270 277 Ungewickell E. 410 Ung Hong Ly 374 Uppstrom B. 264 Urry D. W. 21 Usgaonkar R. N. 272 UskokoviC M.R. 366,367 Usui M.,336 Utawanit T. 360 Utimoto K. 144 315 357 Utley J. H. P. 163 Uwaydah I. M. 51 245 Uziel M.,408 Vakenti J.348 Valenta Z. 197 229 Valkovich P. B. 235 Van Alsenoy C. 38 van Asten ,J. J. A. 203 van Bekkum H. 15 van Berkel T. J. C. 392 Van Binst G. 14 van Boom J. H. 402 van de Graaf B. 4 Van de Kerckhove J. 410 Vandenberghe A. 410 van den Brock P. J. 272 van den Heuvel C. G. 7 Van der Avoird A. 44 van der Gen A. 193,311 Vanderhoeff L. N.,379 Vanderlinden P. 170 van der Lugt W. Th. A. M. 68 van der Plas H. C. 275 Van de Sande C. C. 4 7 van de Sande J. H. 404 van Dijk J. M. F. 41. 93 Van Duyne R. P. 33 Van Ende D. 192,252 Vanest J. M. 238 van Haard P. M. M. 211 338 Vanier N. R. 79 van Knippenburg P. H. 41 1 van Leusen A. M. 14 Van Montagu M. 410 van Pelt F. G. 398 van Rossen A.R. 381 van Straten J. W. 218 van Tamelen E. E. 293 Van Thoai N. 390 391,392 Van Wazer J. R. 37 Varkony T. H. 319 Varsanyi G. 34 Varshavsky A. J. 397 Vasil’eva 1. A. 189 Vastag S. 12 1 Vaughan Griffiths D. 12 Vdovin V. M. 146 Vecera M.,225 Vedejs E. 65 312 Vederas J. C. 425 Veillard A. 39 Venanzi L. M. 133 Veniard L. 185 Venier C. G. 114 Vergoten G. 34 VerhC R. 250 Vermeer H. 45 149,253 Verrneer P. 186 Vermeeren H. P. W. 203 Vernet R. B. 170 Verwiel P. E. J. 351 Vetter W. 6 Vibet A. 209 Vick K. W. 348,355 Vierhapper F. W. 11,272 Vijttewaal A. P. 193 Vil’davskaya A. I. 181 Vilhelmsen K. 18 Villani M. C. 41 Villiersas J. 139,181,316,317 360 Vincent E.-J., 51 Author Index Vincent F.152 Vincent I. G. 37 Vincow G. 102 Vineyard B. D. 121 Vining L. C. 16 Vinograd J. 414 Vinson S. B. 351 Viscontini M.,351 Viswamitra M. A. 404 Vite J. P. 350 Vittorelli P. 67 Vitullo V. P. 202 Vleggaar R.,16.421 Vogeli G. 409 Vogtle F. 230 232 283 Voerman S. 348 Vogel E. 155 237. 246 Vogel T. M. 108 Voigt E. 21 1 252 Voigt R. F. 121 Volckaert G.. 405 410 Volkmer P. 37 Vollhardt K. P. C. 123 185 221,289 Volodarsky L. B. 273 von der Haar F. 2 1,408 Vonderheid C. 138 von Endt D. W. 208 Von Gross B. 244 Von Lehman T. 297 von Minden D. L. 409 von Sonntag C. 9 1 von Zabern I. 387 von Zelewsky A. 103 Vorbruggen H. 286 310,402 Voronenkov V. V. 191 Vosberg H.-P. 414 Vose C.W. 369 Vostrowsky O. 356; 358 359 VrkoE J. 353 Vyazankin N. S. 148 Wada E. 330 Wada Y.,127 Waddell W. H. 168 Wade J. J. 61 Wadhams L. J. 353 Wadt W. R.. 46 Waelder S. 20 Wagemann W. 246 Wagner K. 262 Wagner R. W. 52 Wagner T. E. 22 Wahl G. H. 106 Wahlgren U. 48 Wakelin L. P. G. 407 Wakselman C. 200 Walch S. P. 39 210 Walker G. C. 404 Wallace T. W. 236 Walsh C. T. 391 Walsh K. A. 385 Walsh R. 118 Author Index Walsh T. D. 172 Walter. J. A.. 16 Walter R. 18 Walther W. 6 Walton D. R.M. 181 Walton J. W. 55 Wan J. K. S. 98 Wang A. H. J. 407 Wang C.-L. J. 138 321 Wang D. K. W. 121 Wang J. C. 414 Wang T. C. 80 Wang Y. C. 217 Waniu M. C.. 148 Ward D. C.399,405 Ward J. S. 302 Ward M. A. 114 Ward R. R. 167 257 Ware R. S. 158 Ware W. R. 169 Waring M. J. 407 Warren C. H. 34 Warren P. J. 78 Warren S. 191 311 346 Warrener R. N. 53 254 Warshel A. 52 Washburn N. N. 105 Washtien W. 82 Wasserman H. H. 324 340 Wasylishen R.E. 10 Watanabe Y. 131 324 325 Waters J. A. 405 Watkins B. F. 153 164 Watson C. A, 352 Watson D. 205 Watson H. C.,392 Watson J. W. 83 Watt D. 169 Watt D. S. 324 Watt G. D. 379 Watt R. A. 61 312 Watts D. C. 388 390 Watts G. B. 93 Watts W. E. 120 Weavers R. T. 247 Weber E. 283 Weber H. 66 306 Weber H. P. 6 1 3 I2 Weber W. P.. 235 Webster 0.W. 272 Webster R. G. 266 Weeks P. D. 140 208 Weese G. M. 4 Wege D. 243 Wegener G.55 Wehman A. T. 130 Wehner G. 138 Wei C.-M. 41 1 Wei C.-N. 97 Weidenbruch M. 147 Weigel L. O. 208 360 Weigele M. 204 Weinburger A. J. 408 Weingarten L. 208 Weinkauff D. J. 121 Weinreb S. M. 61 156 312 320 Weinstein H. 49 Weinstock L. M. 206 Weintraub H. 396 Weintraub P. M. 279 Weiss. D. S. 230 Weiss R. 171 217 239 Weiss R. A. 398 Weissberger E. 127 Weissman B. A. 85 Weissman C. 398 Weissman S. M. 41 1 Weith H. L. 413 Welch J. 107 227 Weller T. 47 Wells D. 168 Wells P. P. 175 Wells R. D. 412 Wels C. M. 425 Welti D. 22 Welty P. K. 129 Wemmer C. 208 Wendell P. C. 392 Wendt H. 157 Wenkert E. 11 Wennerstrom H. 43 207 Wennerstrom 0..23 1 Wessels P. L. 16 23,421 West C.T. 147 West J. W. 202 West R. 178 230 Westenberg A. A, 97 Westerman P. W. 10 16,44 Westhead E. W. 391 Westmoreland D. G. 20 Weston J. B. 223 Westphal Y. L. 67 Westwood F. W. 286 Wetmore R. 49 . Weustink R. J. M.. 278 Wexler. A. 399 Weyerstahl P. 237 336 Weyler W. jun. 229 Whangbo M. H. 45 Wheeler D. M. S. 172 Wheeler G. L. 242 Wheeler J. W. 208 350 351 White D. N. J. 294 White D. R. 320 White J. D. 312 White P. S. 25 White R. E. 206 Whitham G. H. 203 Whiting M. C. 76 Whitlock H. W. 16 Whitman D. R.. 37 40 Whitman P. J. 115,427 Whittaker G. 244 Whitten C. E.. 333 341 Whitten J. L. 44 295 Wibberley D. G. 257 Wicha J. 367 Wickens M. P. 404 Widdowson D. A. 415,425 Widiger G.N. 319 Widmer U. 67 Wiech G. 64 Wieland P. 57 Wielesek K. A. 41 Wielesek R. 229 Wielesek R. A, 206 Wiersum U. E. 264 Wieser K. 256 Wife R. L. 281 Wigfield D. C. 66 Wightman R. H.,246,28 1 Wilcox C. F. jun, 215 235 296 Wilcsek R. J. 145 Wilde H. 179 270 Wilke. G. 119 Wilkins. A. L. 371 Wilkins J. M. 341 Will R. A, 420 Willcott M. R. 55 67 Willer R. L. 11 Williams A. 78 205 Williams D. C.. 427 Williams D. H. 6 7 8 Williams D. L. H. 235 Williams G. R. J. 42 Williams J. E. jun. 45 Williams R. J. P. 391 Williams R. M. 145 Williams R. N. 353 Williams T. J. 20 403 Williamson A. D. 8 Willison K. R. 380 Willoughby T. V. 25 Willy W. E. 193 Wilshire C. 244 Wilson C. A. 337 Wilson J.S. 384 Wilson K. E. 338 Wilson M. S. 404 Wilson N. H. 107 118 Wilson S. R. 360 Wilt J. W. 96 Wiltshire H. R. 425 Winkler F. K. 27 Winkler H. U. 8 Winterfeldt E. 181 Wipke W. T. 50 125,299 Wisowaty J. C. 20,403 Witkop B. 405 Witkowski. J. T. 401 Wittig G. 228 Wittlin F. N. 406 Woese C. R. 410 Wohlfeil R. 399 Wojtkowski P. W.. 347 Wolf A. D. 116 147 Wolf H. 409 Wolf J. F.. 45 Wolfbeis 0.. I19 Wolfe S. 45 52 Wolinsky J. 363 Wollenburg R. H. 148 Wollowitz S. 250 Wolochowicz I. 309 Wolovsky. R. 126 Woltermann A. 280 Wong C. M. 358 Wong C. S. 132 Wong J. T. F. 378 Wong P. K. 127 Wong S. 98 Wood D. E. 212 Wood D. L. 355 Wood W. F. 349,353 Woods G. F. 375 Woods T.S. 283 Woodward R. B. 55 Woolfson M. M. 25 Woolhouse A. D. 255 Woollard. J. 271 Wolley P. 82 Wooten J. 21 Wormer P. E. S. 44 Worth G. T. 41 Woznow R. J. 197,229 Wright B. W. 363 Wright J. L. C. 16 415 Wright M. J. 192 315 Wright P. W. 193 31 1 Wright R. H. 355 Wrobliewski A. 186 Wu M. 397 Wu R. 405 Wudl F. 228 Wulff G. 377 Wurziger H. K. W. 426 Wynberg H. 218? 264,291 Yabe A. 112 Yadav J. S. 52 Yamabe S. 42 Yamabe T. 43 Yamada H. 110,231 Yamada K. 182,316 Yamada M. 359 Yamada S. 366,367 Yamada Y. 110,211,222 Yameguchi H. 52 Yamaguchi Y. 229 Yamamoto G. 261 Yamamoto H. 193 201 315 338,357 Yamamoto K. 122 238 Yamamoto S. 116 Yamamoto Y. 132 144 149 309,318 Yamashita A.184 Yamashita M. 131 138 324 325 Yamdagni R. 80 Yanez M. 43 Yang N. C. 169,220 Yang S. L. 17 421 Yano T. 122 Yanofsky C. 41 1 Yansura D. G. 405 Yarger R. G. 349 Yarkony D. R. 55 Yarwood A. J. 67,230 Yasuda A.. 193,357 Yasuhara A. 247 Yatagai H. 144 Yates K. 48 116 Yates M. G. 379 380 381 Yates P. 61 64 112 Yates R. L. 50 64 372 Yavari I. 294 Yeager R. L. 43 Yeshurun A 190 Yokono T. 14 Yonce C. E. 348 Yoneda H. 85 Yoneda S. 267 Yonemitsu O. 279 Yoshida M. 107 Yoshida S. 422 Yoshida T. 144 145,233 Yoshida Z. 240 267 Yoshifuji M. 131 227 Yoshihiro. K. 122 147 Yoshimura J. 122 Yoshimura K. 172 Young D. 124 Young D. W. 426 Young K. 16 Young M. A. 406 Youngs D.S. 307 Ysebaert M.,4 10 Author Index Yu S. H.. 209 Yu T.-C.,30 Yurtsever E. 4 1 Yushima T. 348 Zablen L. 410 Zahradnik R. 52 Zak H. 202 Zamecnik P. C. 409 Zander G. S. 8 Zanno P. R. 352 Zatorski A. 344 Zavelovich E. B. 14 Zeb M. A. 216 Zecchi G. 261 271 279 Zeder D. C. 230 Zeisberg R. 15 Zeitoun Y. 390 Zeller K.-P. 115 265 Zens A. P. 20,403 Zerner M. J. 37 Zhdanova M. P. 275 Zhukov V. P. 41 Ziffef H. 177 Zimmerman H. E. 57,59,17 1. 177 Zimmerman R. A. 410 Zimmerman S. 87 Zimmern D. 41 1 Zinder N. D. 409,412 Zlotogorski C. 291 Zollinger H. 106 224 227 Zon G. 8 108 Zoran A. 291 Zoretic P. A. 334 345 Zsindely J. 61 233 Zubkov V. A. 41 Zubrick J. W. 340 Zuckerman J.J. 148 Ziicher C. 181 Zumft W. G. 383 Zuniga Juaristi G. 11 Zurawski B. 40 Zvezdina E. A. 275 Zvilichovsky G. 242 Zwanenburg B. 21 1,338 Zweifel G. 136 182 313

ISSN:0069-3030    

DOI:10.1039/OC9757200429

出版商:RSC    

年代:1975

数据来源: RSC