2 Physical Methods and Techniques Part (ii) Nuclear Magnetic Resonance Spectroscopy By L. PHILLIPS Chemistry Department Imperial College of Science and Technology London SW7 2AY 1 Introduction This Report as last year is a highly selective review of areas of n.m.r. which are of continuing or growing importance to organic chemists. Inevitably therefore it concentrates upon applications of 13Cn.m.r. Fourier Transform technique selective T,measurements and high-field ’H n.m.r. Many important areas such as ‘other nuclei’ studies of molecules in liquid-crystaI solvents and structure determination by routine methods have been ignored although the author is not blind to their merits! 2 AppIications of ”C N.M.R. Relationships between Structure and Shielding.-These are fundamental to the application of the technique in many fields.In the first volume’ of the important new series ‘Topics in Carbon- 13 N.M.R. Spectroscopy’ there are excellent chapters covering the theory of 13Cchemical shifts and substituent effects. Maciel comments in Chapter 2 of this work,that he ‘hasheard talks introduced with a phrase equivalent to ‘now that I3Cchemical shifts are well understood. . .’ Such speakers have either grossly misjudged the situation or eise have been keeping one of the world’s best guarded secrets!’ The past year’s literature has done little to change this situation aIthough an empirical approach has often proved helpful. There is still widespread faith in a simple relationship between chemical shift and charge density.An examination* of allyl pentadienyl and arylmethyl carbanions suggests a linear relationship to .rr-electrondensity,of 160ppm. per electron. This agrees with an earlier value3 but is twice as large as a recent suggestion for non-ionic specie^,^ which lends weight to Maciel’s assertion that undue importance should not be placed on the values of such slopes.5 Substituent effects in 4-substituted phenylacetylenes,‘ isothiazoles,’ and couma-rim’ have all been examined in this light as have chemical shifts in ionic spe~ies.~”’ I ’Topics in Carbon-13N.M.R.Spectroscopy’ ed. G. C.Levy Wiley-Interscience New York 1974. D. H. O’Brien A. J. Hart and C. R.Russell J. Amer. Chern. Soc. 1975,97,4410. G. A. Olah and G. D. Mateescu J. Amer. Chem. Soc. 1970,92,1430.G. E.Maciel J. L. DalIos R.L. Elliott and H. C. Dorn J. Amer. Chern. Soc. 1973 95 5857. G. E. Maciel ref. 1 p. 74. D.A. Dawsoo and W. F. Reynolds Canad.J. Chem. 1975,53,373. ‘R.E.Wasylishen T.R.Clem and E.D.Becker Canad.J. Chcrn. 1975,53,596. 8 H. Giinther J. Prestien and P. Joseph-Nathan Org. Magn. Resonance 1975,7,339. G. A. OIah P.W.Westerrnan and D.A. Forsyth J. Amer. Chern. Soc. 1975,97,3419. lo E. Dradi and G. Gatti J. Amer. Chem. Soc. 1975 97. 5472. 10 Part (ii) Nuclear Magnetic Resonance Spectroscopy Martin et a/.'' have examined in more general terms the theoretical and empirical calculation of 13C chemical shifts in terms of electronic distribution. In an important paper Roberge and Fliszar" analyse the relationship between 13C chemical shifts and charge-density distributions in cyclohexane and methyl- substituted cyclohexanes.All observed effects are accommodated by the relation- ship 8 = -237.1qc+242.64 where qcis the net charge density (ab initio) on a particular carbon. It is not necessary to consider extra terms for 'sterically crowded' carbons such as the 3-carbon in axial methylcyclohexane. With cyclopropanes such a simple relationship does not hold. Particular attention has been given to the influence of substituents upon carbon shielding at y positions and the idea of a y gauche sterically induced high-field shift is of continued importance in conformational and assignment problems (e.g.ref. 13). It was pointed out last year14 that y truns effects were also important and often paralleled y gauche shifts casting some doubt on their steric origin.This is emphasized by work on 2-substituted bicyclo[3,3,l]nonan-9-0nes'~where y truns high-field shifts of a magnitude comparable with y gauche are observed. Eliel et have examined the universally upfield shifts caused by N 0,and F atoms in a y antiperiplanar relationship to the observed 13C nucleus and it always appears to be significant unless the substituent is at the bridgehead of a bicyclic compound. Elsewhere Eliel" points out an interesting parallel between substituent effects upon 'H and 13C in analogous y positions with respect to substituents. Usually they are interpreted in quite different terms but the implication is that they have a common origin. Batchelor'* points out (in agreement with the assessment in last year's Rep~rt'~) that the theory of the steric origin of y gauche high-field shifts cannot account for low-field S effects in sterically crowded situations.He suggests that in such close proximity the second-order electric field influence of fluctuating dipoles (i.e.van der Waals interactions) may be decisive. In keeping with his earlier assessment of the electric field origin of chemical shifts induced by remote dipolar substit~ents,'~ this same author further develops the theory2* and because of the orientation dependence of electric fields suggests an application to conformational analysis. He also applies these ideas to 13C protonation shifts in amines carboxylic acids and amino-acids.2' Conformational Analysis.-A novel method for observing n.m.r.spectra of high-energy conformations in systems with barriers to conformational interconversion as G. J. Martin M. L. Martin and S. Odiot Org. Mugn.Resonance 1975,7 2. l2 R. Roberge and S. Fliszar Cunud. J. Chem. 1975,53,2400. l3 Y. Senda J. Ishiyama and S. Imaizumi Tetruhedron 1975,31 1601. l4 R. B. Jones and L. Phillips Annual Reports (B) 1974,26. lS A. Heumann and H. Kolshorn Tetrahedron,1975,31 1571. l6 E. L. Eliel W. F. Bailey L. D. Kopp R. L. Willer D. M. Grant R. Bertrand K. A. Christensen,D. K. Dalling M. W. Duck E. Wenkert F. M. Schell and D. W. Cochran J. Amer. Chem. SOC.,1975,97,322. l7 E. L. Eliel V. S. Rao F. W. Vierhapper and G. Zuniga Juaristi Tezruhedron Letfers 1975 4339. J.G. Batchelor J. Mugn. Resonance 1975,18 212. l9 J. G. Batchelor R. J. Cushley and J. H. Prestegard J. Org. Chem. 1974,39 1698. 2o J. G. Batchelor J. Amer. Chem. Soc. 1975,97 3410. 21 J. G. Batchelor J. Feeney and G. C. K. Roberts J. Mugn. Resonance 1975 20 19. 12 L.Phillips small as 30 kJ mol-’ has been described.22 A high-temperature equilibrium in which high-energy conformations become significantly populated is set up and then frozen by cryogenic deposition under high vacuum. The power of the method is illustrated by the direct observation using i.r. techniques of the twist-boat confor- mation of cyclohexane; the n.m.r. study of this system is presently under way.23 A limitation on the use of 13C n.m.r. as a tool for studying conformational rate processes at low temperatures is the absence of a suitable ‘Chemical Shift Ther- mometer’ as widely used in ‘H n.m.r.Two answers to this problem have now appeared. One makes use of the great linewidth variation (in the range 0-80 “C) in the 13Cspectrum of furfural due to the conformational rotation about the ring- aldehydic bond;24 the other ulitizes the temperature dependence (in the range -80 to +40 “C) of the pseudo-contact-shifted 13C carbonyl resonance of acetone in the presence of the Yb(f0d)3 shift reagent.25 When a conformational equilibrium may be ‘frozen out’ at low temperatures 13C n.m.r. offers a very powerful tool for estimating free-energy differences. This has been exploited by Booth for example with some N-substituted decahydro-quinolines..26In such circumstances spectral assignment is often straightforward and the free-energy difference may be obtained directly by integration of the relevant signals.In an averaging situation the observed spectral parameters must be dissected into different contributions from the constituents of the equilibrium and it is necessary to characterize properties which are ‘conformationally dependent’. Much use is made of high-field y gauche effects as mentioned above; for example in six-membered cyclic sulphoxides with the oxygen atom axial the 3-carbon resonance is 7.5 p.p.m. to high field of the analogous equatorial situation.*’ In these compounds the 2-carbon (p to 0)also shows a high-field shift for the same structural change of 5.5 p.p.m.; this is rationalized in terms of intramolecular electric field effects.The same properties of the sulphoxide group have been used to show that conversion of penicillins into their sulphoxides produces a conformational change.28 Pearson believes that the upfield shift in the I3CH3 resonance of toluenes on ortho-methylation is a classical y-gauche (in this case actually syn) effect. Further alkylation of the substituent sometimes increases and sometimes decreases the shielding due to the ‘steric’ origin of the ‘6 effect’ which is to low field; by balancing the two together the conformational behaviour of the u-alkyl substituent may be deduced. 29 Traditionally coupling constants have given the most useful conformational information; one- two- and three-bond couplings between ‘H and 13Chave all been used in this way.Cantacuzene et al.have reported the stereochemical dependence of ‘J(CH) in a-halogen~-ketones,~’ while two papers demonstrate the importance of 22 F. A. L. Anet and M. Squillacote J. Amer. Chem. SOC.,1975,97,3243. 23 M. Squillacote R. S. Sheridan 0.L. Chapman and F. A. L. Anet J. Amer. Chem. SOC.,1975,97,3245. 24 S. Combrisson and T. Prange J. Magn. Resonance 1975 19 108. *5 H. J. Schneider W. Freitag and M. Schommer J.Magn. Resonance 1975,18 393. H. Booth and D. Vaughan Griffiths J.C.S. Chem. Comm. 1975 111. 27 G. W. Buchanan and T. Durst Tetrahedron Letters 1975,1683. 28 K. Tori T. Tsushima Y. Tamura H. Shigemoto T. Tsuji H. Ishiboti and H. Tanida Tetrahedron Letters 1975 3307. 29 H.Pearson J.C.S. Chem. Comm. 1975 912. 30 J. Cantacuzene R. Jantzen M. Tordeaux and C. Chachaty Org. Magn. Resonance 1975,7,407. Part (ii) Nuclear Magnetic Resonance Spectroscopy 13 variation in ,J(C,H) as a conformational The sign of 2J(C,H) may be either positive or negative under different circumstances depending for example upon orientation of the coupled ‘H to an oxygen attached to the 13C; 0 anti to H gives a positive contribution to 2J(C,H) while 0gauche gives a negative contribu- ti~n.~~ This work suggests that 2J(C,H) is a ‘more definitive type of stereochemical descriptor than ,J(C,H)’ but this may not be generally so since two papers show that the more useful parameter for studies on peptides is 3J(C,H).33,34 In 13C-enriched materials 2J(C,C) and 3J(C,C) may be observed in addition to the more obvious ‘J(C,C).Barfield et al. have examined the theory of these3’ by the finite perturbation formulation in the INDO approximation and show that 3J(C,C) shows a strong dihedral angle dependence with 3J(C,C) (cis)apparently larger than 3J(C,C) (trans). This parameter has been used in studies of I3C-enriched amino- Coupling to ‘other nuclei’ also shows stereochemical dependence and 2J(P,C) 3711J(P,C) +2J(P,C)1,38 3J(C,14N),39 and 3J(C,199Hg)40 have all been used as conf ormational probes. Lanthanide shift reagents continue to play their part although their use has been much more limited than in recent years. Their value is shown for example in the conformational study of the valinomycin backbone in CDCI and in the investigation of nicotinamide mononucleotide in aqueous Physical Organic Chemistry.-Donor-acceptor equilibria may be conveniently studied by 13C n.m.r.Forskn et al. have pointed out that on adduct formation ‘H n.m.r. spectra of donor molecules show downfield shifts; 13C resonances may move downfield or upfield in spite of the accompanying electron ~ithdrawal.~~ A report that AsCl is a convenient solvent for 13C n.m.r. studies ‘unless the solute contains OH or NH when the AsCI acts as a shift reagent’ is best considered as an example of donor-acceptor c~mplexation.~~ Similarly the shifts induced by TiCI on the 13C n.m.r. spectra of carbonyl should not be confused with the shifts induced by lanthanide shift The charge-transfer complexes of nitroben-zene with various electron-rich aromatic donors have been examined and association constants and 1 1 complexation shifts evaluated.46 MacNico14’ has made very elegant use of the ability of the cyclodextrins to complex hydrocarbons into their voids; the complexation results in a ‘spreading out’ of the hydrocarbon n.m.r.31 J. A. Schwartz N. Cyr and A. S. Perlin Canad. J. Chem. 1975 53 1872. 32 T. Schaeffer K. Chum D. McKinnon and M. S. Chauhan Canad. J. Chem. 1975,53,2734. 33 P. E. Hansen J. Feeney and G. C. K. Roberts J. Magn. Resonance 1975,17,249. 34 V. F. Bystrov Ya. D. Gavrilov andV. N. Solka J. Magn. Resonance 1975,19 123. 35 M. Barfield I. Burfitt and D. Doddrell J. Amer. Chem. SOC.,1975,97 2631. 36 S. Tran-Dinh S. Fermandjian E.Sala R. Mermet-Bouvier and.P. Frornageot J. Amer. Gem. Soc. 1975,97,1267. 37 J. P. Dutasta and J. B. Robert J.C.S. Chem. Comm. 1975 747. 38 R. K. Harris E. M. McVicker and M. Field J.C.S. Chem. Comm. 1975 886. 39 R. DiBlasi and K. D. Kopple J.C.S. Chem. Comm. 1975 33. 40 W. Kitching D. Praeger D. Doddrell F. A. L. Anet and J. Krane Tetrahedron,1975,31 759. 41 K. L. Servis and D. J. Patel Tetrahedron,1975,31 1359. 42 B. Birdsall N. J. M. Birdsall J. Feeney and J. Thornton J. Amer. Chem. SOC.,1975,97,2845. 43 J. S. Hartman P. Stilbs and S. ForsCn Tetrahedron Letters 1975 3497. 44 A. K. Bose M. Suguira and P. R. Srinivasan Teirahedron Letters 1975 125 1. 45 A. K. Bose and P. R. Srinivasan Tetrahedron Letters 1975 1571. 46 R. C. Griflith D.M. Grant and J. D. Roberts J. Org. Chem. 1975 40,3726. 47 D. D. MacNicol Tetrahedron Letters 1975 3325. 14 L.Phillips spectrum. Although this has sofar been used only for ‘H n.m.r. the extension to 13C studies is obvious. The co-ordination of organic molecules to metal ions in solutions may be observed. Following the assignment of the I3Cn.m.r. spectrum of tetra~ycline,~’ the complexation with metals has been studied and the binding site analy~ed.~~ The complexation between Co”’ and dimethylglyoxime has been studied.” Some molecules have the ability to form adducts with both cations and anions; valinomycin has been examined in this way,s1 as have the nucleosides cytidine and guanosine.52 I3CN.m.r. may be used to study chemical exchange processes.Grant et al. have looked at tautomerism in purine^.^^*'^ A set of 13Cn.m.r. parameters from studies on model compounds having been the tautomeric populations in purine adenine hypoxanthine 6-rnercaptopurine7 and others were quantitatively e~timated.~~ Prototropic tautomerism in the imidazole ring was examined together with lactam-lactim or thione-thiol tautomerism in the pyrimidine ring. Prototropic rearrangements in azoles have also been studied by Russian ~orkers.~~,~~ The importance of competing interactions with solvents is highlighted by the slow reaction of benzimidazole derivatives with acetone which affects the tautomeric eq~ilibriurn.~~ Tourwe et al. have made direct I3C n.m.r. observations upon the tautomeric equilibrium involving enamines and enamino-ketones; they were able to deduce the preferred conformation of the cyclohexanone enamine ta~tomer.~~ The more widely studied keto-enol equilibrium involving p -diketones has also received further at ten tion.’* A different sort of exchange namely fluxion in co-ordination compounds may be usefully studied by 13Ctechniques. Thus Howell et uLS9have examined isocyanide derivatives of tetracarbonylbis-(q-cyclopentadieny1)di-iron and were able to differentiate between derivatives which show fluxion and those which do not. They also demonstrated clear differences between CNR ligands which are bridging or terminally bonded. Studies of stable ionic species continue to be of importance. These are typified by Olah’s and references therein.By this technique Olah has been able to establish the non-classical nature of the secondary and tertiary 7-norbornenyl cations,60 and the technique is described in his recent book.6‘ Other relevant work 48 G. L. Asleson and C. W. Frank J. Amer. Chem. SOC.,1975,97,6246. 4p J. Gulbis and G. W. Everett J. Amer. Chem. SOC.,1975 97 6248. 50 C.Bied-Charreton B. Septe and A. Gaudemar Org. Mugn. Resonance 1975,7 116. 51 D. G. Davis and D. C. Tosteson Biochemistry 1975,14,3962. 52 T.Yokono,S. Shimokawa and J. Sohma J. Amer. Chern. SOC.,1975,97,827. 53 M.T. Chenon R. J. Pugmire D. M. Grant R. P. Panzcia and L. B. Townsend J. Amer. Chem. Soc. 1975,97,4627. 54 M. T. Chenon R. J. Pugmire D. M. Grant R. P. Panzcia and L. B. Townsend J.Amer. Chem. Soc. 1975,97,4636. 55 A. N. Nesmeyanov E. B. Zavelovich V. N. Babin N. S. Kochetkova andE. I. Fedin Tetrahedron,1975 31,1461. 56 A. N. Nesmeyanov E. B. Zavelovich V. N. Babin N. S. Kochetkova and E. I. Fedin Tetruhedron,1975 31,1463. s7 D. Tourwe,G. Van Binst S. A. G. de Graaf and U. K. Pandit Og. Mugn. Resonance 1975,7,433. 58 N. N. Shapet’ko S. S. Berestova G. M. Lukovkin and Yu. S. Bogachev Org.Magn. Resonance 1975,7 237. 59 J. A. S.Howell T. W. Matheson and M. J. Mays J.C.S. Gem. Comm. 1975,865. 6O G.A.Olah and G. Liang J. Amer. Chem. SOC.,1975,% 6803. 61 G. A. Olah ‘Halonium Ions’ Wiley New York 1975. Part (ii) Nuclear Magnetic Resonance Spectroscopy 15 includes studies of cyclopropenylium ions,62 xanthylium ions,l0 and uronium thiouronium and guanidinium The mutarotation of hexoses has been studied by examining the 13C n.m.r.spectrum of a-glucopyranose in D,O solution as a function of the concentration of added potassium hydroxide.& The titration shifts suggest that the results cannot be explained in terms of a simple ionization and a rapid equilibration between cyclic and acyclic ionized species is suggested (Scheme 1). The experiment shows clearly that the rate-determining step for the mutarotation is rotation around the C(H0H)-C(0)R bond in the acyclic form. OH OH CH20H HOo-Scheme 1 The use of 13C labels in arder to give mechanistic information is well established. Rees et al.65have used this technique to investigate the pyrolysis of 1,4-and 1,5- diphenyl-1,2,3-triazoles.Similarly the mechanism of formation of azepines from the reaction of dimethyl ace tylenedicarboxylate with 2-methylquinolines has been elucidated.66A 13C label arising from the methyl group of 2-methylquinoline or its 6-bromo-derivative is found at pgsition 10 or 11 of the azepino[ 1,2-a]quinolines formed in the reaction.This excludes a reaction pathway involving an ester shift but is entirely consistent with a route involving a spiro intermediate. Rummens et aZ.67have compared the utility of 13C and radioactive I4C labels as quantitative probes in scrambling studies. They examined 1,2-phenyl shifts in the reaction between labelled triphenylvinyl bromide and silver acetate. Both techni- ques proved useful but the radiotracer method gives greater precision.In a similar study Oka and Lee68 have used the 13C scrambling label technique for the acetolysis and trifluoroacetolysis of triani~yl[2-~~C]vinyl bromide. The modification of horse-heart cytochrome c by carboxymethylation has been studied using [2-13C]bromacetate. The 13C n.m.r. spectrum clearly reveals that the protein is much more extensively changed than previously In a some- what more detailed study amino-acid side-chains have been derivatized with a 62 E. V. Dehmlow R. Zeisberg and S. S. Dehmlow Org. Magn. Resonance 1975,7,418. H. 0.Kalinowski and H. Kessler Org. Magn. Resonance 1975,7 128. 64 G. de Wit A. P. G. Kieboom and H. van Bekkum Tetrahedron Letters 1975,3943. 65 T. L. Gilchrist C. W. Rees and C.Thomas J.C.S. Perkin I 1975,8. 66 R. M. Acheson and R. F. Flowerday J.C.S. Perkin I 1975 394. 67 F. H. A. Rurnmens R. D. Green A. J. Cessna M. Oka and C. C. Lee Canad.J. Chem.,1975,53,214. 68 M. Oka and C. C. Lee Canad. J. Chem. 1975,53,320. 69 R. T. Eakin L. 0.Morgan and N. A. Matwiyoff Biochemistry 1975,144538. 16 L.Phillips variety of 13C-labelled ele~trophiles,~" namely acrylonitrile 3-bromopropionic acid acrylamide iodoacetic acid and methyl iodide. The technique involves blocking the N-terminus as a t-butoxycarbonyl derivative and the C-terminus by esterification; the side-chain is then electrophilically labelled. With the amino-acids cysteine lyseine histidine and serine there are sufficient differences in the chemical shifts of any one of these labels that they may be used as probes for these acids in enzyme active sites.13 C N.m.r. is a very convenient method of locating deuterium labels since replacement of 'H by 2Hconsiderably increases the spin-lattice relaxation time (Tl) of a bonded I3Cnucleus and reduces the nuclear Overhauser enhancement (NOE) obtained on 'H spin decoupling. This effectively removes the 13Cresonance line from the spectrum under normal conditions. In this way Hanson and Siverns71 have located the site of deuteriation in the aromatization reaction used to synthesize methyloestratrienes. This has enabled a differentiation between two possible reac- tion pathways. Biosynthesis.-The incorporation of I3C labels during biosynthesis continues to be an area of great importance.There have been many studies utilizing singly labelled precursors. For example acetate (labelled 1 or 2) has been used to elucidate the biosynthesis of fusi~occin,~~ aflatoxin B,73leucomycin A3,74 and sterigrnat~cystin.~~ [1-13C]-Pr~pionate and -butyrate have been incorporated into leucomycin A3,74 and [4-'3C]mevalonate has proved a useful precursor for oleanene and ursene-type triterpene~.~~ has been incorporated into chl~ramphenicol~~ [6-13C]-~-GI~~~~e and streptomycin.78 The major development in this field has been the increased use of doubly-labelled precursors including [1,2-13C2]acetate,73*74*7y-87 and [1,3-13C2]phenylalanine,88.89 [1,4-13C2]succinate.76 Last year's Report emphasized that doubly-labelled tracer studies could be difficult to interpret quantitatively because I3C-l3C dipolar relaxa- tion could be important for quaternary carbonsgo and cause intensity anomalies.70 I. J. G. Clunie D. A. Evans and M. Akhtar J.C.S. Chem. Comm. 1975 160. 71 J. R. Hanson and M. Siverns J.C.S. Perkin I 1975 11 10. 72 K. D. Barrow R. B. Jones P. W. Pemberton and L. Phillips J.C.S. Perkin I 1975 1405. 73 P. S. Steyn R. Vieggaar P. L. Wessels and D. B. Scott J.C.S. Chem. Comm. 1975 193. 74 S. Omura A. Nakagawa H. Fakeshima K. Atusmi J. Myazawa F. Pirion and G. Lukacs J. Amer. Chem. Soc. 1975,97,6601. 75 K. G. R. Pachler P. S. Steyn R. Vleggaar and P. L. Wessells J.C.S. Chem. Cornm. 1975 355. 76 S. Seo Y. Tomita and K. Tori J.C.S. Chem. Comm. 1975 270. 77 M. H. G. Munro M. Taniguchi K.L. Rinehart and D. Gottlieb Tetrahedron Letters 1975,2659. 78 M. H. G. Munro M. Taniguchi and K. L. Rinehart J. Amer. Chem. SOC.,1975,97,4784. 79 R. C. Paulick M. L. Casey D. F. Helenbrand and H. W. Whitlock J. Amer. Chem. SOC. 1975,97,5303. 8o A. G. McInnes D. G. Smith J. A. Waiter L. C. Vining and J. L. C. Wright J.C.S.Chem. Comm. 1975 66. 81 T. J. Simpson and J. S. E. Holker Tetrahedron Letters 1975,4693. 82 F. C. Baker C. J. W. Brooks and S. A. Hutchinson J.C.S. Chem. Comm. 1975 293. 83 S. Seo Y. Tomita and K. Tori J.C.S. Chem. Comm. 1975 954. 84 J. Bolonsky G. Lukacs N. Cagnoli-Bellavita and P. CeccherelIi Tetrahedron Letters 1975,481. 85 J. S. E. Holker and K. Young J.C.S. Chem. Comm. 1975 525. 86 A. J. Birch T. J. Simpson and P. W. Westerman Tetrahedron Letters 1975 4173.87 R. E. London N. A. Matwiyoff V. H. Kollman and D. D. Mueller J. Magn. Resonance 1975,18,557. E. Leete N. Kowanko R. A. Newmark L. C. Vining A. G. McInnes and J. L. C. Wright Tetrahedron Letters 1975 4103. 89 E. Leete N. Kowanko and R. A. Newmark J. Amer. Chem. SOC. 1975,97,6826. 90 C. G. Mcxeiand and F. I. Carroll J. Magn. Resonance 1974 15 596. Part (ii) Nuclear Magnetic Resonance Spectroscopy 17 Additionally if the C-resonances are close together the tightly coupled spin system also causes intensity errors to arise in F.T. spectra." Matwiyoff et alqg7 have recently pointed out that singlet (ie. non-coupled) and multiplet resonances will have differentNOE's which can also lead to incorrect enrichment values if the effect is not taken into account.These same workers have made a thorough investigation into the anomalies caused by the ~plittings.~~ They point out also that 13C-13C coupling extends the value of 13C enrichment as a biosynthetic tool in several ways namely (i) as an aid to spectral assignment (ii) as an indicator of dilution of the labelled substrate by unlabelled material (iii) by giving information about intermediate steps in the biosynthesis from the 13C-13Cspin-spin multiplet pattern (iv) where equivalent carbons occur giving rise to a single resonance they may not be labelled equivalently by a biological system; the differential labelling may be studied by observing splitting of adjacent carbon resonances. These aspects are illustrated using labelled carbohydrates to study glycolysis.In contrast in a study of the biosynthesis of bikaverin it was necessary to remove the '3C-*3Csplitting by homonuclear spin decoupling." This overcame the difficulties caused by low 13C incorporation of [1,2-'3CJacetate and enabled the assembly pattern of the precur- sor units in the metabolite to be established In an attempt to ensure equal relaxation rates of 13C coupled and non-coupled resonances Paulick et al.79have used the so-called 'shiftless relaxation reagent' Cr(acac) in their study of the biosynthesis of islandicin from [1,2-'3CJacetate. They were able to differentiate successfully between two alternative pathways by this technique. Following their earlier study of incorporation of [4-13C]mevalonate into oleanene and ursene-type triterpenes (in which they were able to verify Ruzicka's hypothesis for cyclization of squalene-2,3-oxide as the mechanism for formation of the pen- tacyclic triterpene~),~~ Seo at al.have used [1,2-"C;]acetate to elucidate the biosynthesis of ring E in these compounds. The difficult assignment of the relevant spectra was carried out with the aid of lanthanide-induced shifts and this paper corrects an earlier as~ignment.~~ Usually in a double-labelling study it is an alI-or-nothing situation. The directly bonded 13C-13C coupling from the precursor is either retained in the metabolite or is lost. Simpson and Holker however observe the 2J(C,C)values which arise from cleavage and re-assimilation of the fragments of [1,2-13CJacetate in the biosynthesis of a pyrone metabolite of Aspergillus melleus.This is mechanisti- cally useful and is the first example of detection of such a coupling from biosynthetic rearrangement of a doubly labelled precursor. 2J(C,C)is a parameter which has sometimes been observed due to high incorporation of [1-13C]acetate(e.g.ref. 94) and has been seen as a result of an intramolecular rearrangement of [2,11- 13 CJporphobilinogen during protoporphyrin bio~ynthesis.~~ A neat reversal of this occurs when labels in a 1,3 configuration are rearranged during biosynthesis to a 1,2 relationship. The unmistakable directly bonded cou- pling appears in the metabolite and demonstrates a point of mechanism. Leete et al. S. Schaublin A. Hohener and R.R. Ernst J. Magn. Resonance 1974,13 196. q2 R. E. London V. H. KoIlman and N. A. Matwiyoff J. Amer. Chem. Soc. 1975,97,3565. 93 S.Seo Y.Tomita and K. Tori Tetrahedron Letters 1975,7. D. P.H.Hsieh J. N. Seiber C. A. Reece D. L. Fitzell S. L. Yang J. I. Dalezios G. N.La Ma,D. L. Budd and E. Motell Tetruhedron 1975,31,661. 95 A. R. Battersby E. Hunt and E. McDonald J.C.S. Chem. Cornrn. 1973,442. 18 L. Phillips have used [1,3-13CJphenylalanine to demonstrate such a rearrangement in the biosynthesis of tenellins* and tropic acid.89 The determination of relative signs of 'J(CC) in doubly labelled compounds is of some consequence and has been demonstrated by Hansen et ~1.~~ Selective TIMeasurements.-13C Spin relaxation is the subject of Chapter 3 in ref.1. This provides an excellent background to both theory and practice and is mainly concerned with longitudinal or spin-lattice relaxation characterized by the time constant Tl. Levy and Peat have considered the experimental aspects of Tl measurement for 13C nuclei in some detail.97 They compare and contrast the saturation recovery inversion recovery and partial saturation techniques. In addi- tion they provide a useful discussion about the influence of sample geometry sample state transmitter r.f. pulse power computer and data-processing characteristics instrument gain and stability and magnetic field homogeneity. An earlier paper by the same considers the different experimental approaches in greater detail and describes a technique which greatly shortens the length of time required for measurements by the inversion recovery technique.This 'Fast Inversion Recovery Fourier Transform' method (FIRFT) uses the conventional [180"-~-90"-(FID)-T] sequence but with an arbitrary short interval Tbetween the sequences. Normally T is 5( Tlma)and is a limitation of the usefulness of the method; with a much smaller T (which is nevertheless long enough to allow the decay of tranverse magnetization before application of the next 180" pulse) the speed of the other methods may be achieved. In the same context a fast non-linear least-squares method for calculating TIhas been described.99 This also uses short repetition times T,and since it obviates the necessity for determining S (the intensity of lines in a fully relaxed spectrum) it offers considerable savings of time.Perhaps the most widespread use of 13C Tl measurements is in the study of mobility in large molecules particularly in alicyclic species. loo An examination of n-alkyltrimethylammonium halides with polar substituents in the chain demon- strates that molecular motion is reduced in the region of the substituent.lol A study of NOE and Tl values in zinc and nickel insulin as well as in the metal-free system has been carried out using 90% 13C-enriched carbonyl groups attached to A1 glycine B1 phenylalanine and B29 lysine. The state of aggregation of the molecules is revealed by the correlation times for overall tumbling while the correlation times for internal rotation of the carbonyl functions indicate the relative mobilities of their local environments.lo' Deslauriers et al. have examined the conformational properties of cyclic dipep- tides,lo3 the octapeptide hormone [5-isoleucine]angiotensinII,l@' and the conforma- tional flexibility of luteinizing-hormone-releasinghormone (LH-RH) lo' in aqueous 96 P. E. Hansen 0.K. Poulsen and A. Berg Org. Magn. Resonance 1975,7,405. 97 G. C. Levy and I. R.Peat J. Magn. Resonance 1975,18,500. 9a D. Cault G. C. Levy and I. R.Peat J. Magn. Resonance 1975,18 199. 99 D. L.De Fontaine D. K. Ross,and B. Ternai J. Magn. Resonance 1975,18 276. 100 G. C.Levy,R.A. Komoroski and R.E. Echols Org. Magn. Resonance 1975,7,172. 101 J. M.Brown and J. D. Schofield J.C.S. Chem. Cbmm. 1975,434. 102 J. J. Led D. M.Grant W. J. Horton,F. Sundby and K. Vilhelmsen J. Amer. Chem. SOC.,1975,97,5997. 103 R.Deslauriers Z.Gozonka K. Schaumberg T. Shiba and R.Walter J. Amer. Gem. SOC.,1975,97 5093. 104 R.Deslauriers A.C. M. Paiva K. Schaumberg and I. C. P. Smith Biochemistry 1975,14,878. 105 R.Deslauriers G. C. Levy W. H. McGregor D. Sarantakis and I. C. P. Smith Biochemistry 1975,14 4335. Part (ii) Nuclear Magnetic Resonance Spectroscopy 19 solution at various pH values. In this latter work Ti's are evaluated for all hydrogen-bearing carbons at both 25.2 and 67.9 MHz. LH-RH is shown to be a flexible molecule in solution with segmental motion along the backbone as well as in the non-aromatic side-chain. This is one of the rare determinations of 13C Tl's at 67.9 MHz and in another paper Levy sounds a note of caution concerning interpre- tation of such data for unsaturated carbons not bonded to hydrogen.lM At such high field-strength the chemical shift anisotropy mechanism for relaxation becomes import ant .Adenosylcobalamin and alkylcorrinoids selectively enriched with 13C have been examined.lo7 The Tl values of the CH2 carbons of [5-13C]adenosylcobalaminand [2-'3C]carboxymethylcobalamin are similar to that of the methine bridge carbon (C-10) of the corrin ring indicating severely restricted rotation around the C-Co bond. The rotation about the Co-Co bond of methylcobalamin appears to be rapid however. The motional properties of biological macromolecules are conveniently studied by the Tl method. By using 13C enrichment of the oleic acid chains in whole virions it has been possible to investigate their mobility directly.lo8 Examination of the temperature dependence of Tl for 13C nuclei in some fractionated membrane^'^^ has enabled activation energies (-15-24 kJ mol-') for the C-C bond rotations to be evaluated. The details of segmental motion of the chains in these membranes have been elucidated.lW Lyerla and Torchia have studied molecular mobility in elastin' lo and collagen molecules. '11 The determination of the anisotropy in the motion of a molecule in solution gives useful structural information. Roberts et al. have described a computer program to calculate the rotational diffusion tensor for anisotropic motion from 13C T1data."' They show an application to methyl-substituted cycloalkanes and conclude that the degree of anisotropic motion decreases with increasing ring size.A similar study on androstane and cholestane confirms that the preferred axis of rotation in the anisotropic tumbling of these molecules in solution is the 'long' steroid This is shown by the fact that all CH and CH2 carbons except C-3 which is on the axis have the same Tl within experimental error. The correlation times for internal rotation of the C-18 and C-19 methyl groups depend upon the number of 1,3- diaxial methyl-H interactions and so C-19 has a longer Tl than C-18 in androstane. In trans-cholestanol C-19 has a longer TI than in either cis-cholestanol or choles- ter01.l~~ In an important and elegant piece of work Freeman et al.'14 have used motional anisotropy as a probe for transient complex formation in solution.They point out that even transient association alters the degree of motional anisotropy of a molecule by a significant amount. The tumbling rate of an associated pair of molecules is loci G. C. Levy and U. Edlund J. Amer. Chem. Soc. 1975,97,5031. lo' H.P.C. Hogenkamp R. D. Tkachuch M. E. Grant R. Fuentis and N. A. Matwiyoff Biochemistry, 1975,14,3707. lo8 W.Stoffel and K. Bister Biochemistry 1975,14 2841. log R.E.London V. H. Kollman and N. A. Matwiyoff Biochemistry 1975,14,5492. 110 J. R.Lyerla and D. A. Torchia Biochemistry 1975,14 5175. D.A. Torchia J. R. Lyerla and A. J. Quattrone Biochemistry 1975 14 887. 11* S.Berger F.R. Kreissl D. M. Grant and J.D. RobertsJ. Amer. Chem. Soc. 1975,97,1805. 113 J. W.Apsimon H. Beierbeck and J. K. Saunders Canud. J.Gem. 1975,53 338. 114 I. D.Campbell R. Freeman and D. L. Turner J. Mugn. Resonance 1975,20,172. 20 L. Phillips slower than that of the discrete species about an axis perpendicular to the direction of the 'bond' between them. The change in the rotational diffusion tensor may be conveniently followed by 13C Tl measurements. Transient complexes whose lifetimes exceed the correlation time for end-over-end rotation (typically lo-'' s) may be observed in this way. The approach is illustrated for hydrogen-bond formation in a molecule which does not self-associate but forms a hydrogen bond with a second species and thereby acquires anisotropic motion.Changes in the ratio of the Tl values of the carbons in this molecule (termed a 'Motional Anisotropy Probe') give a sensitive test for the size and shape of the complex. A good example is pyridine in which C-2 C-3 and C-4 all have equal Tl values in the neat liquid or in CCI solution. Addition of a hydroxyl-containing compound causes the axis of fastest rotation to be coincident with the N * H-0 bond direction and C-2 and C-3 retain equal Tl while that of C-4 is shortened. The spin-lattice relaxation of protons has been used to study fast proton exchange in aqueous ~olution~,"~ but its main use to date appears to be as a conformational mobility probe. A study of thermally unfolded ribonucIease A' l6 concentrates upon the four histidine residues.Two of these are exposed to solvent interactions [His-48 and (tentatively) His-1051 while two remain in regions of residual (ie. folded) structure [His-12 and (tentatively) His-1191. Rowan and Sykes'l7 have examined the conformational behaviour and flexibility of the polyene chains in retinals using this technique and other workers have studied the conformational dynamics of nicotinamide adenine dinucleotide and nicotinamide mononucleotide.' '* The effect upon 'H Tl values of replacing neighbouring protons by *H1 and thereby removing the dominating dipolar-relaxation mechanism from the system has been put to good use. Akasaka et a[. have christened this technique DESERT (DEuterium Substitution Effect on Relaxation Times) and have used it to estimate inter-hydrogen distances in 2',3'-isopropylidene-3,5'-cycloguanosine whose con- formation is fixed and to study syn-anti conformational equilibria in 2',3'- isopropylidene-adenosine adenosine and 5'-adenosine m~nophosphate."~ Specifically deuteriated L-malate has been used in a study of the aspartate trans- carbamylase catalytic site.'20 Spin-lattice relaxation in the presence of paramagnetic ions has received some attention.The use of Cr(acac) to ensure equal relaxation times of all carbons in a complex molecule (e.g. ref. 79) is not without its difficulties. Levy and Edlund'*' have pointed out that the highly efficient directly bonded 13C-'H dipole-dipole relaxation mechanism can compete successfully with the electron-nuclear spin relaxation due to the paramagnetic CrI'I.The suppression of the NOE in the 'H decoupled spectrum may be incomplete and variable and the spectrum may actually degrade as a result. This work examines the 13C Tl values of cholesteryl chloride at 67.5 MHz and shows that as much as 60% of the relaxation of C-5 (non-hydrogen 115 S. Waelder L. Lee and A G. Redfield J. Amer. Chem. Soc. 1975,97,2927. 116 C. R. Matthews and D. G. Westmoreland Biochemistry 1975,14 4532. 117 R. Rowan and B. D. Sykes J. Amer. Chem. SOC. 1975,97,1023. 118 A. P. Zens T. J. Williams J. C. Wisowaty R. R. Fisher R. B. Dunlop T. A. Bryson and P. D. Ellis J. Amer. Chem. Soc. 1975,97,2850. 119 K. Akasaka T. Imoto S. Shibata and.H. Hatano J. Magn. Resonance 1975,18,328. H. I. Mosberg P. G. Schmidt and S. S.Seaver J. Magn. Resonance 1975,20,82. 121 G. C. Levy and U. Edlund J. Amer. Gem. SOC.,1975,97,4482. Part (ii) Nuclear Magnetic Resonance Spectroscopy 21 bearing olefinic carbon) arises from the chemical shift anisotropy mechanism. 12' In another paper'22 these workers suggest that Cr(dpm) is a more suitable TI-equalizing reagent than Cr(acac),. It is more intert towards organic substrates and produces no observable contact shifts. Cr(acac) actually causes shifts as large as 0.64 p.p.m. for CDC13 and up to 0.4 p.p.m. with nitrogen-containing aromatic From an examination of Tl for 13C nuclei in histidine complexed to paramagnetic Mn" it has been possible to calculate the precise distances between the metal and the carbon atoms in the ligand. These correspond closely to the distances determined in the X-ray crystal structure of the analogous Ni" complex and it is claimed that the structural parameters are comparable in accuracy to X-ray data.124 It has been possible to locate the Ca2' ion binding site in bovine a-and p-trypsin using the relaxation effects of Gd3+ as a probe in metal-inhibitor-trypsin ternary complexes.It is established that Ca2' and Gd3' compete for the same It should not be imagined that paramagnetic species are able to shorten TIof all nuclei. Because of the overwhelming importance of quadrupolar as opposed to dipolar relaxation for 2H a concentration of a paramagnetic species which reduces Tl of 'H in 'H20 by a factor of CQ. 400 will only shorten Tl of 2H,0 by a factor of 3 High-field N.M.R.Studies of 'H Resonance One of the major limitations upon the usefulness of 'H n.m.r. in biological systems has been the narrow range of chemical shifts. This leads to overlapping lines and tightly-coupled spin systems which do not yield easily to analysis. The use of high fields from superconducting magnets has overcome some of these difficulties and although the studies may be of a somewhat routine spectroscopic nature they are important in terms of the results that are now achievable. The 'H n.m.r. spectrum of bovine pancreatic trypsin inhibitor has been examined at 250MHz and all proton resonances of the aromatic rings of the four tyrosine residues have been located and assigned.127 pH titration enables the pK of each of the tyrosines to be measured and a similarly successful study was carried out with mono- and di-nitrated derivatives.The polypeptide antibiotic N-methyl-leucine gramicidin-S dihydrochloride has been examined at 220 MHz and all resonances have been assigned to the specific hydrogens of the constituent amino-acid frag- ments. ,J(H,H) (across C-N bonds) and chemical shifts give conformational information.12' The ability of high fields to spread out 'H n.m.r. spectra makes available new conformational information. Thus fragments from the anticadon loop of baker's yeast phenylalanine transfer ribonucleic acid have been examined in this way.'29 The 270 MHz spectrum of Histone H2A shows variations with ionic strength and pH lz2 G. C. Levy,U. Edlund and J. G. Hexem J.Magn. Resonance 1975,19,259. lz3 P. M. Henricks and S. Gross J. Magn. Resonance 1975,17 399. lZ4 J. J. Led and D. M. Grant J. Amer. Chem. SOC.,1975,97,6962. F. Abbott J. E. Gomez E. R. Birnbaum and D. W. Darnall Biochemistry 1975,14,4935. 126 J. Wooten G. B. Savitzky and J. Jacobus J. Amer. Chem. SOC. 1975,97 5027. G. H. Snyder R. Rowan S. Karplus and B. D. Sykes Biochemistry 1975,14,3765. N. G. Kiemar N. Izumiya N. Miyoshi H. Sugano and D. W. Urry Biochemistry 1975,14,2197. lz9 L. S. Kau P. 0.P. Ts'o F. von der Haar M. Sprinzl and F. Crarner Biochemistry 1975,14,3278. 22 L.Phillips which suggest that an equilibrium exists between largely unstructured coiled molecules and fully structured aggregated molecules. 13* Similar studies of histone IV and some fragments derived by cyanogen bromide cleavage have been reported.l3','32 Markley has used difference spectra at both 100 and 250 MHz to reveal a pH-dependent conformational change in ribonuclease A which affects only the chemical shift of a single tyrosine residue.133 This conformational change is absent in ribonuclease S and is changed in ribonuclease A by the presence of either acetate or cytidine 3'-monophosphate. Prior to this investigation it was necessary to reassign the 'H n.m.r. spectrum of the histidine residues in the ribonuclea~e;'~~ Bradbury and Seng Teh have independently arrived at the same conclusion.'35 Thermally induced conformational transitions have been monitored by the high- field 'Hn.m.r. method. By examining the 300MHz spectrum of the non-exchangeable purine and pyrimidine resonances together with the sugar resonances in a D20 solution of d-ApTpGpCpApT the helix-coil transition has been observed.136 The sequential thermal unfolding of valine transfer RNA has similarly been st~died.'~' The ability of this technique to elucidate minute details of structure is well illustrated by the binding of ethidium bromide to yeast tRNAPhe.At 300 MHz it is possible to observe that just two ring NH protons have moved on complexation; this uniquely determines the binding site as located between the base pairs AU6 and AU of the amino-acid receptor Application and Development of the FTTechnique Perhaps the most remarkable development has been that of 'Zeugmatographic High Resolution N.M.R.Spectroscopy'. 139-141 N.m.r. signals arising from samples subject to magnetic field gradients may be used to produce two- and three- dimensional images of the object from which they arise. The technique has recently been extended by Lauterbur et~Z.'~* and by Ernst A magnetic field gradient is imposed on the sample during a radiofrequency pulse causing resonance to occur only in a limited region. The gradient is then removed and the FID of the remaining transverse magnetization in the selectively excited region is Fourier-transformed. Repetition of this sequence with a slight change in the magnetic field or radiofre- quency gives information from other sections of the sample. The information may then be assembled to give one- two- or three-dimensional images of the distribution of resonating material in the sample.Chemically shifted species may be brought into 130 E. M. Bradbury P. D. Cary C. Crane-Robinson H. W. E. Rattle M. Boublik and P. Sautiere Biochemistry 1975,14 1876. 131 A. E. Pekary H. J. Li S. I. Chan C. J. Hsu and T. E. Wagner Biochemistry 1975,14 1177. 13* A. E. Pekary H. J. Li S. I. Chan C. J. Hsu and T.E. Wagner Biochemistry 1975,14 1184. 133 J. L. Markley Biochemistry 1975,14 3554. 134 J. L. Markley Biochemistry 1975,14,3546. 135 J. H. Bradbury and J. Seng Teh J.C.S. Chern. Comm. 1975,936. 136 D. J. Patel Biochemisrry 1975,14 3984. 137 R. V.Kastrup and P. G. Schmidt Biochemistry 1975,14,3612. 138 C. R. Jones and D. R. Kearns Biochemisfry,1975,14,2660. 139 P.C. Lauterbur Nature 1973.242 190. 140 P. C. Lauterbur PureAppl. Chem. 1974,40 149. 141 W. S. Hinshaw Phys. Letters (A) 1974,48 87. 142 P. C. Lauterbur D. M. Kramer W. V.House and C.-N. Chen. J. Amer. Chem. Soc. 1975,97,6866. 143 A. Kumar D. Welti. and R. R. Ernst J. Magn. Resonance 1975,18 69. Part (ii) Nuclear Magnetic Resonance Spectroscopy 23 resonance individually and the distributions of different materials in an object may be separately examined. It is hoped that there will be useful biological applications such as the differentiation between fat and other tissues in organisms'42 or the examination of the binding of water in biological systems and the differentiation of malignant and normal cells.'43 An exciting development has been the combination of stopped-flow techniques with FT n.m.r.to give spectra of intermediates in moderately fast chemical reactions (half-life greater than ca. 0.15 s typically of the order of seconds). Grimaldi and Sykes have Fourier difference spectro~copy'~~ to detect the weak resonances of the intermediate species in the presence of strong unwanted resonances. They describe a variety of pulse techniques for obtaining stopped-flow FT spectra and discuss in some depth the capabilities of the method. The technique has been applied to the observation of transient intermediates in the tricyanovinylation of NN-dimeth~laniline,'~~ and in the reaction between tris(pentane-2,4-dionato)Co1"and N-chlorosuccinimide.'47 By a somewhat related method Fyfe et al. have directly studied chemical reactions in flowing liquids.14* They have obtained information on intermediates in the reaction between methoxide and l-X-3,5-dinitrobenzenes (X= CN CF, C02Me or C0,Et). The development of double-resonance methods in FT n.m.r. continues. An improved method of completely decoupling ['aspins from I3C has been described,'49 which is claimed to be much more effective than random-noise decoupling giving signal :noise improvements by a factor of two or more. Phase modulation of the decoupler carrier wave with a 50% duty-cycle square wave brings about the bandwidth necessary for broad-band coupling. The combination of double irradiation and a simple two-pulse spin-echo sequence is a powerful method for detection of homonuclear decoupling in complex spectra.150 A 90°-r-1 80"sequence refocuses phase loss due to inhomogeneity and forms an echo at a time 27after the 90"pulse; data are then acquired. Non-selective pulses affect all spins equally and dephasing due to spin-spin coupling is not refocused and the phase loss follows a precise pattern e.g. at a time 1/Ja fist-order doublet of separation J Hz is inverted. Singlets and the central components of triplets do not change phase but outer components are inverted at time 1/2J. If selective double irradiation is applied during the period between the 90" pulse and the start of data acquisition the multiplicity and hence the phase of coupled resonances changes and gives a sensitive method of detection of the phen~rnenon.'~~ This technique is related to methods described earlier for the detection of coupling by 'FTINDOR-like' experi- ments (e.g.refs. 151-153) which have recently found application^."^'^^ 144 J. J. Grimaldi and B. D. Sykes J. Amer. C'hem. Soc.,1975,97 273. 145 R. R. Erst J. Map. Resonance 1971,4,280. 146 M. J. T. Robinson and S. M. Rosenfeld Tetrahedron Letters 1975 1431. D. A.Couch 0.W. Howarth and P. More J.C.S. Chem. Cumm. 1975,822. 14* C. A.Fyfe M. Cocivera and S. W. H. Damji J. Amer. Chem. Soc. 1975,97,5707. 149 J. B.Grutzner and R. E. Santine J. Magn. Resonance 1975,19 173. I5O I. D.Campbell and C. M. Dobson J.C.S. Chem. Comm. 1975,750. J. Feeney and P. Partington J.C.S. Chem. Comm. 1973,611. 152 K. G. R. Pachler and P. L. Wessels J.C.S. Chem. Comm. 1974 1038.Is3 Refs. 156-162 of ref. 14. 154 G. Massiot S. K. Kan P. Gouord and C. Duet J. Amer. Chem. Soc. 1975,97 3277. 155 T.Bungaard and H. J. Jakobsen J. Magn. Resonance 1975,18,209. Is6 K.Kushida K. Aoki and S. Satoh J. Amer. Chem. Soc. 1975,97,443. 14'