2 Physical Methods and Techniques Part (ii) Nuclear Magnetic Resonance By R. B. JONES and L. PHILLIPS Department of Chemistry Imperial College of Science and Technology South Kensington London S W7 2A Y 1 Introduction In this Report we have concentrated attention upon two major areas of continuing importance namely I3C n.m.r. and conformational analysis. Two obvious 'growth points' selective TI measurements and studies of the less-common isotopes of hydrogen are also considered. Needless to say the coverage of each of these areas is highly selective and reflects the authors' impression of what is important; apologies are therefore in order to the originators of all the work which lack of space has forced us to ignore! 2 Conformational Analysis This is one of the largest areas of application of n.m.r.techniques and we cannot cover the many papers of a 'routine' nature which have appeared in the past year. We concentrate upon two relatively new techniques of importance 3C n.m.r. and lanthanide-induced shifts (LIS); in addition we cover just one area to which the techniques of conformational analysis have been applied biological systems. Applications of l3CN.M.R.-HinderedRotation about C-C Bonds. This has been examined in some n-conjugated systems. In 2,5-dimethyh3,4-di-isopropyl-2,4-hexadiene the barrier to internal rotation about the (formally single) C-3--C-4 bond is unusually high with AG* > 1lOkJmol-'. The methyl groups of the isopropyl functions are diastereotopic because of the chirality induced by the hindered rotation and their non-equivalence persists at temperatures up to 195"C.' An analogous study of the s-cisSs-trans equilibrium in some enamino-ketones and -thiones of the form X=C(R)CH=CHNR (X = OS R = alkyl) shows that only in the case ofthe thiones is there significant restriction to rotation resulting in non-planarity.2 The power of the I3C technique in this area is D.S. Bomse and T. H. Morton Tetrahedron Letters 1974 3491. J. Dabrowski K. Kamienska-Frela and L. Kozerski Org. Magn. Resonance 1974 6 499. 17 18 R. B. Jones and L.Phillips illustrated by a study of the diastereomeric modifications of some 2,3-disubsti- tuted succinic acids and a 3,4-disubstituted adipic acid. The diastereomers show significant differences in chemical shift for all carbons and this enables conforma- tional populations to be ded~ced.~ Conformational Properties of Glycosidic Linkages.These have been studied by the 3C n.m.r. method. The '3C-'H coupling between anomeric hydrogens and carbon in the aglycone moiety enables torsion angles to be deduced and the '3C chemical shifts give a measure of relative steric compression at the anomeric ~entre.~ The barrier to internal rotation around this bond in some nucleosides has been measured by using aglycone bases which have C, symmetry to ensure equal popu!ations of the two rotamers. The changes in lineshape of the I3C n.m.r. spectrum of the aglycone enable the barriers to be mea~ured.~ Alicyclic Compounds. Alicyclic compounds have been examined using the concept of relative high-field shifts for sterically compressed carbons.6-* Of particular interest is a study of cyclohexylmercuric derivatives which utilizes the fact that 3J199Hg-L3C is much larger (275 Hz) for equatorial mercury than for axial ( -70 Hz).For bromomercuri- and acetoxymercuri-substituents there is greater preference for axial orientation than equatorial and there is no steric compression shift of C-3,5 in the axial isomer.' The Conformations of Heterocyclic Compounds. ' 3C Studies resolve an ambiguity evident in previously published work on 1,Zdimethylhexahydropyridazines which suggested a significant population of the diaxial conformer ;the '3Cstudy shows that this is not so.'' In a similar study of double inversion at two N atoms this time in a bicyclic hydrazine the process was shown to be trans trans (1) rather than cisccis (2)." The variation of 'J13C-lH with the nature and 3 L.Ernst and W. Trowitsch Chem. Ber. 1974 107 3771. 4 R. U. Lemieux and S. Koto Tetrahedron 1974 30,1933. 5 H. von Voithenberg A. Skrzelewski J. C. Jochims and W. Pfleiderer Tetrahedron Letters 1974 4063. 6 R. J. Abraham C. M. Holden P. Loftus and D. Whittaker Org. Magn. Resonance 1974 6 184. 7 Y. Senda S.Imaizumi S. Ochiai and K. Fujita Tetrahedron 1974 30 539. 8 Y. Senda and S. Imaizumi Tetrahedron 1974 30 3813. 9 F. A. L. Anet J. Kraus W. Kitching D. Doddrell and D. Praeger Tetrahedron Letters 1974 3255. 10 S. F. Nelson and G. R. Weisinan J. Amer. Chem. Soc. 1974 % 711 1.11 Y. Nomura N. Masai and Y. Takeuchi J.C.S. Chem. Comm. 1974 288. Physical Methods and Techniques-Part (ii) Nuclear Magnetic Resonance geometrical arrangement of substituents in the vicinity of the C-H bond has been used to examine some hexahydro-3H-oxazolo[3,4-c]pyridines. l2 Long-range coupling between tin and carbon 5J13C-119Sn has been used as a probe for hyperconjugation from a carbon-metal bond into aromatic n-systems. It is shown to have marked stereochemical dependence and hence is a suitable conformational probe for such systems.' Application of Lanthanide Shift Reagents (LSR's).-An excellent review-type article has appeared which specifically examines the usefulness of LSR's as tools for the study of conformational equilibria in ~olution.'~ The difficulties encoun- tered in the differentiation between contact and pseudo-contact contributions to the observed shift and the location of the chelate with respect to the substrate by means of computerized search-and-fit programmes are discussed in detail.An equally thorough examination of the use of LSR's to determine conformational properties of acyclic alcohols is given by Williamson et al.' This points out the usefulness of Yb(d~m)~* for '3C studies since all contact shifts are eliminated and specifically examines the questions of distortions of the substrate by the complexation and of its influence upon the conformational equilibrium. Other work also affirms the usefulness of Yb(dpm) for 13Cstudies and points out the necessity of correcting for non-paramagnetic 'complexation shifts' using La(dpm) as a probe for this.16 The danger of distortion of structure or of favouring one partner of a conforma- tional equilibrium by shift reagents is obvious.Several pieces of work show that this is sometimes not a problem as in studies on dialkylnitro~amines'~ and NN-dimethylamides. Other work clearly demonstrates that the converse may apply however. Thus complexation of 1-methylcyclohexanol with a LSR causes the equatorial conformer of the alcohol to be more favoured.Ig N-Substituted-4- phenylpiperidines show differential complexation of equatorial and axial nitrogen lone pairs with the shift reagent Co" acetylacetonate; this work also presents evidence that the shift mechanism is pseudo-contact for this reagent.20 Studies in aqueous solution are of potential importance in systems of biological interest.Thus the perchlorates of Pr3 + and Ho3+have been used to examine the l2 Y. Takeuchi P. J. Chivers and T. A. Crabb J.C.S. Chem. Comm. 1974 210. l3 W. Adcock B. D. Gupta W. Kitching D. Dodrell and M. Geckle J. Amer. Chem. SOC. 1974 % 7360. l4 J. D. Roberts G. E. Hawkes J. Husar A. W. Roberts and D. W. Roberts Tetrahedron 1974 30 1833. l5 K. L. Williamson D. R. Clutter R.Emch M. Alexander A. E. Burroughs C. Chua and M. E. Bogel J. Amer. Chem. Sac. 1974 % 1471. l6 D. J. Chadwick and D. H. Williams J.C.S. Perkin 11 1974 1202. R. A. Perry and Y. L. Chow Cunud.J. Chem. 1974 52 315. G. Montaudo P. Maravigna S.Caccamese and V.Librando J. Org. Chem. 1974 39 2806. l9 J. Bouquant M. Wuilmet A. Maujean and J. Chuihe J.C.S. Chem. Comm. 1974 778. I. D. Blackburne A. R. Katritzky and Y. Takeuchi J. Amer. Chem. SOC.,1974 % 682. * dpm = dipivalomethanato. 20 R. B. Jones and L. Phill@s structure of cyclic j?-adenosine 3',5'-phosphate in aqueous solution ;the conform- ation is the same as in the crystalline state.2' Use has been made of edta com- plexes of La"' Pr'll Eu'" and Gd"' to examine the conformations in aqueous solution of adenosine 5'-monophosphate and cytidine 5'-rnonopho~phate.'~ A major advantage of these reagents is that they may be used at much higher pH values than the lanthanide aquo-ion probes which are insoluble above pH 6.23 Conformational Studies of Biological Interest.-The con formational properties of polypeptides are of continuing interest.Two series of macromolecular sequen- tial polypeptides containing up to four y-benzyl-~-glutamyl and one y-benzyl-L- glutamyl or L-leucyl residue in the repeating unit have been examined by 'H n.~.r.'~ The ordered conformations seem to be a distorted form of the a-helix with special types of cross lj-structure. A similar study of random copolymers of benzyl L-aspartate with 0-and p-nitrobenzyl aspartate shows that the presence of nitro-groups induces a reversal of the helix sense from left-hand to right-hand Complete assignment of the peptide NH resonances of angiotensin I1 (Asn' Vals) in aqueous solution has enabled 3JHNCH to be used for conformational analysis; no definite conclusions could be reached but the data exclude many possible arrangemenkZ6 A similar study has been made of the repeating penta- peptide unit of elastin." Following the identification by PerutzZ8 of two different quaternary structures for high-ligand-affinity deoxyhaemoglobins (T and R,conformational isomers) 'H n.m.r.has been used to observe the switch from T to R forms in solution at pH 9-7. The addition of inositol hexaphosphate at pH 7 reverses the change.29 Restricted rotation of a methyl group in a haem side-chain in some ferric myo- globin complexes has been examined. The barrier to this rotation may serve as a sensitive probe for the study of interactions between the haem side-chain and the ap~protein.~' Many molecules of biological importance form associations with metal ions in solution.The conformational implications of such associations have been re-examined for the case of the valinomycin-K+ complex. A 'H n.m.r. study at 220 MHz shows that the 3J, (aCH-CHD) of the valyl residue is 11.0 0.3 Hz rather than the previously suggested 3.54.0 Hz. This necessitates a revision of the hitherto accepted conf~rmation.~ The conformational change which occurs 21 D. K. Lavallee and A. H. Zeltmann J. Amer. Chem. SOC. 1974 96,5552. 22 C. M. Dobson R. J. P. Williams and A. V. Xavier J.C.S. Dalton 1974 1762. 23 C. D. Barry C. M. Dobson R. J. P. Williams and A. V. Xavier J.C.S. Dalton 1974 1765. 24 H. T. Storey R. C. Thompson P. M. Hardy and H. N. Rydon Polymer 1974 15 690.25 M.-H. Loucheux-Lefebvre A. Forchioni and C. Duflot Polymer 1974 15 474. 26 J. D. Gluckson J. Dadok and G. R. Marshall Biochemistry 1974 13 11. 27 D. W. Urry W. D. Cunningham and T. Ohnishi Biochemistry 1974 13 609. 28 M. F. Perutz Nature 1970 228 726. 29 S. Ogawa D. J. Patel and S. R. Simon Biochemistry 1974 13 2001. 30 I. Morishima and T. Iizuka J. Amer. Chem. Soc. 1974 96,7365. 31 D. W. Urry and N. G. Kumar Biochemistry 1974 13 1829. Physical Methods and Techniques-Part (ii) Nuclear Magnetic Resonance 2 1 when carp muscle calcium-binding parvalbumin releases its Ca2 + ion has been studied by a combination of 3C n.m.r. chemical shifts Nuclear Overhauser Effects and TI measurement^.^' There have been studies of small molecules of biological interest.For example in a study of some C-%substituted adenine nucleotides by ‘H and 1H[31P] Fourier transform n.m.r. it has been shown that the bulky C-8 substituent favours the syn orientation about the glycosidic linkage (3) rather than the more usual anti ~rientation.~~ This change is accompanied by distortion of the sugar phosphate backbone conformation about the C-4’-C-5’ bond. In a study of HO OH (3) X = Br or MeS 8-phenylpurines (3 ;sugar replaced by H X = Ph) conformational information concerning the imidazole-phenyl bond is obtained by studying the separation between the ortho proton resonance and the meta/pura resonances as a function of the nature of substituents in the base. The method calculates this separation as a function of the purine ring current the effect of which varies with the torsion angle.34 2 The Use of Spin-Lattice (Longitudinal) Relaxation Times in Structure Determination An excellent introduction to this potentially important area is given by Wehrli,35 and the reader is referred to this for an account of theory and practice up to the beginning of 1974.The most useful parameter is TyD,the contribution to TIobs (the observed longitudinal relaxation time) from dipole-dipole interactions with other dipolar nuclei in the molecule; this may be calculated if TIobs and the Nuclear Overhauser Enhancement* are known. A straightforward application of the method is the investigation of the variation of the 3C TI TyD,and NOE in cycloalkanes of different ring size.As the ring J2 S. J. Opella D. J. Nelson and 0.Jardetzky J. Amer. Chem. SOC.,1974 96,7157. 33 R. H. Sarma C. H. Lee F. E. Evans N. Yathindro and M. Sundaralingham J. Amer. Chem. Sor. 1974 96 7337. 34 F. Bergmann I. Tamir Z. Neiman and D. Lichtenberg Telrahedron 1974 30 3045. 35 F. W. Wehrli ‘Advances in Magnetic Relaxation’ ed. W. J. Orville-Thomas Academic Press London 1974. * NOE the increase in intensity of an observed resonance obtained upon saturation of the resonances in the system which are responsible for its relaxation. 22 R.B. Jones and L. Phillips size increases TlObs and T?D decrease markedly.36 'H Tl measurements may also show important variation with structure as has been demonstrated for the alkaloid vindolene in which each of the individual Tl values has been measured ; they vary from -0.45 to 2.7s and reflect the degree of crowding of each 'H by the others which are responsible for its rela~ation.~~ A rather more sophisticated study of 'H and 13C T,'s in small molecules (cis-and trans-crotonaldehyde) enables internuclear distances involving the methyl-group protons to be calcu- lated to a degree of accuracy comparable to that obtained by microwave methods.An important p~blication~~ studies dipole-dipole relaxation between protons in 3,4,6-tri-O-acetyl-1-O-benzoyl-2-chloro-2-deoxy-~-~-glucopyranose, using TI and NOE measurements based upon a new pulse technique. This enables the dipolar contribution to T of a given line in the 'Hspectrum arising from all other protons to be compared with the contributions from just its nearest neigh- bours and the ratio of the distances of these from the observed proton to be calculated.The specific replacement of hydrogens in a molecule by deuterium provides a useful structural probe for it is much less efficient in relaxing carbon to which it is bonded than is the lighter isotope. This has been exploited in the assignment of the I3C spectrum of thiamine hydr~chloride,~' where use is made of the fact that if all attached protons are replaced by *H the value of TYDfor 3C increases by a factor of ten; the effects are also seen by nearby non-hydrogen-bearing carbons which depend upon neighbouring hydrogens for relaxation. A I3C TIstudy of the back-bone and side-chains of oxytocin and lysin vaso- pressin gives a complete picture of their conformational fle~ibility.~' The suggestion is made that similar measurements on '3C-enriched neurohypophy- seal peptide hormones may allow a study of the conformational changes and dynamic processes which occur on binding to the neurophysins (intracellular protein carriers) A Tl study of the eleven resolvable "F resonances in fluoro- tyrosine alkaline phosphatase (corresponding to the eleven known tyrosines per subunit) has similarly enabled a detailed study of the general tertiary and quater- nary structure of the protein to be made.42 The differences in TI caused by different local environments of particular residues are a very useful probe and it is suggested that the fluorination approach may be of wider applicability.Several papers report the use of paramagnetic relaxation agents as alternatives or complements to lanthanide shift reagents as structural probes in solution. 36 S. Berger F. R. Kreissl and J. D. Roberts J. Amer. Chem. SOC.,1974 % 4348. 37 L. D. Hall and C. M. Preston Cunud.J. Chem. 1974.52 829. 38 R. Rowan tert. J. A. McCammon and B. D. Sykes J. Amer. Chem. SOC.,1974 % 4773. 39 R. Freeman H. D. W. Hill B. L. Tomlinson and L. D. Hall J. Chem. Phys. 1974,61 4466. 40 R. E. Echols and G. C. Levy J. Org. Chem. 1974 39 1321. 41 R. Deslauriers I. C. P. Smith and R. Walter J. Amer. Chem. SOC.,1974 % 2289. *' W. E. Hull and B. D. Sykes Biochemistry 1974 13 3431. 43 G. C. Levy and R.A. Komoroski J. Amer. Chem. SOC.,1974 % 678. Physical Methods and Techniques-Part (ii) Nuclear Magnetic Resonance 23 Their advantage is that the effects upon line-broadening (T,) or Tl show a simple r-6 dependence from the site of the metal. A definitive work by Levy and Komoro~ki~~ considers the effects of Cr and Fe trisacetylacetonates upon 13C T l’s in various organic substrates. The use of Gd(dpm) has also been e~amined.~~.~’ The selective relaxation effects upon 3Cresonance in adenosine 5’-triphosphate (ATP) which occur on binding Mn2 have been used to establish that the binding + site is the N-7 atom of the adenine base.46 Smith et aL4’ make use of the fact that TI relaxation of ,H resonance is entirely by an intramolecular quadrupole mechanism and consequently the interpretation of Tl’s is much easier than for ‘Hor I3C.They illustrate this by considering its implications with regard to stereochemistry intramolecular rotations and anisotropic motions in a variety of deuteriated alkane and aromatic deriva- tive~.~’ A note of caution is sounded in a paper which examines some of the experi- mental problems encountered in making Tl measurements?* From a study of I3C Tl in dioxan-D,O solutions by the progressive-saturation method35 it is noted that the dependence of this parameter on concentration roughly parallels the inverse of solution viscosity. A 2 1 (v/v) dioxan-D,O mixture has Tl of 6.2 0.6 s at 30 “C and is recommended as a standard for checking instrument performance.Using this the authors discuss the effects of dissolved oxygen intermolecular interactions and radiofrequency (H field inhomogeneity upon the experimental value of T ; in addition they discuss the experimental advan- tages of the progressive-saturation technique. Finally two papers deal with the determination of T in spin systems which are not in a Boltzmann equilibrium state. Ernst et al.49 point out that these may arise as a result of CIDNP effects,” Overhauser saturation experiments or radiofrequency pulses during a Tl measurement. They also occur in any repetitive Fourier transform experiment with a close pulse spacing (i.e.normal experimental conditions for I3C n.m.r.). Relative signal intensities vary as a result particularly in systems with homonuclear spin-spin coupling which is second order.No distortion of intensities will arise however in a weakly coupled system without degeneracy if a 90” pulse is used. The second paper5 deals specifically with the determination of TIin species with CIDNP using the polarized products of benzoyl peroxide decomposition for an example. 44 J. W. Faller and M. A. Adams Tetrahedron Letters 1974 699. 45 G. N. La Mar and E. C. Metz J. Amer. Chem. SOC.,1974 96 561 1. 46 Y. F. Lam G. P. P. Kuntz and G. Kotowycz J. Amer. Chem. SOC. 1974 % 1834. ” H. H. Mantsch H. SaitB L. C. Leitch and I. C. P. Smith J. Amer. Chem. SOC.,1974 % 256. 48 I. M. Armitage H. Huber D. H. Live H. Pearson and J. D. Roberts J. Magn. Resonance 1974 15 142. 49 S. Schaublin A. Hohener and R.R. Ernst J. Magn. Resonance 1974 13 196. I. Sadler Ann. Reports (B) 1973 70 22. ” K. A. Christensen D. M. Grant E. M. Schulman and C. Walling J. Phys. Chem. 1974 78 1971. 24 R.B. Jones und L. Phillips 4 The Study of ’H and 3H N.M.R. A survey52 of recent advances in deuterium and tritium n.m.r. covers work up to 1972. The field has since expanded and is obviously a growth point so a Report of progress in 1974 is in order. There have been studies of liquid-crystal structure by 2H In one of these,54 double-quantum transitions are observed in the case of a non-macro- scopically aligned lyotropic mesophase (a situation which is particularly favour- able for such observations). The observed intensities are so high that double- quantum transitions must be taken into account in the analysis and interpretation of 2H n.m.r.spectra of such systems. A complementary study is that of 2H n.m.r. in molecules which are aligned by liquid crystals. One report examines CD,SiH CH,SiD, and germanium analQgues in a nematic-phase liquid crystal. The calculated ratios of interatomic distances and bond angles which result are in good agreement with microwave data.55,56 2H N.m.r. has been used to study lipid bilayer~.~’-’~ In one of these ~tudies,~’ selectively deuteriated lipids are used to build up a liquid-crystalline bilayer and the anisotropic motion of the lipid molecules is studied. The results are compared with those obtained using a nitroxide spin label which the authors assert causes a significant distortion of the structure; the ’Hn.m.r.results represent a real improvement on the spin-label method. The point is reiterated in a second paper,58 and the same workers also examine the dynamic structure of the fatty acyl chains in a phospholipid bila~er.~~ Much the same comments emerge from work by Reeves et who examine the possibility of using selective or massive deuteriation together with 2Hn.m.r. to examine the local degree of micro-orientation in chain segments in membrane systems. The first application of 3H n.m.r. to a biosynthetic investigation has now appeared. [3H]Acetate is incorporated into penicillic acid by Penicillium cyclo-pium and the stereospecificity of labelling of the vinyl methylene group was directly demonstrated.6’ The authors point out that adequate spectra may be obtained from samples containing as little as 1 mCi of 3H,and there is no signifi- cant radiological hazard.The same group has used 3H n.m.r. to study the steric course of the A’-dehydrogenation of testosterone by a highly stereospecific 52 P. Diehl ‘Nuclear Magnetic Resonance Spectroscopy of Nuclei ather than Protons’ ed. T. Axenrod and G. A. Webb Wiley New York 1974 p. 275. 53 Z. Luz. R. C. Hewitt and S. Meiboom J. Chem. Phys. 1974 61 1758 ” H. Weunerstrom N. 0.Persson and B. Lindrnan J. Mugn. Resonance 1974 13 348. 55 R. Ader and A. Loewenstein Mol. Phys. 1974 27 11 13. 56 R. Ader and A. Loewenstein J. Amer. Chem. SOC.,1974 96,5336. ’’ J. Seelig and W. Niederberger J. Amer. Chem. SOC.,1974 96,2069.58 J. Seelig and W. Niederberger Biochemistry 1974 13 1585. ’9 A. Seelig and J. Seelig Biochemistry 1974 13 4839. 6o F. Fujiwara L. W. Reeves A. S. Tracey and L. A. Wilson J. Amer. Chem. SOC.,1974 % 5249. 61 J. M. A. Al-Rawi J. A. Elvidge D. K. Jaisival J. R. Jones and R. Thomas J.C.S. Chem. Comm. 1974 220. Ph-ysical Methods und Techniques-Part (ii) Nuclear Magnetic Resonunce 25 microbial lor,2P-truns elimination.62 In a full paper,63 they demonstrate the equality between 3H and ‘H chemical shifts and from an accurate measurement of the ratio between the gyromagnetic ratios of the two nuclei propose that ‘ghost references’ derived from observed ‘H n.m.r. frequencies may be used for 3H n.m.r. In the same work they give a simple example of a mechanistic application of 3H n.m.r.by a study of the Reimer-Tiemann reaction. 5 Carbon-13 N.M.R. Theory.-Factors influencing I ‘C chemical shifts remain poorly understood and the calculation of ‘useful‘ values is a difficult task. Extensive studies of polar substituent effects in alk~l~~ derivatives have been published but no and meaningful correlation of shieldings with 6-or total electron densities (calculated by extended HMO techniques) was obtained particularly for /3 or y effects where qualitative trends could not be reproduced. The use of the Average Excitation Energy (AEE) approximation AE in calculating paramagnetic* contributions (cP)to carbon shieldings in alkanes has been criticized and other approaches have been proposed allowing (a) variation in AE between different molecules obtained by calculation (modified MIND0/2) of all available excitation energies66 and (b)dependence of a ‘localized AE’ of a given carbon nucleus on substitution elsewhere in the molec~le.~ Both papers describe encouraging correlations with experiment.Good correlation has been described between alkane shieldings calculated within the framework of the AEE approximation by a population analysis modified to allow for polarity of the C-H bond.68 Variation in AE has been invoked to explain the carbon shifts of thiocarbonyl resonances com- pared with their carbonyl analogues69 and the protonation shifts of pterine and its derivative^.^' Deshielding S effects observed in various systems for carbon atoms in sterically crowded situations must lead to re-evaluation of the much-used steric explanation of shielding effects.’ In methyl decalin and androstane derivatives a 1,3-diaxial interaction between Me and OH deshields the former by 24 ~.p.m.’~ 62 J.M. A. Al-Rawi J. A. Elvidge R. Thomas and B. J. Wright J.C.S. Chem. Comm. 1974 1031. h3 J. M. A. Al-Rawi J. P. Bioxsidge C. O’Brien D. E. Caddy J. A. Elvidge J. R. Jones and E. A. Evans J.C.S. Perkin 11 1974 1635. 64 G. Miyajima and K. Nishimoto Org. Magn. Resonance 1974 6 313. 65 G. Miyajima K. Takahashi and K. Nishimoto Org. Magn. Resonance 1974 6 413. 66 I. Ando A. Nishioka and M. Kondo Bull. Chem. SOC.Japan 1974 47 1097. h7 K. Takaishi I. Ando M. Kondo R. Chujo and A. Nishioka Bull. Chem. SOC.Japan 1974 47 1559.6* S. Fliszar A. Goursot and H. Dugas J. Amer. Chem. SOC.,1974 96 4358. h9 H. 0. Kalinowski and H. Kessler Org. Nagn. Resonance 1974 6 305. 70 U. Ewers H. Gunther and L. Jaenicke Chem. Ber. 1974 107 3275. ’I J. B. Stothers ‘Carbon-13 N.M.R. Spectroscopy’ Academic Press New York 1972 p. 112. 72 S. H. Grover and J. B. Stothers Canad. J. Chem. 1974 52 870. * The paramagnetic term in the Ramsey expression for shielding. We consider the frequent use of ‘paramagnetic’ and ‘diamagnetic’ as synonyms respectively for ‘downfieid’ and ‘upfield’ inadvisable. 26 R. B. Jones and L. Phillips Similarly the peri interaction between methyl groups in 1,8-dimethylnaphthalene deshields both by 6.6 p.p.m. relative to 1-meth~lnaphthalene.~ Other examples of downfield 6 and E effects have been described for the methoxy carbon atom in 2,6-dialkylated an is ole^^^ and the methyl carbon in related 2,6-dialkylated methyl phenyl ~ulphides,~ which increase with increasing size of the alkyl substituents for the peri interaction between methyl groups on C-3 and C-4 of ben~ofuran,~ and for interactions between methyl groups in cyclohexanone derivative^.^^ Of possibly related origin are the shielding effects seen on reson- ances from sterically compressed aromatic carbons in 2,2-metacy~lophanes,~~ although the authors suggest that n-electron redistribution due to direct trans- annular repulsion is responsible.Shielding E effects have been seen in 43-dimethylphenanthrene resonances from sterically interacting methyls being to high field (4.6p.p.m.) of the methyl resonance of 4-meth~lphenanthrene,~~ and on the -S(0)CH3 in 2,4,6-tri-isopropylphenylmethyl sulphoxide (relative to phenyl methyl sulphoxide) although deshielding E effects were seen in both the related sulphides and sulphones.75 Further understanding of the origin of the y effect must derive from data obtained from aliphatic molecules of rigid conformation where both Ygauche and yfranseffects may be simultaneously studied. The study of a number of 'frozen-out' cyclohexane derivatives" illustrates not only the application of 3Cn.m.r. to this type of conformational problem but also provides direct comparison of Ygaucfte (i.e.substituent axial) and ytrans (equatorial) effects. The significant size of the latter and its correlation with electronegativity (increasing electronegativity f increasing shielding) suggests that electronic (through bond?) contributions cannot be ignored in explaining the y effect.Further ygouche correlates with ytrans (ygauche = -2.5ylrans) for small electronegative substituents behaviour seen in other systems where steric interactions are thought to be unimportant.81 The relationship persists even where similarly sized substituents (i.e.CN and NC) produce greatly differing Ygauehe effects and may therefore be evidence for pre- dominance of (stereospecific?) electronic ygouche effects in these systems. yrrans Effects have also been measured in 1-substituted adamantanes,82 where electro- negativity dependence in the series Me NH, OH and F was also observed.However the trend for the halogens differs in sign from that of the equatorial cyclohexyl halides. Attempts to separate steric from electronic shielding contri- 73 N. K. Wilson and J. B. Stothers J. Magn. Resonance 1974 15 31. 74 G. W. Buchanan G. Monlaudo and P. Finocchiaro Canad. J. Chem. 1974,52 767. 75 G. W. Buchanan C. Reyes-Zamora and D. E. Clarke Canad. J. Chem. 1974,52,3895. 76 N. Platzer J. J. Basselier and P. Demerseman Bull. SOC.chim. France 1974 905. 77 J. B Stothers and C. T. Tan Canad. J. Chem. 1974,52 308. 78 T. Sato T. Takemura and M. Kainosho J.C.S. Chem. Comm. 1974 97. 79 J. B. Stothers personal communication quoted in ref. 75. H. J. Schneider and V. Hoppen Tetrahedron Letters 1974 579.' R. B. Jones and L. Phillips unpublished results. 82 G. E. Maciel H. C. Dorn R. L. Greene W. A. Kleschick M. R. Peterson and G. H. Wahl Org. Magn. Resonance 1974 6 179. Physical Methods and Techniques-Part (ii) Nuclear Magnetic Resonance 27 butions in 9cc-substituted cortisolss3 illustrate further the inadequacy of present shielding theories. The failure of CND0/2 to estimate electronic shielding effects and the inverse correlation of ygauchewith substituent size were noted although for a given substituent correlation of ygauckeshielding with internuclear distances (X-ray data) was seen. The downfield Y~,~,,~ effect noted for the C-19 methyl group on the introduction of the 9a-F has also been observed in other related systems" and contrasts with the upfield effects seen at methylene and methine centres described in the above This illustrates the structural dependence of the y effect and emphasizes the caution with which 13C substituent additivity rules must be treated.Studies on unsaturated fatty acids have shown the importance of n-bond polarization by intramolecular dipolar electric fields on olefinic chemical shifts ;84 substituent effects were interpreted in terms of steric and linear electric field effects. Contributions to coupling constants between directly bonded '3C nuclei arising from various mechanisms have been calculated and good agreement is claimed with e~periment.~' Vicinal 3J13c-,3c couplings were found to follow closely a calculated 'Karplus-type' curve.86 Applications.-Applications of 3Cn.m.r.are growing rapidly with a consequent enormous increase in the volume of data published although it is disturbing to note cases in which assignment of 13C spectra of available compounds has become an end in itself. The use for full unknown structure determination remains impossible in the absence of closely related models because of the general lack of understanding of l3C shielding but information available from noise- decoupled and 'off-resonance' spectra (carbon types and numbers of bonded protons) has frequently proved useful in structural problems. Physical Organic Chemistry. Emphasis here is given to selected applications of 13Cn.m.r. to physical organic problems and much work has been directed to protonation-deprotonation effects on carbon resonances.Predominant 0-protonation of HCONMe in aqueous acidic media has been confirmeds7 by the observation of discrete I3C methyl resonances from the protonated species. Slow isomerization (uia N-protonation) sufficient to cause coalescence of the 'H methyl resonances because of the small chemical shift differences limited the application of 'H n.m.r. to this problem. Rates of hindered rotation and acid- base exchange of benzaldehydes in superacid media have been determined by analysis of I3C spectra with a four-site exchange programme the proton 83 D. D. Giannini P. A. Kollman N. S. Bhacca and M. E. Wolff J. Amer. Chem. SOC. 1974 96 5462. J. G. Batchelor R. J. Cushley and J. H. Prestegard J. Org. Chem. 1974 39 1698.'* J. M. Schulman and M. D. Newton J. Amer. Chem. SOC.,1974 96,6295. 86 D. Doddrell I. Burfitt J. B. Grutzner and M. Barfield J. Amer. Chem. SOC.,1974 96. 1241. " R. A. McQelland and W. F. Reynolds J.C.S. Chem. Comm. 1974 824. 88 T. Drakenberg S. Forsen and J. M. Sommer J.C.S. Perkin ZZ 1974 520. 28 R. B. Jones and L. Phillips spectra were far too complex to be handled in this way. I3C Chemical shifts have been used to estimate n-electron densities in several mesoionic sydnones and related compoundsg9 and confirm calculated predictions. Protonation of 3-aryl-sydnones in strong acid media has been studied by * 3C and 15N n.m.~.~O Changes in 13C shifts as a function of pH obtained by titration have much application for analysis of spectra of peptides and proteins.” Titration curves for amino-acids have been de~cribed.~~,~~ The ionization shifts on deprotonation at adjacent sites have been attributed to competition between deshielding re- sulting from a decrease in AE and shielding by increased electron density whereas at remote sites only the latter effect is important.92 Some additivity of protonation shifts has been ~laimed.’~ Both 3513c-lH coupling9’ and the pH dependence of ’ 3C resonances93 have been used to determine side-chain configurations in amino-acids.The co-ordination of organic molecules to metal ions can be examined where discrete carbon resonances are visible from both bound and free molecules. The solvation of A13+ and Mg2+ by alcohols96 and the complexation of A13+ with amides” have been thus studied.I3C and I5N spectra of chlorophyll u and its magnesium-free derivative pheophytin have been used to study the binding of the central magnesium atom ; quaternary carbons were assigned by their 2J, and 3JcHcouplings to visible protons by INDOR techniques. The effect of the magnesium atom was analysed in terms of perturbation of the interaction of the nitrogen lone pairs with the unoccupied n*-orbitals leading to variation in AE(n,n*)and thereby alteration of op,and a qualitative picture was obtained of the nature of the Mg-N bond-ing.’” ’ 3C Studies on coproporphyrin-type molecules and their zinc@) and thallium(w) derivatives have been used to support the existence of an 18-atom 18-n-electron delocalization system in these molecules over the alternative proposal of a 16-atom 18-n-electron system.’” Significant broadening of the signal (15-20 Hz) of the a-pyrrole carbon atoms was attributed to tautomerism of the NH protons between two available sites at a rate corresponding to coales- cence of the carbon signals from each distinct environment.The unpaired- 89 M. T. W. Hearn and K. T. Yotts J.C.S. Perkin II 1974 875. 90 A. J. Buglass J.C.S. Chem. Comm. 1974 313. 91 J. Feeney P. Partington and G. C. K. Roberts J. Mugn. Resonance 1974 13 268. ” A. R. Quirt J. R. Lyerla I. R. Peat J. S. Cohen W. F. Reynolds and M. H. Freedman J. Amer. Chem. Soc. 1974 % 570. 93 S. Tran-Dinh S. Ferrnandjian E. Sala R. Mermet-Bouvier M. Cohen and P. Fromageot J. Amer.Chem. Soc. 1974 % 1484. 94 K. F. Koch J. A. Rhaades E. W. Hagaman and E. Wenkert J. Amer. Chem. Sot. 1974 % 3300. 95 J. Feeney P. E. Hanson and G. C. K. Roberts J.C.S. Chem. Comm. 1974 465. 96 G. W. Stockton and J. S. Martin Canad. J. Chem. 1974,52 744. 97 D. Canet J.-J. Delpuech M. R. Khaddar and P. Rubini J. Magn. Resonance 1974 15 325. 9* T. T. Nakashima D. D. Traficante and G. E. Maciel J. Phys. Chem. 1974 78 124. 99 I. D. Gay J. Phys. Chem. 1974 78 38. S. G. Boxer G. L. Gloss and J. J. Katz J. Amer. Chem. Soc. 1974 % 7058. lo’ R. J. Abraham G. E. Hawkes and K. M. Smith J.C.S. Perkin II 1974 627. Physical Methods and Techniques- Purr (ii) Nuclear Magnetic Resonance electron spin density in low-spin iron(m) complexes with protoporphyrin has been determined by measurement of the 13C hyperfine shifts.lo2 The data have been used to prove the interaction of peripheral substituents on the molecule with the delocalized n-system to assist understanding of the relationship between electronic structure and the biological role of these molecules.The association of acetone and phenol in CCl solution has been studied by observation of the carbonyl 3C re~onance.~~ Thermodynamic data for self- association of these species were also obtained. 13CShifts and 1J13c-lH couplings have been measured for simple organic molecules adsorbed on to silica ;99 carbon atoms adjacent to probable binding sites generally showed downfield shifts relative to the neat liquid (0-10 p.p.m.) whereas other carbons showed upfield shifts (0-2 p.p.m.).Extensive work on the 13C n.m.r. of onium ionslo3 and carbocation~'~~ in superacid media has been completed by Olah. Organometallic Chemistry. Extensive use has been made of 3C n.m.r. in organo- metallic chemistry to aid structure determination and deduce information about C-metal bonding. As frequently admitted the latter deductions are handicapped by ignorance of the factors influencing the observed shieldings. Studies on car- bony1 complexes are widespread and early work has been reviewed;'05 reson- ances usually lie in the region 150-250 p.p.m. below TMS with a general trend to lower field with increased bridging. The application of the relaxation agent Cr(acac) is widespread as is the use of 13C-enriched CO to overcome sensitivity problems caused by low solubility at the low temperatures frequently required.Application to structure determination is illustrated by the study on [Rh 2(CO),o(p2-CO)2(p3-CO)8]2 -in the presence of Cr(acac) ,where resonances due to terminal and doubly and triply bridged carbonyls could be distinguished and integrated. lo6 'JRhXcouplings are inversely related to Rh-C bond lengths. O6 The qualitative solution dynamics of M3(CO),2 systems (M = Fe Ru or 0s) have been studied by variable-temperature '3C n.m.r.lo7 The activation energy for the isomerization of the [(Cp)(OC)Fe(p-CO),Fe(CO)(Cp)]molecule has been estimated by simulation of the temperature dependence of the carbonyl signals. lo* Severe quadrupolar broadening of carbonyl resonances has been demonstrated for carbonyl derivatives of 59C0(I = 7/2) and 55Mn (I = 5/2) which is reduced considerably by recording spectra at lower temperatures.'02 K. Wuthrich and R. Baumann Helv. Chim. Acra 1974 57 336. Io3 G. A. Olah G. Liang and J. Staral J. Amer. Chem. Soc. 1974 % 81 12 and references therein. lo' G. A. Olah G. Liang A. Babiak and R. K. Murray J. Amer. Chem. SOC. 1974 96 6794 and references therein. '05 L. J. Todd and J. R. Wilkinson J. Organometallic Chem. 1974 77 1. lob P. Chini S. Martinengo D. J. A. McCaffey and B. T. Heaton J.C.S. Chem. Comm. 1974 310. lo' A. Forster B. F. G. Johnson J. Lewis T. W. Matheson B. H. Robinson and W. G. Jackson J.C.S. Chem. Comm. 1974 1042. lo*D. C. Harris E. Rosenberg and J.D. Roberts J.C.S. Dalton 1974 2398. Io9 L. J. Todd and J. R. Wilkinson J. Organometallic Chem. 1974 80 C31. 30 R.B. Jones and L. Phillips Several attempts have been made to examine the nature of the metal-C=O bonds from I3C measurements. It is argued"' that correlation of I3C shifts with carbonyl i.r. stretching frequencies and metal-C bond lengths leads to the hypothesis that increasing metal-warbonyl n-back donation causes progressive deshielding of the carbonyl resonance. The shielding of the carbon nucleus is inversely related to the carbon electron density and the authors therefore attribute the observed trends to variation in cp,but were unable to justify this assumption qualitatively. Studies' ' in some thiocarbonyl derivatives demonstrated the con- siderable deshielding of thiocarbonyl resonances over carbonyl resonances [by 218 p.p.m.for (Z-C,H,)M~(CO)~(CS) and 105 p.p.m. for (7t-C5H5)Fe(CO),(CS)PF6]. This was attributed to changes in both AE in cpand increased metal n-back donation to the thiocarbonyl ligand. The interpretation of chemical shifts of metal-bound carbonyls purely in terms of changes in oPalone has been strongly and it has been pointed out that variation in the calculated diamag- netic shielding terms (ad)of carbonyl groups bound to different heavy metals can amount to several hundred p.p.m. Further evidence for the importance of c,,has come from studies of the carbonyl shifts of Ni(CO) and Fe(CO) relative to those of free C0.113Independent attempts to calculate both cdand cpterms have led to the conclusion that the carbonyl shifts on binding to Ni (12.5 p.p.m.) could be explained by changes in either cdor CT, (or both) whereas the shift on binding to Fe (31 p.p.m.) could only be explained by changes in cP.I3C Studies on both carbonyl and aryl nuclei on complexation of Cr(CO) with monosubstituted benzenes were interpreted as indicating a net withdrawal of electron density by the Cr(CO) fragment from the framework of the arene ring.114 Several studies have appeared on 13C shifts and 19sPt-'3C couplings in organo-plati-num(Ir),' 15-i17 and organo-platinum(1v)' ' derivatives and the 'cis and trnns n.m.r.influences' of other co-ordinated ligands have been discussed. The former is tentatively attributed to steric interactions"7 and the latter which follows the same qualitative relationship in both Pt" and Pt'" derivatives,"' to a-orbital rehybridization.Studies on the complexation of Ag' with ketones have suggested that the carbonyl group acts as an n-rather than a n-donor."' Simple olefins bound to Ag' have been studied both in solution120 and absorbed on to an AgNaX-type G. M. Bodner and L. J. Todd Inorg. Chem. 1974 13 1335. ]I1 G. M. Bodner Inorg. Chem. 1974 13 2563. I I J. Evans and J. R. Norton Inorg. Chem. 1974 13 3042. *I3 H. Mahnke R. K,Sheline and H. W. Spiess J. Chem. Phys. 1974 61 55. 'I4 G. M. Bodner and L. J. Todd Inorg. Chem. 1974 13 360. 'I5 H. C. Clark and J. E. H. Ward J. Amer. Chem. SOC.,1974 96 1741. H. C. Clark L. E. Manzer and J.E. H. Ward Canad. J. Chem. 1974,52 1165. H. C. Clark and J. E. H. Ward Canad. J. Chem. 1974,52 570. H. C. Clark t.E. Manzer and J. E. H. Ward Canad. J. Chem. 1974 52 1973. D. R. Crist Z.-H. Hsieh G. J. Jordan F. P. Schinco and C. A. Maciorowski J. Arner. Chem. Soc. 1974 96,4932. 120 D. M' ichel W. Meiler and E. Angele 2.phys. Chem. (Leipzig) 1974 255 389. Physical Methods and Techniques-Part (ii) Nuclear Magnetic Resonance 3 1 zeolite.12' The olefinic carbon resonances showed high-field shifts on binding of similar magnitude in each case. Biochemistry.* Uses of 13C as biosynthetic label are growing the feeding of doubly labelled acetate ['3CH3'3C02Na] becoming increasingly popular for obtaining information about bond fission during biosynthesis.Incorporations required for visible peak enhancements (generally >0.2%)have largely limited the application of '3Cn.m.r. to micro-organisms and fungal metabolites although incorporation of a I3C-labelled precursor into a higher plant has been detected.' 22 The incorporation of singly labelled acetate@) alone has been used to study the biosynthesis of hirsutic acid C,123 taji~anthone,'~~ cytochalasinsB and D,125and metacyclo- and undecyl-prodigiosin.'26 Feeding of doubly labelled acetate (with the subsequent observation of 'Jcc satellite lines arising from acetate subunits which remain intact during biosynthesis) has been used to prove methyl migration during the biosynthesis of the triprenylphenol asc~chlorin,'~' to suggest oxidative fission of a preformed aromatic precursor during the biosyn- thesis of multicolic and multicolosic acids,*28 to prove C-C bond fission during the biosynthesis of the fungal sesquiterpenoid o~alicin,'~~ to distinguish between alternative folding patterns of the polyketide chains prior to cyclization in the biosynthesis of the metabolite ~terigmatocystin,'~~ to study the polyketide origin of the metabolite ~chrephilone,'~' and to determine the partial acetate origin of the metabolite tenellin.'32 The latter authors argue that the 'doubly labelled acetate' method is superior to the singly labelled technique where only low 3C incorporations are achieved if it may be assumed that contributions to spin-lattice relaxation from bonded '3C are negligible.This has been disproved for non-protonated carbon atoms,133 where I3C-' 3Cdipolar relaxation provides a very important contribution to the overall relaxation rate (1/Tl).Further where coupled carbon resonances are close together they constitute an AB system and signal intensity errors (a function of relaxation rates and flip angles) are seen in tightly coupled homonuclear systems in non-Boltnnann equilibrium states as is the case for rapidly pulsed FT experiment^.'^^ l2 I D. Michel W. Meiler and D. Hoppach Z. phys. Chem. (Leipzig) 1974 255 509. A. R. Battersby P. W. Sheldrake and J. A. Milner Tetrahedron Letters 1974 3315. lZ3 T. C. Feline G. Mellows R. B. Jones and L. Phillips J.C.S. Chem. Comm. 1974 63. IZ4 J. S. E. Holker R. D. Lapper and T. J. Simpson J.C.S.Perkin I 1974 2135. W. Graf J. L. Robert J. C. Vederas C. Tarnm P. H. Solomon I. Miura and K. Nakanishi Hefu. Chim. Acta 1974 57 1801. '26 H. H. Wasserrnan C. K. Shaw R. J. Sykes and R.J. Cushley Tetrahedron Letters 1974 2787. M. Tanabe and K. T. Suzuki J.C.S. Chem. Comm. 1974,445. J. A. Gudgeon J. S. E. Holker and T. J. Simpson J.C.S. Chem. Comm. 1974 636. M. Tanabe and K. T. Suzuki Tetrahedron Letters 1974 4417. I30 H. Seto L. W. Cary and M. Tanabe Tetrahedron Letters 1974 4491. 13' H. Set0 and M. Tanabe Tetrahedron Letters 1974 651. 13' A. G. McInnes D. G. Smith J. A. Walter L. C. Vining and J. L. C. Wright J.C.S. Chem. Comm. 1974,282. C. G. Moreland and F. I. Carroll J. Magn. Resonance 1974 15 596. S. Schaublin A. Hohener and R.R. Ernst J. Magn. Resonance 1974 13 196. * See also Ch. 15. 32 R. B. Jones and L. Phillips Examples of the use of other 13C precursors in biosynthetic studies have been described. [2-13C]Mevalonic acid has been synthesized and incorporated into the sequiterpenoid trichothecolone. (2RS,3S)-[4-'3C]Valine has been syn- thesized and incorporated into penicillin V.'36 [1-' 3C]Glucosamine and [6-'3C]Glucose have been incorporated into the antibiotic neomycin. 37 I3C-Labelled methionine baminolevulinic acid porphobilinogen and uroporphy- rinogens I-IV have been incorporated into vitamin B ,.'38 Selective bio- synthetic enrichment studies with labelled amino-acids have been used to assign all carbon resonances seen from the decapeptide gramicidin S-A.139 13Chas been utilized in many other diverse areas of biochemistry. An n.m.r. probe designed to take sample tubes of 20 mm 0.d. has been developed and has enabled observation of 13Cresonances from some minor bases in tRNA which were assigned by comparison with models.'40 '3C7'''s have been used to confirm the random coil conformation of the linear pentadecapeptide gramicidin A in E2H6]DMS0 and the existence of its double-helical dimer in CD30D.14' The binding of '3C0 to various haemoglobins has been monitored by means of the carbonyl resonances. The two discrete resonances seen were specifically assigned to 13C0bound to either Q or /Isubunits and both the thermodynamic stability and the ease of replacement of CO by O2were found to differ for each site.'42 An unusually high pK value of '3C-enriched carboxymethyl-cobalamin (HO '3C'3CH,-cobalamin) was determined by the titration shifts of both methylene and carboxyl ~arb0ns.l~~ The interaction of the Cu2 + ion with pyrimi- dine nucleosides and nucleotides has been studied by observation of the changes in relaxation rates (estimated by conventional inversion-recovery techniques and from signal linewidths) of 13C nuclei due to the paramagnetic ion.144 This technique has been criticized.14' The specific co-ordination of Ca2+ and Na' (but not K+ or Zn2 +)with the macrocyclic antibiotic rifamycin S has been proven by the magnitudes of the shifts on the 13C resonances observed on addition of the metal ion to the antibi~tic.'~~ Assignment ofCarbon Resonances.Several novel assignment techniques have been described in the period of this Report. 'Partially Relaxed Fourier Transform' 135 J. R. Hanson T. Marten and M. Siverns J.C.S. Perkin I 1974 1033. 13' D. J. Aberhart and L. J. Lin J.C.S. Perkin I 1974 2320. 13' K. L. Rinehart J. M. Malik R. S.Nystrom R. M. Stroshane S. T. Truitt M. Tani- guchi J. P. Rolls W. J. Haak and B. A. Ruff J. Amer. Chem. Soc. 1974 96,2263. 13' A. I. Scott C. A. Townsend K. Okada M. Kajiwara R. J. Cushley and P. J. Whitman 1. Amer. Chem. SOC.,1974 96,8069. 139 J. A. Sogn L. C. Craig and W. A.Gibbons J. Amer. Chem. SOC., 1974 96,3306. laa R. A. Komoroski and A. Allerhand Biochemistry 1974 13 369. la' E. T. Fossel W. R. Veatch Yu. A. Ovchinnikov and E. R. Blout Biochemistry 1974 13 5264.R. B. Moon and J. H. Richards Biochemistry 1974 13 3437. 143 T. E. Walker H. P. C. Hogenkamp T. E. Needham and N. A. Matwiyoff J.C.S. Chem. Comm. 1974 85. ja4 G. Kotowycz Canad. J. Chem. 1974,52 924. Ia5 W. G. Esperin W. C. Hutton S. T. Chow and R. B. Martin J. Amer. Chem. SOC. 1974 96,8111. D. Leibfritz Tetrahedron Letters 1974 4125. Physical Methods and Techniques-Part (ii) Nuclear Magnetic Resonance 33 spectra (PRFT) have been used as an alternative to off-resonance double ir- radiation for determination of numbers of directly bonded protons during the assignment of the sesquiterpenoid ~uanhternone'~~ and the polyketides cyto- chalasins €3 and D.125 The use of paramagnetic relaxation agents to complement lanthanide shift reagents has been mentioned above.The interaction of Cr(acac) and Fe(acac) with a number of various functionalized substrates has been studied and the advantages and disadvantages of their use have been discussed.43 The lanthanide relaxation agent Gd(fod) has been used to assign olefinic carbons in the conjugated side-chain of all-tr~ns-retinal.'~~ The Gd(fod) binds to the terminal aldehyde function and addition of successive aliquots causes the se- quential disappearance (through line-broadening) of resonances from carbon atoms at increasing distances from the binding site. The use of TiCl as an alternative shift reagent to Eu(fod) in 13Cn.m.r. has been ~uggested.'~~ Observed shifts are generally similar although TiCI appears to produce greater shifts of carbonyl resonances; its smaller size may also be of advantage in sterically hindered systems.An attempted use of long-range 13C-'H couplings to assign quaternary atoms in polynuclear aromatic hydrocarbons failed because of the difficulties of corre- lating the observed couplings with structure.' 50 The assignment of carbonyl resonances in large molecules (especially in peptides and proteins) is particularly difficult and several approaches have been made to this problem. The pH dependence of carboxylate functions is useful where pK,'s are known or accurately estimable." In cases where carboxylate resonances are hidden they may be detected and assigned by a subtraction of a spectrum obtained at one pH from another obtained at a different pH.pH- insensitive resonances cancel and therefore disappear revealing the hidden carboxylate resonance. This technique was used during a study on the basic pancreatic trypsin inhibitor.' 5' Two further general techniques are the solvent dependence of carbonyl resonances and their assignment by solvent-mixture titration' 52 and the detection and specific collapse by secondary irradiation of long-range C-H coupling^.^^ Amide resonances may be assigned by recording spectra in a 50 50mixture of H20and D,O. The NH/ND exchange is sufficiently slow to show two lines in the I3C spectrum (corresponding to -CONH- and -COND-species) separated by a typical deuterium isotope effect ofO.1 p.p.m9' The well known 'second-order 'H off-resonance fingerprint' effects,' 53,1 54 of considerable value in interpreting off-resonance spectra have been quantitatively explained for a AA'X system.' '4i K.Nakanishi R. Crouch I. Miura X. Dominguez A. Zamudio and R. Villareal J. Amer. Chem. SOC.,1974 % 609. 148 R. Rowan and B. D. Sykes J. Amer. Chem. SOC.,1974 96,7000 149 A. K. Bose and P. R. Srinvasan J. Magn. Resonance 1974 15 592. R. S. Ozubka G. W. Buchanan and I. C. P. Smith Cunad. J. Chem. 1974,52 2493. 151 W. Maurer W. Haar and H. Ruterjans Z. phys. Chem. (Frankfurr) 1974 93 119. '" D. W. Urry L. W. Mitchell and T. Ohnishi Biochemistry 1974 13 4083. Is' G. Jikeli W. Herrig and H. Gunther J. Amer. Chem. SOC. 1974 96,323. J. B. Grutzner J.C.S.Chem. Comm. 1974 64. ' 55 H. Fritz and H. Sauter J. Magn.Resonance 1974 15 177. 34 R .B. Jones and L. Phillips Several very closely related techniques have been described for the assignment and sign determination of '3C-'H coupling constants involving pulsed hetero- nuclear decoupling to produce total' 56-' 58 or partial population in~ersion'~~-'~~ of selected proton transitions seen as intensity variations in the carbon spectrum. Total population inversion produces FT spectra analogous to CW INDOR spectra. 156 K. G. R. Pachler and P. L. Wessels J. Magn. Resonance 1973 12 337. 57 A. A. Chalmers K. G. R. Pachler and P. L. Wessels Org. Magn. Resonance 1974 6 445. ls8 A. A. Chalmers K. G. R. Pachler and P. L. Wessels J. Magn. Resonance 1974 15 415. i59 J. Runsink J. de Wit and W. D. Weringa Tetrahedron Letters 1974 55.160 A. Kiewiet J. de Wit and W. D. Weringa Org. Magn. Resonance 1974 6 461. 16' S. Sorensen R. S. Hansen and H. J. Jakobsen J. Magn. Resonance 1974 14 243. '62 H. J. Jakobsen S. A. A. Linde and S. Sorensen J. Magn. Resonance. 1974.15. 385.