The 1H, 13C, and 15N high field nuclear magnetic resonance spectra of the cyclic peptide viomycin have been fully assigned using homo- and heteronuclear double resonance experiments and pH effects. In addition it is shown how the two- and three-bond H-D isotope effects upon carbonyl resonances may assist in their assignment. The resistance to exchange with solvent water of the amide proton involved in the transannular hydrogen bond is observed directly in the 1H spectra, via the isotope effect on a carbonyl resonance in the 13C spectra, and via the one-bond 1H couppling in the 15N spectra.

Medium effects upon the 15N chemical shifts of some small peptides are reported, to allow the relative importance of solvent, pH, and sequence effects to be delineated. A change in the pH of a solution of carnosine (P-alanylhistidine) from 0.4 to 11 .O causes the His 15N resonance to shift downfield by 8.5 p.p.m. Changing the solvent from dimethyl sulphoxide (DMSO) to trifluoroacetic acid (TFA) causes downfield shifts of ca. 4 p.p.m. for the peptide nitrogen resonances of some N-acetyldipeptides, and explains why the 15N shift observed for polyglycine in TFA solution is to low field of that predicted earlier for a glycine residue of a peptide in DMSO solution. The magnitude of these pH and solvent effects is such that they may mask any sequence effects upon peptide 15N shifts, which are reported to be 1-4 p.p.m. for dipeptides in aqueous solution.College, Mile End Road, London E l 4NS MAGNETIC resonance spectra of the 15N nucleus are being used increasingly in the study of peptides.1-9 Two important features of these studies are the conclusions of Roberts and his co-workers5 and conclusions from this l a b ~r a t o r y . ~ The former group found that the chemical shifts of the peptide 15N nuclei in simple dipeptides (aqueous solutions; pH range 5.0-6.1) were slightly sensitive to the nature of both C-terminal and N-terminal units. Thus it is formally possible that sequence information may be obtained by 15N n.m.r., a non-destructive method. Our ~t u d i e s , ~ however, indicated that there was no detectable sequence information from 15N chemical shifts of N-acetyldipeptides, at least in dimethyl sulphoxide (DMSO) solution. Accordingly we have undertaken a study of medium effects (including both solvent and pH effects) upon peptide 15N chemical shifts for some simple model systems, to clarify the situation.Assignment of 15N Resonances.-The first stage in the investigation of peptides by 15N n.m.r. is the assignment of the resonances to specific amino-acid units. We shall assume initially that sequence effects upon the l5N shifts are small and that in the absence of any differential effects upon the amide nitrogens in the peptide (caused by solvation, hydrogen bonding, or conformation changes), the relative I5N shifts of the amide nitrogens are controlled mainly by the nature of the amino-acid unit itself.Previously published 15N chemical shifts of aminoacids,l N-formyl a r n i n o -a ~i d s , ~, ~~ N-acetyl amino-acid~,~ and amino-acid methyl ester hydrochlorides allow estimation of the effect of substitution at the amino-acid a-carbon atom upon the 15N shifts. In the manner previously adopted 1*11 we assume an additivity relationship for these substituent effects similar to that of Grant and Pau112 for 13C chemical shifts. The approach is

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The proton magnetic resonance spectra of such complexes provide therefore a very sensitive test for the steric arrangement of the ligand. We have used this method to establish the conformation of trimethylthiourea (tmtu) in its complexes with cobalt(n), EXPERIMENTAL Prepamtions.-TlieComplexes of general formula Co-(tmtu),X, (X = C1, Br, or I) have been prepared by literature methods 49 and recrystallized from ethanollight petroleum mixtures.The ligand was deuteriated a t the nitrogen by dissolving it in D,O and drying it under vacuum several times. The degree of deuteriation was checked by n.m.r.: the N H signal had completely disappeared and the methyl attached to i t appeared as a sharp singlet.

N.m.r. spectra of some potentially bidentate thiosemicarbazones, NR3H*CS*NH.N:CR2R1 (L: R1,R2 = H, Me, Ph, or R1R2 = CI HI 1; R3 = H or Ph), and their complexes [ZnC12L] or [ZnCI,L,] have been measured. The results are compared with semiempirical CN D 0 / 2 total-energy calculations. Only one conformer, containing an intramolecular hydrogen bond, is present in solution. All the zinc(ii) complexes are tetrahedral. The possible formation of complexes in which the ligand is bidentate is discussed. THE synthesis and physicochemical characterisation of EXPERIMENTAL transition-metal complexes with thiosemicarbazone-typeThe starting carbonyl compounds and the thiosemib a n d s is being undertaken in Our laboratory-The carbazidc were Merck reagent-grade products ; 4-phenylinterest in these ligands is connected with their flexithiosernicarbazide was a I< and I< reagent and ZnC1, a n bility which leads to extremely variable behaviour in Erba R P product. Thiosemicarbazones were prepared their reactions with transition and non-transition metals. following tlie procedures of Sah and Daniels.6 TThe crude TABLE 1Analytical data for the thiosemicarbazones Analysis (%I Compound Acetone thiosemicarbazone, (I) C4HgN,S