High-resolution nitrogen-15 NMR spectra of six nucleosides and six nucleotides have been obtained at the naturalabundance level by high-resolution NMR spectroscopy. All of the nitrogen resonances have been assigned on the basis of comparison of their chemical shifts and nitrogen-hydrogen coupling constants with those of related compounds. Studies of the effect of protonation on the nitrogen chemical shifts of four of these compounds enabled the site of protonation to be detected directly.

The 15N chemical shifts of eight aliphatic tripeptides have been measured at the natural-abundance level. For a given tripeptide, the resonances of the COOH-terminal and NHlrterminal amino acids can be identified by measurements at low or high pH. The shifts of the NH~rterminal amino acid nitrogens are essentially independent of the amino acids in the rest of the peptide. The shifts of the other nitrogens are characteristic of the amino acids themselves and of the immediately preceding amino acid toward the NH2 terminus. Nonterminal amide nitrogens have shifts of about 6 ppm upfield of COOH-terminal amide nitrogens at the isoelectric point of measurement. 15N chemical shifts appear to have considerable potential value for peptide sequencing. Although carbon-13 nuclear magnetic resonance spectra can be used to distinguish between NH2-terminal, COOH-terminal, and nonterminal residues in small peptides (1, 2), the observed shifts are independent of the nature of the adjacent amino acid residue (3). For this reason, 13C chemical shifts are unable to provide much sequencing information for peptides containing more than three residues. In a recent communication (4), we have shown that resonance signals of nitrogen-15 are not subject to this limitation and show a distinct neighboring residuedependence for simple aliphatic dipeptides. The individuality of these shifts indicates that 15N nuclear magnetic resonance spectra might play a useful role as a nondestructive method for sequence analysis of peptides and, except for sensitivity problems, should be superior to 'H and '3C nuclear magnetic resonance spectroscopy. We provide here a further test of this expectation through studies of the 15N nuclear magnetic resonance spectra of some aliphatic tripeptides and also of dipeptides containing more complex amino acid residues than investigated earlier. EXPERIMENTAL SECTIONAll of the peptides were commercially available materials and were used without further purification. The spectra were taken of 0.1-0.2 M peptide solutions in water, and unless otherwise noted, the pH values were those of the isoelectric points. The spectra were recorded at 18.25 MHz on a Bruker WH-180 pulse spectrometer described in detail elsewhere (5). Reasonable signal-to-noise ratios could be obtained over 2-6 hr with the aid of proton noise decoupling, a 30 ltsec pulse width (300 flip angle), and a repetition rate of 2 sec. The chemical shifts reported are in ppm upfield from 1 M external D15NO3, which also was used as the external field-frequency lock.

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SummaryThe lSN chemical shifts of a number of simple aliphatic dipeptides have been determined in aqueous solution and while the amine nitrogen shift is independent of the nature of the neighbouring residue, the peptide nitrogen shift shows a marked dependence upon the nature of the adjacent amino-acid.STUDIES on simple peptides using 13C n.m.r. spectroscopy have shown that there is a distinct effect on the carbonyl chemical shift depending upon whether an amino-acid residue is N-terminal, C-terminal, or non-termina1.l However, this shift is independent of the nature of the adjoining residues2 and, as such, cannot be used to obtain full sequence information in peptides containing more than three aminoacids. Very little information is available on nitrogen chemical shifts in amino-acids and pep tide^.^ Consequently, we investigated the lW chemical shifts in a series of simple dipeptides, to see if a neighbouring residue effect could be discerned.I n order t o minimize possible complicating factors due to differential solvent4 and substituents effects the study was restricted to dipeptides composed of the simple aliphatic amino-acids glycine, alanine, valine, and leucine. Since in this case we are restricting ourselves t o observations of the effect of varying the size of the aliphatic side chain substituent, the variations observed in this series might be expected to be amongst the smallest observed and, as such, give a simple indication of the potential usefulness of such a technique. All samples used were commercially available and were made up, unbuffered, as 0 . 2~ aqueous solutions in the p H range 5.0-6-2.Spectra were obtained at natural abundance on a Bruker WH-180 spectrometer operating at 18.230MHz for 16N. Typical running conditions were a pulse delay of 2 s for a 3 0~s (38') pulse angle, giving an accumulation time of about 6 h. The results are given in the Table. An important question posed by these studies is the intrinsic sensitivity of the nitrogen chemical shifts to the p H

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