Solvent-induced and temperature-induced ' 0 chemical shifts of , LeuSI-enkephalin and solvent-induced spectral modifications of the amide-I' stretching vibrations of [1-13C-Gly2, Leu51-enkephalin and [1-13C-Gly2, Leu51-enkephalin are reported and correlated with the spectroscopic characteristics of model amides. It is demonstrated that both Gly2 and Gly3 peptide oxygens are motionally equivalent and form solvation species which are essentially monohydrated in aqueous solution, contrary to several simple amides and model peptides in which water largely forms dihydrates. It is shown that the combined use of 170-NMR and Fourier transform infrared is a unique methodology for studying the hydration state of specific peptide oxygens in peptide hormones.Peptides and proteins in biological systems are usually surrounded by a predominantly aqueous solvent. Their small size, large dipole moment and high capacity for forming hydrogen bonds means that the water molecules have unique effects on peptide and protein conformations [l -31. Hydrogen-bonding interactions between the oxygen atom of the peptide groups and the molecules of water undoubtedly contribute to the overall conformational stability of peptides and proteins in aqueous medium. The knowledge of these solvent interactions is of importance for any conformational study since it is known that biomolecules often change their conformation as the solvent is varied.As a first step towards the solution of hydration of peptides and proteins, numerous experimental and theoretical investigations on the most probable sites for binding of the water molecules have been undertaken on amides [4-lo]. These molecules are considered as the simplest representatives of the peptide bond; therefore, the determination of the possible monohydrated and multihydrated species has been considered as crucial in gauging the magnitude of specific hydration of more complex peptides and proteins. Furthermore, several theoretical calculations of model peptides [11 -201 and numerous X-ray structural investigations of several peptide hydrates and proteins [21-281 revealed thermodynamical and geometrical aspects of peptide-backbone hydration.

The synthesis of Leu-enkephalin selectively 17O-enriched in Gly2 and Gly3 is reported. The 17O-nmr chemical shifts of [17O-Gly2, Leu5]- and [17O-Gly3, Leu5]-enkephalins in H2O are almost identical and independent of the pH. Since hydrogen bonding is the dominant factor governing the chemical shifts of the peptide oxygen, it can be concluded that the hydration state of both oxygens is identical and independent of the pH. The 17O chemical shifts of the [17O-Leu5]-enkephalin terminal carboxyl group at pH approximately 1.9 and 5.6 are very different in H2O but very similar in CH3CN/DMSO (4:1) solution. This suggests that the protonation state of the carboxyl group at both pH values in CH3CN/DMSO solution is the same and consequently that Leu-enkephalin exists in the neutral form at pH approximately 5.6. In this organic mixed solvent system both Gly2 and Gly3 oxygen resonances exhibit a significant shift to high frequency by the same extent (delta delta approximately 30 ppm). It is concluded that both peptide oxygens are not hydrogen bonded to an appreciable extent and that no specific 2----5 hydrogen bonding exists to an appreciable extent. This conclusion is in agreement with the energy of activation for molecular rotation, as determined from T1 measurements, which was found to be almost identical for both [17O-Gly2, Leu5]- and [17O-Gly3, Leu5]-enkephalins in CH3CN/DMSO (4:1) mixed solvent.

17O- and 14N-nmr chemical shifts and line widths of the carboxyl and amino terminal groups of Leu-enkephalin--Tyr-Gly-Gly-Phe-[17O]Leu-Oh--and enkephalin-related fragments--[17O]Leu-OH, Phe-[17O]Leu-OH, Gly-Phe-[17O]Leu-OH, and Gly-Gly-Phe-[17O]Leu-OH--were measured in aqueous solution over the entire H pH range. Enrichment in 17O was achieved by saponification of the corresponding O-methyl esters. Ionization constants and titration shifts were obtained by nonlinear least-squares fits to one-proton titration curves. [17O]Leu-OH exhibits a profound pH-dependent solvation change on deprotonation of the carboxyl group, as shown by 17O- and 14N-nmr line widths. In contrast, the peptides studied do not exhibit pH-dependent conformational (solvation) changes on deprotonation of the carboxyl group, and a head-to-tail intramolecular association between the ionic terminal groups should be excluded. It is shown that the peptides do not exhibit isotropic overall molecular motion and that segmental motion rather than fast internal motion influences the effective correlation times at the sites of the carboxyl oxygens and the amino nitrogen.

The ionization state of Leu-enkephalin in DIMSO and MeCNlDMSO (4/l) solution was studied by the combined use of "0 NMR and FT-IR spectroscopy. After lyophilization of an aqueous solution at nearly neutral pH, Leu-enksphalin essentially exists in the uncharged state in MeCN/DMSO (4/l) solution. In pure DMSO, only 40% of the Leu-enkephalin molecules are in the zwittcricnic state under Ihe same conditions. FT-IR; Ionization state of peptide; Leu-enkephalin: "0 NMR 1, INTRODUCTION 2. MATERIALS AND METHODSMany conformational studies of peptide hormones, and particularly of enkephalins (Tyr-Gly-Gly-Phe-Leu/ Met), have been carried out in the organic DMSO (Me,SO) solvent [l]. In practically all the cases, it was assumed that, after lyophilization of a nearly neutral aqueous solution, enkephalins (and more generally peptides with ionizable termini) retain the zwitterionic state after dissolving in DMSO. However, this hypothesis was already questioned on the basis of "C NMR data and 1R and Raman spectroscopy [2,3] and it is known that the pK scale in DMSO differs drastically from that in water [4]. In particular, and in contrast with aqueous solutions of carboxylic acids and primary amines, the pK values for acetic acid and butylamine are not very different in DMSO (12,6 and 11.1, respectively) [5].In view of the importance of establishing the ionization state of peptides prior to conformational analysis, we report here on combined results of an "0 NMR and FT-IR study of Leu-enkephalin in pure DMSO and in MeCN/DMSO (4/l) solution. Leu-enkephalin with selectively "O-enriched C-terminial carboxyl group, Tyr-Gly-Gly-Phe-["O]Leu-01-L was obtained by suponifkation of the methyl ester wiWsodium ethanolate in Hz"0 at 40% enrichment [6,7]. For spectroscopic experiments, aqueous Leu-cnkephalin solutions wcrc adjusted to lhrce pH values (I .9,5,8 (isoelectric point) and X2), lyophilized IO dryness, and the residual solid was taken up in the organic solvent.In order to consider the influence of the dielectric constant and polarity on the spectroscopic data, we have added MeCN to DMSO to a composition compatible with pcptide solubility, i.e. the MeCN/ DMSO (4/l) mixture [8]."0 NMR spectra were run at 4O*C on a Bruker AM-400 spcctrometcr (54.48 MHz) under the following experimental condilions: concentration 0.01 M; 10 nm sample tubes; spectral width 50 kHz; 90" pulse 30 flus; in quadrature phase detection. Acoustic ringing effects were alleviated by using either a pre-acquisition delay or special pulse sequences [9]. Chemical shifts (ppm) are reported relative to 1.4. dioxanc.The experimental conditions for the FT-IR experiments on a BrUkcr IFS-85 spectrometer were as follows: room tcmpcrature; concentration 0.01 M; cell path length 100 pm; 512 scans; subtraction of the solvent spectrum from that of the solution. RESULTS AND DISCUSSIONThe "0 resonance of Leu-enkephalin, Tyr-Gly-GlyPhe["O]Leu-OH, in the MeCN/DMSO (4/l) mixture is illustrated in Fig. 1 as a function of the original pH of the aqueous solution. The chemical shift value r...

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