¹⁷O NMR and FT‐IR study of the ionization state of peptides in aprotic solvents Application to Leu‐enkephalin

Abstract

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...

CD and FTIR spectroscopic studies of Amadori compounds related to the opioid peptides

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Conformational determination of [Leu]enkephalin based on theoretical and experimental VA and VCD spectral analyses

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