Nuclear‐Magnetic‐Resonance Study of Aggregations and Conformations of Melanostatin and Related Peptides

Abstract

The H and 13C nuclear magnetic resonance spectra of melanostatin (Pro-Leu-Gly-NH2) and related peptides (Pro-Leu-Gly, Z-Pro-Leu-Gly, Z-Pro-Leu-Gly-NH2 and Z-Pro-Leu-Gly-OCH3, where Z = benzyloxycarbonyl) were analysed in a variety of solvents. At physiological pH, the melanostatin molecule is N-protonated in aqueous solution. The concentration dependences of the chemical shifts of amide-proton and carbonyl-carbon resonances and of proton spin-lattice relaxation times were observed in relation to molecular aggregations. In dimethylsulfoxide solution, aggregations were observed for N-protonated melanostatin and Pro-Leu-Gly prepared with HCI and for the Na salt of Z-Pro-Leu-Gly but not for N-protonated melanostatin prepared with HC104 or HN03, unprotonated melanostatin, Z-Pro-Leu-Gly-NH2, or Z-Pro-Leu-Gly-OCH3. The leucine NH and glycine CO groups of N-prolonated melanostatin are involved in the intermolecular hydrogen bonds of aggregates. The leucine NH group of N-protonated Pro-Leu-Gly also forms the intermolecular hydrogen bond. The solvent and temperature dependences of the chemical shifts of amide-proton and carbonyl-carbon resonances were measured to determine intramolecular hydrogen bonding, In dimethylsulfoxide solution, N-protonated melanostatin molecules in part take the &turn structure and the trans carboxamide NH proton and carbonyl oxygen of the proline residue form an intramolecular hydrogen bond.Melanostatin, Pro-Leu-Gly-NH2, is a tripeptide hormone inhibiting the release of pituitary melanotropin (melanocyte-stimulating hormone) [l -31, and is also found to possess additional extrapituitary activities [4 -61. An X-ray analysis [7] has shown that the melanostatin molecule takes a ten-membered hydrogen-bonded structure, namely the type I1 pturn structure [8,9]. An intramolecular hydrogen bond is formed between the carbonyl of the proline residue and the trans carboxamide proton of glycinamide. Conformational energy calculations have shown that this p-turn structure is one of the most stable conformations of melanostatin [lo, 111. Conformation studies on melanostatin in solution are also important for elucidating the correlations

Nuclear‐Magnetic‐Resonance Study on Met‐enkephalin and Met‐enkephalin

“…These observations appear to suggest that the amide protons of the Gly-2 and Gly-3 residues are involved in intermolecular hydrogen bonding in (C2H3)2S0 solution. If this is the case, the temperature coefficients of the chemical shifts of these amide protons will be decreased as the concentration is raised, as observed previously for melanostatin [24] and related peptides [25].…”

“…In our previous studies on melanostatin (Pro-Leu-Gly-NH2) [24,25], the cationic form prepared with HCl has been found to form molecular aggregate in (C2H3)2S0 solution. The chemical shifts of the leucine amide proton and the glycine carboxamide carbon resonances exhibit significant concentration dependences.…”

“…Our previous studies on melanostatin and related peptides indicate the importance of taking the molecular association and ionization into account before analyzing molecular conformations of linear oligopeptides [24,25]. In fact, large concentration dependences of the amide proton and carbonyl carbon resonances of Met-enkephalin and Leu-enkephalin have been observed, indicating that intermolecular association is significant in (C2H3)2SO solution [17].…”

“…The temperature coefficients of amide proton chemical shifts are shown in Table 2, for Met-enkephalin and related peptides. In contrast to the amide proton chemical shifts, the chemical shifts of C-a proton resonances of gramicidine S [27], melanostatin [24] and related peptides [25] are independent of temperature. In the present study, however, significant temperature dependences were found for the first time for the C-a proton chemical shifts of the dipolar form of Metenkephalin in (C2H3)2S0 solution and in C2HC13/ (C2H3)2S0 (2/1) solution, as shown in Table 3.…”

Aggregation of pro‐leu‐gly‐NH2 in aqueous solution

“…We reported evidence suggesting that Pro-Leu-Gly-NH,, but not Pro-Leu-Gly-N(CHa)z, can aggregate in dimethylsulfoxide [7] ~ an observation further expanded upon in [8,9].…”