H-VAL-PHE-OH

The crystal structure of Boc-(L-Phe-D-Phe)4-OMe has been determined by x-ray diffraction analysis. The peptide crystallizes in the triclinic system, space group P1 with a = 15.290 A, b = 15.163 A, c = 19.789 A, alpha = 102.49 degrees, beta = 96.59 degrees, gamma = 74.22 degrees, and Z = 2. The structure has been solved by coupling of the molecular replacement technique and expansion by tangent formula refinement of the set of known phases. Several cycles of Fourier calculations and least-squares refinement led to the location of 194 atoms of the two independent octapeptide chains and few molecules of cocrystallized solvent (chloroform, water, and methanol). The isotropic refinement converged to R = 0.13 for the 3077 "observed" reflections. The two independent octapeptide molecule form a dimer in the solid state: the two chains are associated by interstrand hydrogen bonds (12 of the type N-H ... O = C) with the formation of a double-stranded antiparallel right-handed -- beta 5.6-helix. These double helices can be represented as a cylinder with a hydrophilic inner core represented by the peptide units and an hydrophobic exterior constituted by the aromatic moieties. The dimensions of the cylinder are equal to those observed for Boc-(L-Val-D-Val)4-OMe. In the solid state the dimers pack with each other in an hexagonal fashion with the formation of layers; between the layers, solvent molecules fill empty spaces.

The pseudoproline residue (Psi Pro, L-2,2-dimethyl-1,3-thiazolidine-4-carboxylic acid) has been introduced into heterochiral diproline segments that have been previously shown to facilitate the formation of beta-hairpins, containing central two and three residue turns. NMR studies of the octapeptide Boc-Leu-Phe-Val-(D)Pro-Psi Pro-Leu-Phe-Val-OMe (1), Boc-Leu-Val-Val-(D)Pro-Psi Pro-Leu-Val-Val-OMe (2), and the nonapeptide sequence Boc-Leu-Phe-Val-(D)Pro-Psi Pro-(D)Ala-Leu-Phe-Val-OMe (3) established well-registered beta-hairpin structures in chloroform solution, with the almost exclusive population of the trans conformation for the peptide bond preceding the Psi Pro residue. The beta-hairpin conformation of 1 is confirmed by single crystal X-ray diffraction. Truncation of the strand length in Boc-Val-(D)Pro-Psi Pro-Leu-OMe (4) results in an increase in the population of the cis conformer, with a cis/trans ratio of 3.65. Replacement of Psi Pro in 4 by (L)Pro in 5, results in almost exclusive population of the trans form, resulting in an incipient beta-hairpin conformation, stabilized by two intramolecular hydrogen bonds. Further truncation of the sequence gives an appreciable rise in the population of cis conformers in the tripeptide Piv-(D)Pro-Psi Pro-Leu-OMe (6). In the homochiral segment Piv-Pro-Psi Pro-Leu-OMe (7) only the cis form is observed with the NMR evidence strongly supporting a type VIa beta-turn conformation, stabilized by a 4-->1 hydrogen bond between the Piv (CO) and Leu (3) NH groups. The crystal structure of the analog peptide 7a (Piv-Pro-Psi(H,CH3)Pro-Leu-NHMe) confirms the cis peptide bond geometry for the Pro-Psi(H,CH3)Pro peptide bond, resulting in a type VIa beta-turn conformation.

A new synthetic route to (E)-beta-phenyl-alpha,beta-dehydroalanine (delta(E)Phe)-containing peptide was presented via photochemical isomerization of the corresponding (Z)-beta-phenyl-alpha,beta-dehydroalanine (delta(Z)Phe)-containing peptide. By applying this method to Boc-Ala-delta(Z)Phe-Val-OMe (Z-I: Boc, t-butoxycarbonyl; OMe, methoxy), Boc-Ala-delta(E)Phe-Val-OMe (E-I) was obtained. The identification of peptide E-I was evidenced by 1H-nmr, 13C-nmr, and uv absorption spectroscopy, elemental analysis, and hydrogenation. The conformation of peptide E-I in CDCl3 was investigated by 1H-nmr spectroscopy (solvent dependence of NH chemical shift and difference nuclear Overhauser effect). Interestingly, peptide E-I differed from peptide Z-I in the hydrogen-bonding mode. Namely, for peptide Z-I, only Val NH participates in intramolecular hydrogen bonding, which leads to a type II beta-turn conformation supported by hydrogen bonding between CO(Boc) and NH(Val). On the other hand, for peptide E-I, two NHs, delta(E)Phe NH and Val NH, participate in intramolecular hydrogen bonding. In both peptides, a remarkable NOE (approximately 11-13%) was observed for Ala C(alpha) H-deltaPhe NH pair. Based on the nmr data and conformational energy calculation, it should be concluded that peptide E-I takes two consecutive gamma-turn conformations supported by hydrogen bonding between CO(Boc) and NH(delta(E)Phe), and between CO(Ala) and NH(Val) as its plausible conformation.