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N-(2,4-Dinitrophenyl)-(Gly)n–d-Ala–l-Pro–(Gly)np-nitroanilides (7b, n=2; 7c, n=3) were synthesized in order to study the effect of chain length on their conformations. The CD spectra of both 7band 7csnowed similar patterns to that of 7a(n=1), but their Cotton effects were much weaker. Therefore, both compounds seem still to have some populations of folded conformers because their Cotton effects near 350 nm were significantly larger than those of the tetrapeptide derivatives in random conformation. However, the population of the folded conformers must be quite small.

Empirical conformational energy calculations were performed on Ac–l-Pro–d-Ala–NHCH3and Ac–d-Ala–l-Pro–NHCH3. In order to study the turn tendency of the dipeptide sequences turn probabilities and average interterminal distances were calculated. Much larger turn preference of d-Ala containing sequences was indicated compared with the corresponding l-Ala containing sequences. The turn tendency of the dipeptide sequences were shown to be in the order: l-Pro–d-Ala>d-Ala–l-Pro>l-Pro–l-Ala>l-Ala–l-Pro. This order coincides with the order of the magnitude of the Cotton effects in the CD spectra of the chromophoric derivatives having 2,4-dinitrophenylglycyl and glycine-p-nitroanilide at the N- and the C-termini, respectively, of the corresponding dipeptid esequences. This agreement supports the validity of the chiroptical method to study the turn tendency of the peptide sequences using chromophoric derivatives reported earlier.

To investigate the importance of the orientation of side chains of amino acid residues in gramicidin S (GS) for the antibacterial activity, a dimerized analog of GS, namely S,S'-bi([1-l-hemicystine]-GS) (13) was synthesized viaremoval of p-methoxybenzyl group of [1-S-p-methoxybenzyl-l-cysteine]-GS (12) and subsequent oxidation. Compound 12showed the same antibacterial activity as GS, whereas 13was inactive. The resemblance of ORD curves of the two analogs to that of GS suggested the similarity of peptide-backbone structure of these three compounds. Strong activity of 12was explained by similar conformation of 12to that of GS. To explain the inactivity of 13, a molecular model was proposed; four hydrophilic side chains are located on surface of the molecule, whereas six hydrophobic are burried.

Two analogs of gramicidin S, [1-N-methyl-l-valine]–gramicidin S and [1,1'-di-N-methyl-l-valine]–gramicidin S, were prepared for the purpose of investigating the contribution of NH group of l-valyl residue in gramicidin S to antibacterial activity. They exhibit weak activity as compared with gramicidin S in the antibacterial test toward microorganisms. The optical rotatory dispersions of these analogs and gramicidin S were measured in ethanol and 8 M urea. It was suggested that both analogs have a different molecular conformation from that of gramicidin S, and that the NH group of valyl residues is necessary for stabilizing the conformation to exhibit biological activity.

[d-Pro5,5']-Gramicidin S was synthesized to confirm a prediction that d-dsequence at the corner position had low preference for ß-turn. The analog showed much weaker activity than gramicidin S and also showed different CD spectra from those of gramicidin S. The results indicate that the analog can not take stable, gramicidin S-like, ß-sheet conformation.

N-Acetyl-l-Leu–X–l-Pro–l-Val methylamides (IIa: X=d-Ala, IIb: X=Gly, IIc: X=l-Ala) were synthesized and subjected to CD and 1H NMR measurements to study the β-turn preferences of these peptides. Temperature dependences of l-Val4–NH proton chemical shifts in CD3OH solution were in the order of IIaIIb>IIc. This order of β-turn preferences coincides with the order of the magnitudes of the Cotton effects in the CD spectra of the chromophoric derivatives, N-(2,4-dinitrophenyl)tetrapeptide p-nitroanilides, having the same sequences. This agreement supports the validity of the chiroptical method to study turn tendency of tetrapeptide sequences using chromophoric derivatives reported previously.

Two analogs of gramicidin S (GS), [l-Pro4,4′, l-Asn5,5′]-GS (10a) and [l-Pro4,4′, d-Ala5,5′]-GS were synthesized to investigate the possibility of replacing the β-turn part of GS by a different type of β-turn keeping the biological activity. For the synthesis of 10a, three procedures were examined and satisfactory results were obtained by the active ester method applied to cyclization of linear decapeptide with l-Pro at the C-terminus. Neither analog showed antibiotic activity indicating that the β-turn part of GS could not be replaced with l-Pro-l-Asn or l-Pro-d-Ala sequences without affecting on its activity. The CD and ORD spectra of the analogs and their 2,4-dinitrophenyl derivatives showed weaker Cotton effects than those of GS and its derivative, respectively. So, the analogs were considered not to take GS-like β-sheet conformation. The reason why the analogs did not take such conformations was investigated by the model tetrapeptides with chromophoric substituents.

The conformations of N-(2,4-dinitrophenyl)-l-Leu–X–l-Pro–l-Val p-nitroanilides (I, X=D-Ala; II, X=Gly; III, X=l-Ala) were studied by nuclear magnetic resonance and circular dichroism spectroscopies. The l-Val-NH group of compound Itakes part in an intramolecular hydrogen bonding, and the folded conformation is considered to be type II′ β-turn structure as in the case of [d-Ala4,4′]-gramicidin S. The populations of β-turn conformer were considered to be in the order of I>II>III≈0 in methanol and N,N-dimethylformamide solutions, and decreased in dimethyl sulfoxide solution. Compound IIin dimethyl sulfoxide and IIIin all the three solvents do not take a β-turn conformation. The data obtained from NMR and CD experiments showed a good correlation between the magnitudes of the Cotton effects (250–400 nm) and the populations of the β-turn conformation of these peptides. The proximity of the two chromophores was ascertained directly for compound Iin N,N-dimethylformamide solution by nuclear Overhauser effects experiment. In addition, the β-turn preference of these peptides were in good accordance with the antibiotic activities of the gramicidin S analogs having the same tetrapeptide sequences at their β-turn parts.

In order to examine the usefulness of a fragment condensation procedure in solid-phase peptide synthesis, gramicidin S (GS) was synthesized by three different procedures such as stepwise elongation, fragment condensation with dipeptide fragments, and fragment condensation with tripeptide fragments. The two fragment condensation procedures gave intermediate peptide derivatives in high yields and in high purity. Such differences in the purity of the intermediate peptides disappeared in the final product, GS dihydrochloride. The GS synthesized by the three procedures exhibited the same antibacterial activities as those of natural GS.

To confirm the general applicability of CD spectra of N-(2,4-dinitrophenyl(Dnp))tetrapeptide p-nitroanilides (pNA’s) to the analysis of ß-turn preference of the tetrapeptide sequences, Dnp-pNA derivatives of some tetrapeptides related to ß-turn preferring sequences in proteins were synthesized and subjected to CD and 1H NMR measurements. CD spectra of Dnp–Gly–l-Pro–l-Asn–Gly–pNA (5a) were just the mirror image of those of gramicidin S-model peptide, Dnp–Gly–d-Ala–l-Pro–Gly–pNA (6). This suggested that 5astrongly preferred the ß-turn and the type of turn was different from that of 6. The relative intensities of Cotton effects of the tetrapeptide with the general structure Dnp–Gly–l-Pro–Y–Gly–pNA (Y=l-Asn, Gly, l-Ala, or l-Gln) roughly agreed with the bend potentials of the amino acid residues at the third position of ß-turns in proteins reported by Chou and Fasman. The effects of changing the amino acid residues at the second position of ß-turn were also examined.

N-(2,4-Dinitrophenyl)tetrapeptide p-nitroanilides related to the ß-turn part of gramicidin S were synthesized and subjected to CD measurements. The ß-turn preference of the tetrapeptide derivatives had correlation with the antibiotic activities of the gramicidin S analogs containing similar tetrapeptide sequences at their ß-turn part. So, conformational preference of partial sequences of gramicidin S analogs seem to play a significant role for determining their biological activities.

Two analogs of gramicidin S, [2-l-histidine]-gramicidin S (14) and [2,2'-di-l-histidine]-gramicidin S (19), were synthesized to compare the biological role of d-amino groups in l-ornithine residues in the antibiotic with that of weakly basic imidazolyl groups in l-histidine residues. Compound 14exhibited identical activity to that of gramicidin S, but 19was only 1/8 as active. The optical rotatory dispersions of these analogs and gramicidin S were nearly the same: molecular conformation of these peptides must be very similar. Compounds 14and 19showed esterolytic activity against p-nitrophenyl acetate. The activity of 14was higher than that of imidazole, 14being more active than 19.

CD spectral analysis of chromophoric derivatives of tetrapeptides related to the β-turn part of [d-Val1,1′]-and [d-Val1,1′, l-Phe4,4′]-gramicidin S indicated that they had very low preference for β-turn formation. The results suggested that neither analog could take gramicidin S-like β-sheet conformation. It seems noteworthy that the side-chain bulkiness of the 4th amino acid of a tetrapeptide affects the stability of β-turn.