Design and Synthesisof Gramicidin S Analogs with High Antibiotic Activity

N-(2,4-Dinitrophenyl)tetrapeptide p-nitroanilides (Dnp-tetra-peptide-pNA) exhibit characteristic CD spectra when they take on β-turn conformation. Exciton coupling of electric transition dipoles of the two chromophores is thought to be the origin of the CD pattern observed, and intensities of the band near 310 and 350mm were shown to reflect well the β-turn preferences of the parent tetrapeptides. In a series of studies on the β-turn of peptides, CD spectral analysisof Dnp-tetrapeptide-pNA showed that a) L­D­L­L (or D­L­D­D) sequence preferred β-turn, that b) only when Pro was at the 2nd position L­D­D­L (or D­L­L­D) sequence couldalso take β-turn, and that c) each amino acid (AA) in a turn-preferring sequence could be replaced with Gly with a little decrease of turn preference of its original sequence. Especially, it is of interest that tetrapeptide sequence with L-Pro at 2nd position and D-amino acid at 1st and 4th has the highest potential to take onβ-turn conformation.

Based on the results mentioned above, the authoe designed a series of compounds as the analogs of an anti- biotic gramicidin S (GS). GS, cyclo(-Val1,1’-Orn2,2’-Leu3,3’-D-Phe4,4’-Pro5,5’-)2, is a typical amphiphilic peptide known to take on intramolecular antiparallel ¢-sheet conformation with twoβ-turns at D-Phe­Pro sequences. The characteristic feature of this conformation is the orientation of side chains in which charged Orn side chains are on one side and the hydrophobicVal and Leu side chains are on the other side of the molecule. From the study of many synthetic analogs in which these three residues were substituted, it was concluded that this orientation of side chains was essential for the activity of GS.

All the analogs were synthesized by conventional solution phase method. For the synthesis of 1a, a pentapeptide with Pro at the C-terminus was chosen as a precursor in a cyclization reaction, because many GS analogs had been synthesized satisfactorily by a similar strategy. Boc­Asn­D-Leu­D-Lys(Z(Cl))­D-Leu­Pro­OH was prepared by active ester coupling of Boc­Asn­ONp and H­D-Leu­D-Lys(Z(Cl))­D-Leu­Pro­OH which was prepared by stepwise chainelongation, and converted to its active ester. Cyclization of H­Asn­D-Leu­D-Lys(Z(Cl))­D-Leu­Pro­OSu derived from the active ester by acid treatment was carried out in anhydrous pyridine at the final concentration of 3mM. After usual work-up, a mixture of the crude cyclized peptides was applied to a column of Sephadex LH-20. The product obtained from the major peak was determined to be cyclicdimer by FAB-MS measurement (M+, 1219) after hydrogenolysis that afforded 1a.It is interesting that cyclization of pentapeptide active ester afforded much dimeric products and almost no cyclic monomer, since there are few papers which reported the absence of cyclic monomerization of pentapeptides. Analogs were synthesized by a similar procedure as described for 1a.

Based on the β-turn preference of tetrapeptide sequences, the author designed two series of analogs of GS inwhich D­L­D­D and D­L­L­D sequence would be located at theβ-turn position. In

the case of the analogs with D­L­D­D sequence, the analogs were suggested to take on theβ-sheet conformation that is antipodal to that of GS and showed antibacterial activity that is closely related to the hydrophobicity of component amino acid. In the case of the analogs with D­L­L­D sequences, it is not proved that they hadβ-sheet conformation from their CD spectra. However, the fact that some of the analogs showed strong antibacterial activity suggesting that they had amphiphilicβ-sheet conformation. It wasnoteworthy that retention time on HPLC had some relation to the activity of GS analogs. The overall molecular hydrophobicity caused by the difference of conformation may be reflected in HPLC retention time. For the precise conformational analysis of the analogs with D­L­L­D sequence, it would be necessary to apply NMR spectroscopy.



Kazuki Sato, Bull. Chem. Soc. Jpn. Vol. 86, No. 1, 112­120 (2013)