Synthesis of Cyclo-(D-Phe-D-Ala-Ile-Val-Leu-Gly), A Bacicyclin Analogue, Using A Combination of Solid- and Solution-Phase

Abstract

Bacicyclin is a cyclic peptide isolated from the bacterial culture of Bacillus sp. strain BC028 derived from blue mussel (Mytilus edulis) with a cyclo-(D-Phe-D-Ala-Ile-Val-Leu-Gly) sequence, which has antibacterial activity against Enterococcus faecalis and Streptococcus aureus with MIC values of 8 and 12 µM. The analogue was designed through the replacement of Ala residue to cationic residue, Lys, that is theoretically known to increase antibacterial activity to give an analogue of cyclo-(D-Phe-Lys-Ile-Val-Leu-Gly). The synthesis of the analogue was initiated by the synthesis of linear peptide using the solid phase method on 2-chlorotritylchloride (2-CTC) resin. All amino acid precursors were protected by fluorenylmethyloxycarbonyl (Fmoc) group, and a combination of coupling reagents 1-[bis(dimethyllamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and 1-hydroxy-7-azabenzotriazole (HOAt) were employed to facilitate all amide formations. The linear peptide was then removed from the resin using a trifluoroethanol (TFE):acetic acid (AcOH):dichloromethane (DCM) ratio (2:2:6) by maintaining the protecting group in the sidechain. Cyclization was carried out in solution phase by using benzotriazole-1-yloxytripyrrolidinophosphine hexafluorophosphate (PyBOP) as the cyclization agent and N,N-diisopropylmethylamine (DIPEA) as base. Deprotection of the tertier-butyl-oxylcarbonyl (Boc) protective group was carried out using trifluoroacetic acid (TFA):triisopropylamine (TIS): DCM with the ratio of 95:2.5:2.5. The synthetic product was successfully purified using semi-preparative RP-HPLC using acetonitrile (AcN) (0.1% TFA):water (0.1% TFA) as eluent with a combination of isocratic and gradient method starting from 20% until 80% AcN for 30 minutes. The target peak was found at tR = 18.443 minutes with the yield of 29,41%. Analysis of product purity using analytical RP-HPLC gives single peak at tR = 10.624 minutes.  The synthesis results were characterized using HR-ToF-ESI-MS with [M+H]⁺ = 658.4294 and [M+Na] = 680.4080.