Preparation Method Of Anti-HIV Fusion Peptide CP32M

HIV fusion inhibitor is the third class of anti-HIV drugs, following reverse transcriptase inhibitors and protease inhibitors. Since the first fusion inhibitor Fuzeon was approved for clinical use in 2003, this kind of inhibitor has been greatly concerned in the anti-HIV drug design. CP32M (Ac-VEWNEMTWMEWEREIENY TKLIYKILEESQEQ-NH2) was designed by our laboratory and showed highly effective in inhibiting different subtypes of HIV-1 viruses, especially for those highly resistant to Fuzeon, C34 and T-1249.CP32M contains 32 amino acids residues and is difficult to synthesize using linear SPPS method. In the present study, the strategy of combined solid-solution method was employed and a series of explorations on the synthesis of CP32M at large scale were carried out, including the division of peptide fragments, the selection of resin and the condition optimization of condensation, cleavage and purification. Based on the general rule of fragment division, the characteristics of CP32M and the synthetic method of related peptide (T-20, etc), the synthestic strategy of two and three fragments with variant length were investigated. In comparison with the synthestic yield of fragments and coupling efficiency, CP32M was synthesized with the three optimized fragments (Ac-1-9-OH, Fmoc-10-21-OH, H-22-32-NH2) at large scale.The above three peptide fragments were synthesized using linear SPPS respectively, and then resin is removed at the mild condition (1% TFA) to give the side-chain-protected fragment. First, the fragment Fmoc-10-21-OH was coupled to the fragment H-22-32-NH2, the Fmoc protecting group in the intermediate was then removed to give H-10-32-NH2 which was coupled to the fragment Ac-1-9-OH to give side-chain-protected CP32M. Finally, the three batches of crude CP32M (10mmol, 15mmol, 15mmol) were obtained after removing the side-chain-protecting groups, and the total yield could reach to 19.2%.In order to elevate the yield of the N-terminal's fragment and its coupling efficiency with the intermidate fragment, we further optimized the division of fragments and obtained the other three fragments (Ac-1-6-OH, Fmoc-7-21-OH, H-22-32-NH2). 20mmol of the above fragments were synthesized using the same method. The yield of Ac-1-6-OH (75.9%) is much higher than Ac-1-9-OH (39%), and the yield of Fmoc-7-21-OH is 56%.Because CP32M contains nine negatively charged Glu and more impurities in the combined solid-solution synthesis are smaller peptide fragments, the ionic exchange chromatograph (IEC) was used to purify the crude CP32M. Through the optimization for the purification conditions of weak IEC (DEAE) and strong IEC (Q), weak IEC (DEAE) was used to purify the crude CP32M. After final purification by C18-HPLC, CP32M with the purity of more than 98% was obtained.The above research on preparation method of CP32M has made a foundation to the following research of quality study and preclinical trial of CP32M.