Solid Phase Total Synthesis And Cytotoxicity Studies Of Phakellistatin 13 And Analogues

Phakellistatin 13 [cyclo(-Leu-Trp-Pro-Phe-Gly-Pro-Thr-)], a cycloheptapeptide isolated from marine sponge Phakellia fusca Thiele which collected at Yongxing Island of China, exhibits potent cytotoxicity against the hepatoma cell line BEL-7404 (ED₅₀<10^-2 μg/mL).Phakellistatin 13 was synthesized by the SPS-CC method on Oxime resin starting from Thr(OBzl), with 25% TFA/DCM for deprotection and HBTU-HOBt ·H₂O as coupling reagent, resulting in 31.6% final yield. The synthetic cyclic peptide was proved to be chemically identical with the natural counterpart by the ESIMS and NMR data.A structure-activity relationship investigation was continued by Ala scan approach, which consists of the systematic replacement of each amino acid residue in the peptide sequence by Ala, in order to investigate the role and relative importance of each amino acid residue in the sequence of phakellistatin 13 for further design of the structure.Comparing the seven Ala modified analogues of phakellistatin 13 accoridng to the yield and HPLC purity, it was concluded that Pro played an important part in the cyclization of phakellistatin 13 because of its constraint to the peptide backbone, while Leu contributed least to the cyclization.The growth inhibition activities of the synthetic phakellistatin 13 and its analogues were studied on four cancer cell lines: BEL-7404 (human hepatoma), Hela (cervical carcinoma), MCF-7 (breast adenocarcinoma) and HT-29 (colon adenocarcinoma). The IC₅₀ values were always >20 μg/mL. The analogue 1 and 5 showed higher cytotoxicity against the cancer cell lines than the other compounds. In addition, all of the synthetic products performed better growth inhibitory activities against Hela than the other three cancer cell lines. But compared with natural phakellistatin 13, the synthetic compounds showed no activity.The results suggest that Leu residue contributes least to cyclization and growth inhibition of phakllistatin 13, as a result, substitutions of other amino acids can be evaluated at this position for further design of bioactive phakellistatin 13 analogues.