The c(RGDfK) synthesis on a solid‐phase utilizing a removable methyl ester group by lithium hydroxide

Abstract A cyclic RGD peptide, c(RGDfK), has a remarkable selectivity to bind the α v β 3 receptor to inhibit angiogenesis in tumors. Previously, two methods were used to synthesize the c(RGDfK) peptide. The first method involved cyclization of DfKRG in the solution phase. The second method used a removable allylic protecting group (‐OAll) on the C‐terminal backbone of the Asp residue prior to cyclization on the solid phase. The first method produced a low yield and required multiple separation processes. In contrast, the second approach required expensive reagents and hydrogen gas to remove the allylic group. Therefore, the objective of this study was to develop a simplified, accelerated, and cost‐effective synthesis of the c(RGDfK) peptide. This approach employed the methyl ester (‐OMe) protecting group on the C‐terminal of the Asp residue that was removed using lithium hydroxide (LiOH). The 2‐chlorotrityl chloride resin was used for the solid‐phase peptide synthesis (SPPS) of the linear fKRGD with the Fmoc strategy and a mixture of HATU/HOAt/DIPEA as coupling reagents. Then, the methyl ester group on the Asp residue was removed with LiOH, followed by the N‐to‐C‐terminus cyclization at the Asp‐Phe peptide bond using the EDC/HOAt reagent. The peptide was subsequently removed from the resin using a mixture of TFA/TIS/water (95:2.5:2.5). The crude product c(RGDfK) was purified using semi‐preparative RP‐HPLC for 60 min with a gradient eluent of water:acetonitrile (0.1% TFA) from a ratio of 95:5 to 20:80. The pure c(RGDfK) peptide was identified at a retention time ( t R ) of 23,995 min with a purity of 95.7% using analytical RP‐HPLC (gradient eluent of Mili‐Q:ACN + 0.1% TFA from 99:1 to 10:90, 1.0 mL/min, 60 min) with a yield of 20.4%. This identification was subsequently confirmed using HRToF‐ESIMS [M+H] + : 604.3207 with a calculated m/z of 604.3207 for the molecular formula C 27 H 42 N 9 O 7 . A combination of NMR and HRTOF‐ESIMS data confirmed the identity of the synthesized c(RGDfK) peptide.