Poster Presentation 10th Australian Peptide Conference 2013

Fmoc-Cys(Ddm) and Fmoc-Cys(MBom) prevent the risk of racemization in solid phase peptide synthesis (#179)

Hajime Hibino 1 , Yuji Nishiuchi 1 2
  1. Peptide Inst. Inc., Ibaraki-Shi, Osaka, Japan
  2. Graduate School of Science, Osaka University, Toyonaka-Shi, Osaka, Japan
In Fmoc chemistry, the Trt group is widely accepted as offering protection for Cys. However, the use of Fmoc-Cys(Trt) in peptide synthesis occasionally entails serious obstacles: (1) racemization of Cys arising during its incorporation mediated by phosphonium or uronium reagents such as PyBOP or HBTU, respectively, and (2) racemization of the C-terminal Cys esterified to the solid support during repetitive base treatment. These may hamper purification of the products including those obtained after the NCL and disulfide formation reactions. Therefore, the carbodimide-mediated coupling method has been recommended to reduce the racemization rate to acceptable levels (<1.0%), although the DIC/HOBt method is considered to have no advantage in terms of coupling efficiency over that using PyBOP/HBTU. In addition to this, racemization of the C-terminal Cys likely to occur even when employing a Trt-type resin with the aid of its steric hindrance. In the present study, we reviewed the acid-labile protecting groups on Cys from the perspective of suppressing racemization both when Cys incorporation is conducted with PyBOP/HBTU and when Cys is linked to a resin via ester linkage. The base-catalyzed racemization of Cys is considered to proceed via enolization due to stabilization of the carbanion formed on α-proton abstraction. To destabilize this enol form, therefore, an S-protecting group that possesses an electron-donating and/or a sterically-hindered effect(s) would be essential to prevent racemization of Cys. As had been expected, the 4-methoxybenzyloxymethyl (MBom) and 4,4’-dimethoxydiphenylmethyl (Ddm) groups were found to effectively prevent the risk of Cys racemization during its incorporation and that of the C-terminal Cys esterified to the solid support. Even when performing the 1-min preactivation procedure of coupling with Fmoc-amino acid/HCTU/6-Cl-HOBt/DIEA (4/4/4/8 euiv) in DMF, Fmoc-Cys(Ddm) and Fmoc-Cys(MBom) caused a significant reduction in the level of racemization (0.6% and 0.4%, respectively) while Fmoc-Cys(Trt) led to a considerable level of racemization (8.0%).