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%).