Synthesis and application of cleavable and hydrophilic crosslinking reagents

Bifunctional imidoesters are due to their mild reaction especially suitable for the crosslinking of proteins. Most often used are diimidates with a medium span (4 or 6 CH2-groups). Reagents with a wider span might be of interest. In such a case, however, the bridge should be more hydrophilic. Bifunctional imidoesters have, therefore, been prepared from dinitriles NC-(CH2)2-X-(CH2)2-CN; X being -O-, -O-(CH2)n-O- or -O-(CH2)m-O-(CH2)n-O-. The bridge of diimidoesters can also be labelled by coloured or fluorescent groups. Diimidoesters containing -S-S-bonds in the bridge can be cleaved more easily after their reaction with proteins. Dimethyl-3.3' -(8- delta -dithiahexamethylenedioxy)-dipropionimidate, a "long", cleavable and more hydrophilic diimidate, is a promising new reagent. In cases, where HS-(CH2)3-C(=NH)-OCH3- HC1 is is used for crosslinking of proteins, this reagent can possibly be replaced by 2-iminothiolane hydrochloride, a cyclic thioimidate, which is easier to prepare and to handle.