Disuccinimidyl esters as bifunctional crosslinking reagents for proteins

Bifunctional crosslinking reagents have been used in studies of the spatial arrangement of muscle contractile proteins, such as myosin and actin, either in their soluble forms [ 1,2], or in synthetic filaments [3-51, or even in myofibrils [3]. The most commonly used reagents are the bisimidates, which are very reactive, but also quite unstable in aqueous solution; incomplete substitution and unexpected side reactions furthermore occur if the crosslinking reaction is at pH -8 [6,7]. A disuccinimidyl ester, the dithiobis (succinimidyl propionate), DSP, that does not have these drawbacks and contains, moreover, an easily cleavable disulfide bond, has been described [8]; unlike the bis-imidates, this reagent allowed the crosslinking of the two heads of a myosin molecule [ 21. Owing to the high chemical reactivity of DSP and its stability in water, we thought it interesting to synthesize a series of disuccinimidyl esters of various chain lengths (table 1). These include non-cleavable reagents (compounds I-IV), and also reagents with either a uic-glycol (compounds V, VI) or an ethylenic bond (compound VII); the crosslinks formed by these last compounds can in principle be cleaved,