A novel biotinylated suicide inhibitor for directed molecular evolution of lipolytic enzymes Original Research Article

A bifunctional activity label for directed molecular evolution of lipolytic enzymes has been designed and synthesized. The structure is composed of a 4-nitrophenyl activated phosphonate, that is, a suicide substrate of lipases/esterases, connected to a biotin moiety through a spacer containing a disulfide bridge. The phosphonate was prepared by Michaelis–Arbuzov reaction of trimethylsilyl-protected 11-bromoundecanol with triethyl phosphite. The deprotected ω-hydroxyalkylphosphonate was transformed into an active N-hydroxysuccinimide carbonate followed by 4-nitrophenyl activation of the phosphonate using standard procedures. The biotinylated phosphonate inhibitor was then synthesised by coupling the phosphonate inhibitor to the ϵ-amino-caproic acid and cystamine containing biotinyl spacer . The function of all relevant groups of the final activity label (biotin-label, cleavable disulfide bridge, phosphonate-inhibitor) have been successfully tested with the commercial lipase Lipolase® (Novo Nordisk). Hence, a tool for directed molecular evolution of lipolytic enzymes has been developed.