A novel biotinylated suicide inhibitor for directed molecular evolution of lipolytic enzymes.
A bifunctional activity label (8) 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 (3) was prepared by Michaelis-Arbuzov reaction of trimethylsilyl-protected 11-bromoundecanol (2) with triethyl phosphite. The deprotected omega-hydroxyalkylphosphonate (4) was transformed into an active N-hydroxysuccinimide carbonate (5) followed by 4-nitrophenyl activation of the phosphonate using standard procedures. The biotinylated phosphonate inhibitor (8) was then synthesised by coupling the phosphonate inhibitor (6) to the epsilon-amino-caproic acid and cystamine containing biotinyl spacer (7). The function of all relevant groups of the final activity label (8) (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.
Deussen
H J
HJ
Protein Discovery, Novo Nordisk A/S, Bagsvaerd, Denmark.
Danielsen
S
S
Breinholt
J
J
Borchert
T V
TV
D000345:Affinity Labels / Q000138:chemical synthesis; D019921:Biotinylation; D019020:Directed Molecular Evolution; D004791:Enzyme Inhibitors / Q000138:chemical synthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D018189:Immunomagnetic Separation; D007700:Kinetics; D008049:Lipase / Q000037:antagonists & inhibitors; D008066:Lipolysis; D009596:Nitrophenols / Q000138:chemical synthesis / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis; D013329:Structure-Activity Relationship
D000345:Affinity Labels; D004791:Enzyme Inhibitors; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D015842:Serine Proteinase Inhibitors; C023602:O-4-nitrophenyl phenylphosphonate; D008049:Lipase