Monitoring of native chemical ligation on solid substrate by surface plasmon resonance.
During the last years native chemical ligation (NCL) gained in popularity as a method allowing the chemical synthesis of large peptides and entire proteins. NCL is particularly well-suited for chemoselective and nondenaturing attachment of biomolecules on solid substrates. In the present work, we show the feasibility of monitoring of peptide synthesis, NCL and its catalysis on silicon oxide modified gold surfaces by surface plasmon resonance (SPR). NCL of a model peptide-bradykinin thioester-was carried out and monitored with a custom-built SPR apparatus. Solid-phase produced bradykinin thioester was ligated to the surface in the presence of variable concentrations of 4-mercaptophenylacetic acid as transthioesterification catalyst. At catalyst concentration of 48 mM and above, the NCL reaction was maximal and identical to the reaction of the purified peptide-mercaptophenylacetic acid thioester. SPR curves indicate typical first-order kinetics with t(1/2) of 81 s for this aryl thioester, but of 104 min for the primary alkyl thioester.
Wieczerzak
Ewa
E
Département de Pharmacologie, Faculté de Médecine, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, Québec, J1H 5N4, Canada.
Hamel
Raymond
R
Chabot
Vincent
V
Aimez
Vincent
V
Grandbois
Michel
M
Charette
Paul G
PG
Escher
Emanuel
E
D000595:Amino Acid Sequence; D001920:Bradykinin / Q000138:chemical synthesis / Q000737:chemistry; D004952:Esters; D007700:Kinetics; D010455:Peptides / Q000138:chemical synthesis*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance / Q000295:instrumentation* / Q000379:methods*
D004952:Esters; D010455:Peptides; D001920:Bradykinin