Acetonitrile → 4-Nitrobenzyl alcohol

Participants

Reaction Steps

To a solution of antibody 7G12 in 50 mM Tris-buffered solution, pH 8.0 (90 μL), was added a solution of a substrate compound, N-(benzyloxycarbonyl)-L-phenylalanine 4-nitrobenzyl ester (L-1) (R=PhCH2), synthesized in Preparation 3, in 2mM DMSO solution (10 μL) at 25° C., and they were mixed with stirring to obtain the reaction mixture containing 10.8 μM antibody and 200 μM substrate. By means of HPLC (YMCAM-303: C-18, 10 mm in diameter×250 mm in length, an aqueous solution of acetonitrile/0.1% trifluoroacetic acid=35/65 (0-7 minutes), 35/65-90/10 (7-15 minutes), 90/10 (15-17 minutes), 1.0 mL/minute, 278 nm), the amount of 4-nitrobenzyl alcohol (retention time: 5.9 minutes) produced in the reaction mixture was traced in time course for the purpose of determing initial rate. Next, the same procedure as mentioned above was repeated using N-(benzyloxycarbonyl)-D-phenylalanine 4-nitrobenzyl ester (D-1) (R=PhCH2) instead of N-(benzyloxycarbonyl)-L-phenylalanine 4-nitrobenzyl ester (L-1) (R=PhCH2) to determine the initial rate. Background hydrolytic reaction rate was also determined based on the amount of 4-nitrobenzyl alcohol produced by spontaneous degradation in the reaction solution free from antibody 7G12.

Provenance & Evidence