Fig. 2.

Reaction of Leu-NCA (25 mM overall added in 5 portions over 4 h) with 1 mM 5′-AMP in a 100 mM MES buffer (pH 5.5) containing 50 mM MgCl₂ at room temperature. HPLC monitoring of the reaction progress (Method C, detection 248 nm): A reaction medium after the addition of the last portion of Leu-NCA at 4 h; B reaction medium at 24 h (1.9% total yield of esters 4d); C reaction medium at 24 h after addition of ester 4d. Identification of HPLC peaks: 1.3 min, 5′-AMP, 3.5 min leucyl adenylate 6d, and 4.1 and 6.4 min, 2′- and 3′-regioisomers of ester 4d, respectively