To a solution of biotin-maleimide 1 (1 mg, 0.0022 mmol) in 100 μl DMF, a solution of coenzyme A disodium salt (1.79 mg, 0.0022 mmol, 1 eq.) in 90 μl DMF and 10 μl 50 mM Tris-Cl pH 7.5 is added. The mixture is stirred for 4 hours at room temperature. It is then diluted with CH3CN/H2O 1:4, and aliquots of 500 μl are injected on a preparative HPLC column: Gradient (A=H2O 99%, CH3CN 1%, 50 mM NH4OAc/B=CH3CN) from A/B 95:5 to A/B 80:20 in 2 min, to A/B 68:32 in 7 min, to A/B 20:80 in 2 min, then back to A/B 95:5. The retention time of the CoA-biotin is 6.5 min. Fractions containing the desired product are concentrated in vacuo, dissolved in DMSO, and an analytical amount injected to control the purity. The pure fractions are combined. The concentration of CoA-biotin is determined by absorption at 260 nm (ε(adenine, 260 nm)=15'300 [M−1 cm−1]). The yield of CoA-Bt is 0.895 mg (33%).
