Lamprey_methods for Br-routine_2012-10-21.docVIP

Performylation of bile acid Refs. from Tserng Klein, 1977. Page 3–4; Iida et al., 1993. Page 2 Synthesis of performylated bile acid. A stirred solution or suspension of bile acid (5 g) in 20 mL of 90% formic acid containing 4 drops of 70% perchloride acid was heated at 55°C for 1.5 hr. The solution was removed from the bath and allowed to cool to about 40°C. Acetic anhydride was then added dropwise at a rate that maintained the temperature between 55–60°C until a large quantity of bubbles appeared (ca. 16 mL of acetic anhydride was required). The solution was then cooled to room temperature and poured into 200 mL of water, with stirring. The precipitate was collected, washed with water, and dried to yield formylated bile acids. The product (quantitative yield) was chromatographically pure and could be recrystallized by dissolving it in boiling ethanol (50 mL) and diluting slowly with 50 mL of boiling water. They crystallized as needles in over 95% yield. Cholic acid triformate (3α, 7α, 12α-triformyloxy-5β-cholan-24-oic acid), mp 209–210°C (the mp reported by other literatures: mp 205–209°C [8], 208–210°C [14], 206–207°C [6]). Reagents for this step Formic acid perchloride acid Acetic anhydride Ethanol Acetone  Method 1 3-hydroxyl-7α, 12α-diformoxy -5β-cholan-24-oic acid Ref. from Leppik, 1983. Page 7 To a solution of performoxy-5β-cholan-24-oic acid (22.43 g, 50 mmol) in acetone (250 mL) at room temperature was added sodium hydroxide solution (0.2 mol/L, 500 mL) dropwise with stirring over a 2 hr interval. After a further 0.5 hr, the solution was acidified (approx. 200 mL 0.5 mol/L hydrochloric acid), extracted with chloroform (400 mL, 2×200 mL), each chloroform extract washed (100 mLwater), and evaporated on a rotary evaporator. The product obtained was dissolved in acetone (200 mL), cooled in ice. The yield of the crude product was quantitative, and TLC showed it contained only trace amounts of starting material and dideformylated bile acids. The crude pr