Mechanism of hydroxylation at C-2 during the biosynthesis of ecdysone in ovaries of the locust, Schistocerca gregaria

Abstract

The stereochemistry of hydroxylation at C-2 during the biosynthesis of ecdysone in the ovaries of Schistocerca gregaria was investigated by incorporation of [1 alpha,2 alpha-3H(n)]cholesterol in admixture with [4-14C]cholesterol into oöcyte 2-deoxyecdysone and ecdysone conjugates in maturing adult female S. gregaria. Extraction of the eggs followed by enzymic hydrolysis of the ecdysteroid conjugate fraction yielded free ecdysteroids, from which 2-deoxyecdysone and ecdysone were purified. The 3H/14C ratios in the 2-deoxyecdysone and ecdysone were similar, suggesting that the 2 alpha hydrogen of cholesterol was retained during hydroxylation at C-2. This was corroborated by oxidation at C-2 of the 3,22-diacetate derivative of the ecdysone, yielding the corresponding 2-oxo compound with removal of essentially all the 3H originally present at the 2 alpha position of cholesterol. The results indicate that the 2 beta hydrogen of cholesterol has been eliminated during the hydroxylation at C-2. Thus, during ecdysone biosynthesis, hydroxylation at C-2 is direct and occurs with retention of configuration.