Новые подходы к синтезу стероидов

Abstract

A variant of cholesterol side chain cleavage system (fusion protein F2), containing in a single polypeptide chain three protein domains with necessary prostetic groups (heme, FAD, 2Fe-2S-cluster) was synthesized. Selective affinity sorbents in a combination with HPI.C were applied for isolation and purification F2. It is established, that a rather low expression level of full-length F2 and decreasing of its functional activity are caused by incomplete polypeptide chain synthesis, suboptimal domains folding, proteolytic degradation and restrictions at prostetic groups embedding. Incubation of progesterone with of transformed Saccharomices cerevisiae YEp51/17a, expressed bovine adrenocortical P450cl7 , yielded, in addition to 17a-hydroxyprogesterone, a new end steroid product. This substance was purified by repeated TLC and HPLC and structure as 17a,20a -dihydroxypregn-4-ene-3-one was determined by mass spectrometry and 'H NMR analyses. The two 20-epimeric dihydroprogesterones was converted to progesterone via a 20-oxidase reaction followed 17a-hydroxylation and 20-reduction. Introduction of a 17a-hydroxy group strongly enhanced 20a-reduction, whereas an additional 21-hydroxy group decreased 20-reduction and lack steroid 17a-hydroxylation. An additional 11- epimeric hydroxy groups decreased the turnover for the 17a-hydroxylation.