Method for the hydroxylation of steroids

A method of preparing a steroid having the general formula (I):

• wherein
• X1 and X2 are independently H, Cl, F, Br, I, CF3, a C1 to C6 alkyl radical, OH, a C1 to C6 alkoxy radical, CN, NO2, N(R6)2, an epoxy group, CHO, or a CO2R6 radical, wherein
• R6 is —C(O)H, —C(O)CH3, —C(O)CH2CH3, —C(O)(CH2)2CH3, —C(O)CH(CH3)2, —C(O)(CH2)3CH3, —C(O)CH(CH3)CH2CH3, —C(O)CH2CH(CH3)2, —C(O)C(CH3)3, —C(O)Ph, or —C(O)CH2Ph,
• R1 and R2 are independently H, OH, OR7 or O, wherein
• R7 is —C(O)H, —C(O)CH3, —C(O)CH2CH3, —C(O)(CH2)2CH3, —C(O)CH(CH3)2, —C(O)(CH2)3CH3, —C(O)CH(CH3)CH2CH3, —C(O)CH2CH(CH3)2, —C(O)C(CH3)3, —C(O)Ph, or —C(O)CH2Ph,
• R3 is H, OH, OR8, a C1 to C10 alkyl radical, a C1 to C10 alkenyl radical, —CHO, —C(O)(CH3), —C(O)(CH2OH), —CH(CH3)C(O)CH3, —CH(CH3)((CH2)2CO2R9), or —CH(CH3)((CH2)2CONHR9), wherein
• R8 is —C(O)H, —C(O)CH3, —C(O)CH2CH3, —C(O)(CH2)2CH3, —C(O)CH(CH3)2, —C(O)(CH2)3CH3, —C(O)CH(CH3)CH2CH3, —C(O)CH2CH(CH3)2, —C(O)C(CH3)3, —C(O)Ph, or —C(O)CH2Ph, and
• R9 is —CH3, —CH2COOH, —CH2CH3, —CH(CH3)2, —(CH2)2CH3, —(CH2)2SO3H, C(CH3)3, —(CH2)3CH3, —CH(CH3)CH2CH3, —CH2CH(CH3)2, an aryl group, or an alkylaryl group,
• R4 is H, OH, or —OR10, wherein
• R10 is —C(O)H, —C(O)CH3, —C(O)CH2CH3, —C(O)(CH2)2CH3, —C(O)CH(CH3)2, —C(O)(CH2)3CH3, —C(O)CH(CH3)CH2CH3, —C(O)CH2CH(CH3)2, —C(O)C(CH3)3, —C(O)Ph, or —C(O)CH2Ph, and
• R5 is H, CF3, a C1 to C6 alkyl radical, a C1 to Co alkenyl radical, OH, O, or a C1 to C6 alkoxy radical, wherein the dashed line denotes an optional double bond, with the proviso that the B ring has no double bond if the A ring has a C4-C5 double bond, and the C ring has no double bond if X1 and X2 form an epoxy group, or
• wherein the steroid having general formula (I) is selected from the group consisting of 3α,7β, 12α-trihydroxy-5β-cholane-24-acid, 3α,7β, 12β-trihydroxy-5β-cholane-24-acid, 3β, 7β, 12α-trihydroxy-5β-cholane-24-acid, 3β, 7β, 12β-trihydroxy-5β-cholane-24-acid, 7β, 12α-dihydroxy-3-keto-5β-cholane-24-acid, 7β, 12β-dihydroxy-3-keto-5β-cholane-24-acid, 3α,7β-dihydroxy-12-keto-5β-cholane-24-acid, 3β, 7β-diydroxy-12-keto-5β-cholane-24-acid, 7β-hydroxy-3,12-diketo-5β-cholane-24-acid, 3α,7β-dihydroxy-5β-cholane-24-acid, 7β-hydroxy-3-keto-5β-cholane-24-acid and 3β, 7β-dihydroxy-5β-cholane-24-acid,
• the method comprising the step of converting a 7-deoxysteroid selected from the group consisting of 3α,12α-dihydroxy-5β-cholane-24-acid, 3α,12β-dihydroxy-5β-cholane-24-acid, 3β,12α-dihydroxy-5β-cholane-24-acid, 3β,12β-dihydroxy-5β-cholane-24-acid, 3β-hydroxy-12-keto-5β-cholane-24-acid, 3-keto, 12β-hydroxy-5β-cholane-24-acid, 3α-hydroxy-12-keto-5β-cholane-24-acid, 3-keto, 12α-hydroxy-5β-cholane-24-acid, 3,12-diketo-5β-cholane-24-acid, 3α-hydroxy-5β-cholane-24-acid, 3-keto-5β-cholane-24-acid and 3β-hydroxy-5β-cholane-24-acid or having the general formula (II) with a cytochrome P450 hydroxylase or a functional variant thereof in the presence of at least one redox partner system and a system for regenerating the redox partner system:
• wherein X1, X2, R1, R2, R3, R4, and R5 in Formula II are as defined in Formula I,
• wherein the cytochrome P450 hydroxylase comprises an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID No. 1 or SEQ ID No. 2.