Biocatalytic synthesis of 4-pregnen-20,21-diol-3-one, a selective inhibitor of human 5α-reductase type II

Figures & data

Figure 1 Whole cell biocatalysts for steroid biotransformation as obtained by Autodisplay of bovine adrenodoxin (according to references [3] and [6]). In case that the P450 constituent consist of the enyzme CYP11B1, the whole cell biocatalysts efficiently converts the substrate 11-deoxycorticosterone (DOC) to the product corticosterone (B).

Figure 2 HPTLC screening of steroid converting enzyme activity in strains of the E. coli strain collection and subsequent phosphoimager analysis using ³H-labeled DOC as a substrate. DOC = 11-deoxycorticosterone, B = corticosterone, P = product. lane 1: E. coli strain E132, 2: E133; 3: 134; 4: E135; 5: E144; 6: E151; 7: E153; 8: E152; 9: negative control without cells; 10: positive control containing all the constituents as described in Figure 1, products of the biotransformation of ³H-DOC with CYP11B1.

Figure 3 HPTLC and phosphoimager analysis of conversions using ³H-labeled DOC as a substrate in order to verify the biotransformation reaction of E. coli strain E132. The lanes contain the following samples: 1: control reaction of the mitochondrial CYP11B1 system, 2: E. coli E132 combined with the control reaction of 1), 3: components of 2) without adrenodoxin, 4: components of 2) without adrenodoxin reductase, 5: components of 2) without adrenodoxin and adrenodoxin reductase, 6: components of 2) without adrenodoxin and CYP11B1, 7: substrate conversion with E. coli E127 (negative control). The product of the biotransformation is marked on the right with a P and the products of the mitochondrial CYP11B1 system are designated on the left as B (corticosterone), aldo (aldosterone), 18(OH)B and 18(OH)DOC.

Figure 4 MALDI spectrometry analysis of the product 4-pregnen-20,21-diol-3-one (P) and the substrate DOC (inset) of the biocatalytic reaction as described. Signals at 143.11 and 284.04 correspond to the matrix 5-aminochinolin, whereas the signal at 315.06 represents an unknown impurity caused during the MS sample preparation.

Table I.  ¹³C NMR data in CDCl₃ for substrate DOC and the product of biotransformation by E. coli E132, 4-Pregnen-20, 21-diol-3-one.

C	Substrate	product
1	35.78	35.48
2	33.95	33.96
3	199.37	199.63
4	124.06	123.82
5	170.64	171.38
6	32.76	32.86
7	31.95	32.04
8	35.64	35.47
9	53.67	53.80
10	38.63	38.62
11	20.99	20.88
12	38.46	39.47
13	44.71	42.52
14	56.16	55.09
15	24.52	24.50
16	23.01	24.56
17	59.14	52.30
18	13.48	12.44
19	17.42	17.38
20	210.12	74.50
21	69.42	66.44

Scheme 1 (A): converting DOC to 20-dihydro-DOC (4-pregnen-20,21-diol-3-one), a whole cell biotransformation performed by E. coli E132. (B): Progesterone and the 20-oxime derivatives thereof as potent dual inhibitor of 5α-reductase types I and II and 17α-hydroxylase-C_17,20-lyase (according to Ling et al. [18]).

Table II.  Inhibition of human and rat 5α-reductases by 4-pregnen-20,21-diol-3-one (20-dihydro-DOC).

	Human 5α-red. I	Human 5α-red. II	Rat 5α-red. I	CYP17	CYP19
Inhibition %^a	49.0	81.8	82.5	0	0
IC50 value	–	1.56	–	–	–

^a at 10 μM final concentration for 5α-reductases, 2.5 μM for CYP17, and 20 μM for CYP19.

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