Enzymatic method to produce 7-dehydropregnenolone, vitamin D3-like compounds and derivatives thereof

• US 20050277171A1
• Filed: 01/06/2005
• Published: 12/15/2005
• Est. Priority Date: 01/06/2004
• Status: Active Grant

• Current Assignee: University of Tennessee Research Foundation (University of Tennessee)
• Original Assignee: University of Tennessee Research Foundation (University of Tennessee)
• Inventors: Tuckey, Robert, Slominski, Andrzej, Zjawiony, Jordan, Wortsman, Jacobo, Stewart, D. Jeremy
• Granted Patent: US 7,253,293 B2
• Time in Patent Office: Days
• US Class Current: 435/52
• CPC Class Codes: C12P 33/00 Preparation of steroids
• CPC Class Codes: C12P 33/005 Degradation of the lateral chains at position 17
• CPC Class Codes: C12P 7/02 containing a hydroxy group

Abstract

Provided herein are enzymatic methods of producing steroid compounds such as 7-dehydropregnenolone, hydroxy derivatives thereof and vitamin D2- and D3-like compounds and derivatives thereof. Also provided are the derivatives of the vitamin D3-like compounds so generated via the action of cytochrome P450scc enzyme on substrates 7-dehydrocholesterol, vitamin D2 or vitamin D3.

Claims

17. A method of producing a vitamin D3-like compound, comprising:

enzymatically hydroxylating at least a C17 side chain of ergosterol, vitamin D2, vitamin D3 or a combination thereof via a cytochrome P450scc enzyme system and, wherein ergosterol is hydroxylated, optionally, thermophotolytically converting said hydroxylated ergosterol to said C17 hydroxylated vitamin D2 via UVB radiation and thermal intramolecular rearrangement at C9-C10; and

enzymatically shortening the hydroxylated C17 side chain via the cytochrome P450scc enzyme system and, wherein said C17 side chain is shortened on said hydroxylated ergosterol, thermophotolytically converting said ergosterol via UVB radiation and thermal intramolecular rearrangement at C9-C10.

1. A method of producing 7-dehydropregnenolone, comprising:

enzymatically shortening a C17 side chain of 7-dehydrocholesterol via a cytochrome P450scc enzyme system to produce said 7-dehydropregnenolone.

2. The method of claim 1, further producing a vitamin D3-like compound, comprising:

thermophotolytically breaking a C9-C10 bond in 7-dehydropregnenolone via UVB radiation; and

converting 7-dehydropregnenolone to said vitamin D3-like compound via thermal intramolecular rearrangement around the broken bond.

3. The method of claim 2, wherein said vitamin D3-like compound is 5Z,7E-3β

-hydroxy-9,10-secopregna-5,7,10(19)trien-20-one.

4. The method of claim 1, further producing a hydroxy derivative of 7-dehydropregnenolone, comprising:

enzymatically hydroxylating 7-dehydropregnenolone in at least one position.

5. The method of claim 4, wherein said 7-dehydropregnenolone hydroxy derivative is 3β

,11α

-dihydroxypregna-5,7-dien-20-one, 3β

,11β

-dihydroxypregna-5,7-dien-20-one, 3β

,17β

-dihydroxypregna-5,7-dien-20-one, 3β

,21-dihydroxypregna-5,7-dien-20-one, 3β

,17β

,21-trihydroxypregna-5,7-dien-20-one, 3β

,11α

,17β

-trihydroxypregna-5,7-dien-20-one, 3β

,11β

,17β

-trihydroxy pregna-5,7-dien-20-one, 3β

,11α

,21-trihydroxypregna-5,7-dien-20-one, 3β

,11β

,21-trihydroxy pregna-5,7-dien-20-one, 3β

,11α

,17β

,21-tetrahydroxypregna-5,7-dien-20-one, or 3β

,11β

,17β

,21-tetrahydroxy pregna-5,7-dien-20-one, 3β

,20α

-dihydroxypregna-5,7-diene or 3β

,20β

-dihydroxypregna-5,7-diene.

6. The method of claim 4, wherein said 7-dehydropregnenolone is hydroxylated via a P450c11 enzyme, a P450c17 enzyme, a P450c21 enzyme, or 20 hydroxysteroid dehydrogenase or a combination thereof.

7. The method of claim 4, further producing an hydroxy derivative of a vitamin D3-like compound, comprising:

thermophotolytically breaking a C9-C10 bond in said 7-dehydropregnenolone hydroxy derivative via UVB radiation; and

converting said hydroxy derivative to said vitamin D3-like compound derivative via thermal intramolecular rearrangement around said broken bond.

8. The method of claim 7, wherein said vitamin D3-like hydroxy derivative is 5Z,7E-3β

,11α

-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11β

-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3,17β

-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3,β

,21-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,17β

,21-trihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11α

,17β

-trihydroxy-9,10-secopregna-5,7,10(19) trien-20-one, 5Z,7E-3β

,11β

,17β

-trihydroxy-9,10-secopregna-5,7,10(19) trien-20-one, 5Z,7E-3β

,11α

,21-trihydroxy-9,10-secopregna-5,7,10(19) trien-20-one, 5Z,7E-3β

,11β

,21-trihydroxy-9,10-secopregna-5,7,10(19) trien-20-one, 5Z,7E-3β

,11α

,17β

,21-tetrahydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11β

,17β

,21-tetrahydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,20α

-dihydroxy-9,10-secopregna-5,7,10(19)triene or 5Z,7E-3β

,20β

-dihydroxy-9,10-secopregna-5,7,10(19)triene.

9. The method of claim 1, further producing an androstadiene-like compound, comprising:

enzymatically converting 7-dehydroprognenolone via P450c17 to 3-hydroxy-5,7-androstadiene-17-one or 3,17-dihydroxy-5,7-androstadiene.

10. The method of claim 9, further producing a vitamin D3-like compound, comprising:

thermophotolytically breaking a C9-C10 bond in said 3-hydroxy-5,7-androstadiene-17-one or 3,17-dihydroxy-5,7-androstadiene via UVB radiation; and

converting the androstadienes to said vitamin D3-like compound via thermal intramolecular rearrangement around said broken bond.

11. The method of claim 10, wherein said vitamin D3-like compound is 17-ketoetiocalciferol or 17β

-etiocalciferol.

12. The method of claim 10, further comprising:

enzymatically or chemically oxidizing an hydroxy group present at C3, C17 or a combination thereof on said vitamin D3-like compound.

13. The method of claim 10, wherein said further oxidized compound is 3-keto-17-hydroxyetiocalciferol or 3,17-diketoetiocalciferol.

14. The method of claim 1, wherein said cytochrome P450scc enzyme system is an in vitro system, comprising:

cytochrome P450scc enzyme;

adrenodoxin;

adrenodoxin reductase; and

NADPH.

15. The method of claim 14, further comprising a detergent or a phospholipid.

16. The method of claim 14, wherein NADPH is generated within the enzyme system.

18. The method of claim 17, wherein said C17 side chain is hydroxylated at least at position C20 within the chain.

19. The method of claim 17, wherein said C17 side chain comprises vitamin D3 and is hydroxylated at positions C20 and C22.

20. The method of claim 17, wherein said vitamin D3-like compound that is 5Z,7E-3β

-hydroxy-9,10-secopregna-5,7,10(19)trien-20-one.

21. An ergosterol derivative produced by the cytochrome P450scc enzyme system of claim 17 hydroxylated at least at position C20 within the C17 side chain.

22. A vitamin D2 derivative produced by the cytochrome P450scc enzyme system of claim 17 hydroxylated at least at position C20 within the C17 side chain.

23. The method of claim 17, further producing hydroxy derivatives of said vitamin D3-like compound, comprising:

enzymatically hydroxylating said vitamin D3-like compound in at least one position.

24. The method of claim 23, wherein said vitamin D3-like hydroxy derivative is 5Z,7E-3β

,11α

-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11β

-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3,17β

-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3,β

,21-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3,β

,17β

,21-trihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11α

,17β

-trihydroxy-9,10-secopregna-5,7,10(19) trien-20-one, 5Z,7E-3β

,11β

,17β

-trihydroxy-9,10-secopregna-5,7,10(19) trien-20-one, 5Z,7E-3β

,11α

,21-trihydroxy-9,10-secopregna-5,7,10(19) trien-20-one, 5Z,7E-3β

,11β

,21-trihydroxy-9,10-secopregna-5,7,10(19) trien-20-one, 5Z,7E-3β

,11α

,17β

,21-tetrahydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11β

,17β

,21-tetrahydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,20α

-dihydroxy-9,10-secopregna-5,7,10(19)triene or 5Z,7E-3β

,20β

-dihydroxy-9,10-secopregna-5,7,10(19)triene.

25. The method of claim 23, wherein said vitamin D3-like hydroxy derivative is hydroxylated via a P450c11 enzyme, a P450c17 enzyme, a P450c21 enzyme, or 20 hydroxysteroid dehydrogenase or a combination thereof.

26. The method of claim 17, wherein said cytochrome P450scc enzyme system is an in vitro system, comprising:

cytochrome P450scc enzyme;

adrenodoxin;

adrenodoxin reductase; and

NADPH.

27. The method of claim 26, further comprising a detergent or a phospholipid.

28. The method of claim 26, wherein NADPH is generated within the enzyme system.

31. The compound of claim 29, wherein

R₂ is —

CH(R₄)—

CH₂(R₅);

R₁, R₃, and R₅ are independently hydrogen or hydroxy such that at least one of R₁, R₃ or R₅ is hydroxy;

R₄ is a carbonyl bond formed with C20; and

R₆ is —

OH;

or

R₁, R₃, and R₅ are hydrogen; and

R₄ and R₆ are hydroxy.

32. The compound of claim 31, wherein said compound is 5Z,7E-3β

,11α

-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11β

-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3,17β

-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3,β

,21-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,17β

,21-trihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11α

,17β

-trihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11β

,17β

-trihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11α

,21-trihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11β

,21-trihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11α

,17β

,21-tetrahydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11β

,17β

,21-tetrahydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 17-ketoetiocalciferol, 17β

-etiocalciferol, 3-keto-17-hydroxyetiocalciferol or 3,17-diketoetiocalciferol.

29. A compound having a chemical structure

30. The compound of claim 29, wherein said compound is 5Z,7E-3β

,11α

-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11β

-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3,17β

-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3,β

,21-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,17β

,21-trihydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11α

,17β

-trihydroxy-9,10-secopregna-5,7,10(19) trien-20-one, 5Z,7E-3β

,11β

,17β

-trihydroxy-9,10-secopregna-5,7,10(19) trien-20-one, 5Z,7E-3β

,11α

,21-trihydroxy-9,10-secopregna-5,7,10(19) trien-20-one, 5Z,7E-3β

,11β

,21-trihydroxy-9,10-secopregna-5,7,10(19) trien-20-one, 5Z,7E-3β

,11α

,17β

,21-tetrahydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,11β

,17β

,21-tetrahydroxy-9,10-secopregna-5,7,10(19)trien-20-one, 5Z,7E-3β

,20α

-dihydroxy-9,10-secopregna-5,7,10(19)triene, 5Z,7E-3β

,20β

-dihydroxy-9,10-secopregna-5,7,10(19)triene, 17-ketoetiocalciferol, 17β

-etiocalciferol, 3-keto-17-hydroxyetiocalciferol or 3,17-diketoetiocalciferol.

31-1. The compound of claim 27, wherein

R₁ is hydrogen;

R2 is hydrogen;

R3 is hydroxy;

or

R₂ and R₃ form a carbonyl group with C17; and

R₆ is —

OH or forms a carbonyl group with C3.

32-2. The compound of claim 31, wherein said compound is 17-ketoetiocalciferol, 17β

-etiocalciferol, 3-keto-17-hydroxyetiocalciferol or 3,17-diketoetiocalciferol.