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

#1. Field of the Invention

#The present invention relates generally to the fields of enzymology and steroid chemistry. More specifically, the present invention relates to an enzymatic method of producing 7-dehydropregnenolone, vitamin D3-like compounds and derivatives thereof.

#2. Description of the Related Art

#So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention are briefly summarized. The above may be better understood by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted; however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

#FIG. 1A depicts the metabolic pathway of 7-dehydropregnenolone production and its sequential transformation to vitamin D3 and the following hydroxy derivatives (#3-10) and/or to secosteroids (#12-20) generated by the action of UVB radiation: 1. cholesta-5,7-dien-3β-ol (7-dehydrocholesterol) 2. 3β-hydroxypregna-5,7-dien-20-one(7-dehydro pregnenolone) 3. 3β,11α or β-dihydroxypregna-5,7-dien-20-one 4. 3β,17β-dihydroxypregna-5,7-dien-20-one 5. 3β,21-dihydroxypregna-5,7-dien-20-one 6. 3β,17β,21-trihydroxypregna-5,7-dien-20-one 7. 3β,11α or β,17β-trihydroxypregna-5,7-dien-20-one 8. 3β,11α or β,21-trihydroxypregna-5,7-dien-20-one 9. 3β,11α or β,17,β,21-tetrahydroxypregna-5,7-dien-20-one 10. 3β,20α or β-dihydroxypregna-5,7-diene 11. 5Z,7E-9,10-secocholesta-5,7,10(19)-trien-3 1-ol (vitamin D3) 12. 5Z,7E-3β-hydroxy-9,10-secopregna-5,7,10(19)trien-20-one 13. 5Z,7E-3β,11α- or β-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one 14. 5Z,7E-3,17β-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one 15. 5Z,7E-3β,21-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one 16. 5Z,7E-3β,17β,21-trihydroxy-9,10-secopregna-5,7,10(19)trien-20-one 17. 5Z,7E-3β,11α or β,17β-tetrahydroxy-9,10-secopregna-5,7,10(19) trien-20-on 18. 5Z,7E-3β,11α or β,21-tetrahydroxy-9,10-secopregna-5,7,10(19) trien-20-one 19. 5Z,7E-3β,11α or β,17β,21-tetrahydroxy-9,10-secopregna-5,7,10(19)trien-20-one 20. 5Z,7E-3β,20α or β-dihydroxy-9,10-secopregna-5,7,10(19)triene.

#FIG. 1B depicts the 7-dehydropregnenolone enzyme conversion product 3-hydroxy-5,7-androstadiene-17-one (#21) and the following hydroxy and/or keto derivatives (#22-26) generated by the action of UVB radiation and/or subsequent chemical or enzymatic oxidation of the hydroxy groups: 22. 3β,17β-dihydroxy-5,7-androstadiene 23. 17β-hydroxyetiocalciferol 24. 17-ketoetiocalciferol 25. 3-keto-17β-hydroxyetiocalciferol 26. 3,17-diketoetiocalciferol

#One embodiment of the present invention provides a method of producing 7-dehydropregnenolone, comprising enzymatically shortening a C17 side chain of 7-dehydrocholesterol via a cytochrome P450scc enzyme system to produce 7-dehydropregnenolone. In all aspects of this embodiment the P450scc enzyme system may be an in vitro system comprising cytochrome P450scc enzyme, adrenodoxin, adrenodoxin reductase, and NADPH. The P450scc enzyme system further may comprise a detergent or a phospholipid. In the NADPH may be generated within the enzyme system.

#In an aspect of this embodiment, the method further produces a vitamin D3-like compound where the method comprises thermophotolytically breaking a C9-C10 bond in 7-dehydropregnenolone via UVB radiation; and converting 7-dehydropregnenolone to the vitamin D3-like compound via thermal intramolecular rearrangement around the broken bond. An example of the vitamin D3-like compound is 5Z,7E-3β-hydroxy-9,10-secopregna-5,7,10(19)trien-20-one

#In another aspect to this embodiment, the method further produces a hydroxy derivative of 7-dehydropregnenolone where the method comprises enzymatically hydroxylating 7-dehydropregnenolone in at least one position. In this further embodiment 7-dehydropregnenolone maybe hydroxylated via a P450cc11 enzyme, a P450c17 enzyme, a P450c21 enzyme, or 20-hydroxysteroid dehydrogenase or a combination thereof. Examples of 7-dehydropregnenolone hydroxy derivatives are 7-dehydropregnenolone hydroxy derivative is 3β,11α-dihydroxypregna-5,7-dien-20-one, 3β,11β-dihydroxypregna-5,7-dien-20-one, 3β,17β-dihydroxypregna-5,7dien-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-on 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.