Biosynthesis of paclitaxel intermediate

Patent metadata

Field	Value
Publication number	US-10501764-B2
Application number	US-201715671882-A
Country	US
Kind code	B2
Filing date	Aug 8, 2017
Priority date	Jun 18, 2012
Publication date	Dec 10, 2019
Grant date	Dec 10, 2019

Abstract

The invention relates to methods of making compounds useful for production of paclitaxel and analogs or derivatives thereof.

First claim

What is claimed:

1. A method of making paclitaxel or a derivative thereof, comprising contacting an aminopropanoate with a Tyrocidine synthetase A (TycA) to produce an aminopropanoyl-CoA and producing said paclitaxel or a derivative thereof from said aminopropanoyl-CoA.

2. The method of claim 1 , comprising reacting a substrate represented by formula I with a Tyrocidine synthetase A (TycA) to produce an aminopropanoyl-CoA of formula II:

wherein:
X is hydrogen;
Y is hydrogen or OH; and
Ring is an unsubstituted or substituted aryl, heteroaryl, (C4-C10)cycloalkyl, or (C4-C9)heterocycloalkyl.

3. The method of claim 2 further comprising mixing said aminopropanoyl-CoA of formula II with Baccatin III of the formula:

to form a mixture; and
reacting said mixture with a Baccatin III O-phenylpropanoyltransferase (BAPT) to produce N-debenzoyl-2′-deoxypaclitaxel of the formula:

wherein:
X is hydrogen;
Y is hydrogen or OH; and
Ring is an unsubstituted or substituted aryl, heteroaryl, (C4-C10)cycloalkyl, or (C4-C9)heterocycloalkyl.

4. The method of claim 2 , wherein said substrate of formula I comprises a structure represented by formula III or IV:

wherein:
X is hydrogen;
Y is hydrogen or OH;
Z is CH, CH 2 , oxygen (O) or nitrogen (NH or NH 2 ); and
R 1 is selected from the group consisting of hydrogen, alkyl, amino, hydroxyl, cyano, carboxy, nitro, thio, alkoxy, and halogen.

5. The method of claim 2 , wherein said aminopropanoyl-CoA of formula II comprises a structure of formula V or VI:

wherein:
X is hydrogen;
Y is hydrogen or OH;
Z is CH, CH 2 , oxygen (O), or nitrogen (NH or NH 2 ); and
R 1 is selected from the group consisting of hydrogen, alkyl, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen.

6. The method of claim 1 , wherein said Tyrocidine synthetase A is selected from the group consisting of:
a. a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3; SEQ ID NO: 5, or SEQ ID NO: 6;
b. a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with the exception of a substitution of a serine at an amino acid position corresponding to position 563 of the amino acid sequence of SEQ ID NO: 1, wherein said substitution does not have a hydroxy in its side chain;
c. a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with the exception of a substitution of a serine at an amino acid position corresponding to position 563 of the amino acid sequence of SEQ ID NO: 1, wherein said substitution is an alanine, valine, isovaline, leucine, isoleucine, proline, glycine, arginine, lysine, histidine, tryptophan, phenylalanine, methionine or cysteine;
d. a polypeptide comprising an amino acid sequence that shares at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6;
e. a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7;
f. a polypeptide encoded by the nucleotide sequence that shares at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7; and
g. a combination thereof.

7. The method of claim 2 , wherein said reacting of said substrate of formula I comprises a mixture of said Tyrocidine synthetase A, said substrate of formula I, ATP and a divalent cation.

8. The method of claim 1 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

9. The method of claim 3 , wherein the method further comprises reacting said N-debenzoyl-2′-deoxypaclitaxel with N-debenzoyl-2′-deoxypaclitaxel: N-benzoyltransferase (NDTBT) to produce a paclitaxel of the formula:

wherein:
X is hydrogen;
Y is hydrogen or OH; and
Ring is an unsubstituted or substituted aryl, heteroaryl, (C4-C10)cycloalkyl, or (C4-C9)heterocycloalkyl.

10. The method of claim 9 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

11. The method of claim 3 , wherein said method further comprises reacting a 10-deacetyl Baccatin III of the formula:

with a 10-deacetylbacctin III acetyltransferase (DBAT) to produce said Baccatin III.

12. The method of claim 11 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

13. The method of claim 11 , wherein said method further comprises reacting a 2-debenzoyltaxane of the formula:

with a taxane 2α-O-benzoyltransferase (TBT) to produce said 10-deacetyl Baccatin III.

14. The method of claim 13 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

15. The method of claim 13 , wherein said method further comprises reacting a taxa-4(5),11(12)-diene-5α-ol of the formula:

with a taxadienol 5α-O-acetyl-transferase (TAT) to produce said 2-debenzoyltaxane.

16. The method of claim 15 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

17. The method of claim 15 , wherein said method further comprises reacting a taxa-4(5),11(12)-diene of the formula:

with a taxadiene 5α-hydroxylase to produce said taxa-4(5),11(12)-diene-5α-ol.

18. The method of claim 17 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

19. The method of claim 17 , wherein said method further comprises reacting a geranylgeranyl diphosphate of the formula:

with a taxadiene synthase to produce said taxa-4(5),11(12)-diene.

20. The method of claim 19 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

21. The method of claim 19 , wherein method further comprises reacting an isopentenyl diphosphate of the formula:

Assignees

• Univ Michigan State

Inventors

• Walker Kevin

Classifications

• C12P17/02 Primary

  Oxygen as only ring hetero atoms · CPC title

• C12Y501/01011

  Phenylalanine racemase (ATP-hydrolyzing) (5.1.1.11) · CPC title

• C12N9/90

  Isomerases (5.) · CPC title