Process for the synthesis of modified P-chiral nucleotide analogues

• US 6,407,223 B1
• Filed: 10/14/1999
• Issued: 06/18/2002
• Est. Priority Date: 04/25/1997
• Status: Expired due to Fees

• Current Assignee: Polska Akademia Nauk Centrum Badan Molekularnych I Makromolekularnych
• Original Assignee: Polska Akademia Nauk Cenirum Badan Molekularnych I Makromlekularnych
• Inventors: Stec, Wojciech J., Chworoś, Arkadiusz, Woźniak, Lucyna A., Pyzowski, Jaroslaw
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US09/402,974
• Time in Patent Office: 978 Days
• Field of Search: 536/25.3, 536/25.31, 536/25.32, 536/25.33, 536/25.34, 536/25.6, 536/26.26, 536/26.7, 536/26.71, 536/26.8
• US Class Current: 536/25.3
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Abstract

The process of the instant invention is drawn to the synthesis of modified P-chiral nucleotide analogues in the form of pure diastereomers possessing preselected configuration at the P-atom. Oligonucleotides prepared by the method of the invention containing P-chiral compounds have enhanced hybridization and transporting properties.

Claims

1. A process for the synthesis of modified P-chiral nucleotide analogues of Formula 1:

wherein;

R₁ is a protecting group;

R₂ is selected from hydrogen, protected hydroxyl group, vinyl, halogen, nitrile, azide, protected amine group, chloroalkyl, perfluoroalkyl, perfluoroalkoxyl, alkoxyalkyl, ethynyl, OQ₁, SQ₁, NHQ₁, where Q₁ stands for alkyl, aryl, alkenyl or alkynyl;

B is a purine or pyrimidine base;

Z is selected from alkyl, aryl, alkenyl, alkynyl, vinyl, ethynyl, aminomethyl or aminoethyl substituents;

X is an oxygen, sulfur or selenium atom;

R_x is a protecting group;

said process comprising the steps of;

(a) providing the following compound of Formula 2;

wherein

R₁, R₂ and B are as defined above;

Y is an XR₃ substituent, wherein X is an oxygen, sulfur or selenium atom and R₃ is an acyl group of formula COR₄, in which R₄ is an alkyl group, perfluoroalkyl group, or an aryl substituent containing six to fifteen carbon atoms and having an aromatic ring comprising mono-, di- or tri-substituted aromatic substituents activating the aromatic ring;

(b) providing the following compound of Formula 6;

wherein;

B, R₂ and R_x are as defined above;

(c) reacting the compound of Formula 6 with the compound of Formula 2 under anhydrous conditions, in an aprotic organic solvent, in the presence of at least one activating reagent, to yield the compound of Formula 1; and

(d) isolating the compound of Formula 1.

2. The process according to claim 1, wherein R_x is selected from aroyl, acyl, alkoxycarbonyl, benzenesulfonic, alkyl, trialkylsilyl group and an elongated oligonucleotide chain.

3. The process according to claim 1, wherein X is a sulfur or selenium atom and the process comprises oxidizing the compound of formula 1 with an oxidizing reagent, prior to the step of isolating the compound of Formula 1.

4. The process according to claim 1, wherein X is an oxygen atom.

5. The process according to claim 1, wherein the compound of Formula 2 is obtained by:

(i) phosphorylating with a phosphorylating reagent, a substrate of Formula 3;

wherein

R₁, R₂ and B are as defined previously, said phosphorylating reagent comprising a compound of Formula 4;

wherein;

Z is as defined previously, and W is a halogen; and

(ii) followed by hydrolysis without isolation of the intermediate of Formula 5;

where R₁, R₂, B, Z and X are as defined in claim 1, and Y is an oxygen atom, to yield the compound of Formula 2.

6. The process according to claim 1, wherein the aprotic organic solvent comprises tetrahydrofuran or acetonitrile.

7. The process according to claim 1, wherein the at least one activating reagent is an organic base amine.

8. The process according to claim 1, wherein the reacting in step (c) comprises an additional activating reagent which is a lithium salt.

9. The process according to claim 1, wherein the compound of Formula 2 is a diastereoisomer which possesses an absolute configuration at the P-atom identical with that desired the analogues of Formula 1, said diastereoisomer being reacted with the compound of Formula 6 under anhydrous conditions, in the aprotic organic solvent, in the presence of the at least one activating reagent, to yield the compound of Formula 1.

10. The process according to claim 9, wherein the compound of Formula 2 is obtained by phosphorylation of a substrate of Formula 3:

with a phosphorylating reagent of Formula 4;

followed by hydrolysis without isolation of an intermediate of Formula 5;

to yield a compound of Formula 2 wherein R₁, R₂, B, Z and X are as defined previously, and Y is an oxygen atom.

11. The process according to claim 9, wherein the aprotic organic solvent is tetrahydrofaran or acetonitrile.

12. The process according to claim 9, wherein the at least one activating reagent is an organic base.

13. The process according to claim 9, wherein the reacting in step (c) is in the presence of an additional activating reagent which is a lithium salt.

14. A process for the synthesis of modified P-chiral nucleotide analogues of Formula 1 in the form of a pure diastereoisomer possessing a preselected configuration at the P-atom:

wherein;

R₁ is a protecting group;

R₂ is selected from hydrogen, protected hydroxyl group, vinyl, halogen, nitrile, azide, protected amine group, chloroalkyl, perfluoroalkyl, perfluoroalkoxyl, alkoxyalkyl, vinyl, ethynyl, OQ₁, SQ₁, NHQ₁, where Q₁ stands for alkyl, aryl, alkenyl or alkynyl;

B is a purine or pyrimidine base;

Z is selected from alkyl, aryl, alkenyl, alkynyl, vinyl, ethynyl, aminomethyl or aminoethyl substituents;

R_x is a protecting group;

Y is an XR₃ substituent, where X is an oxygen, sulfur or selenium atom and R₃ is an acyl group of formula COR₄, in which R₄ is an alkyl group, perfluoroalkyl group, or an aryl substituent containing six to fifteen carbon atoms and having an aromatic ring comprising mono-, di- or tri-substituted aromatic substituents activating the aromatic ring;

said process comprising the steps of;

(a) hydrolyzing a diastereoisomer of the compound of Formula 2;

where;

R₁, R₂ and B are as defined above;

Y is an oxygen;

X is a sulfur or selenium atom, wherein the compound of Formula 2 possesses an absolute configuration at the P-atom opposite to that desired for the analogues of Formula 1, said hydrolyzing being done in the presence of an activator that inverts the absolute configuration of the P-atom resulting in a product of Formula 2,

(b) reacting the product of step (a) with a compound of Formula 7;

wherein

R₄ is an alkyl, perfluoroalkyl, or a aroyl substituent containing six to fifteen carbon atoms, and having an aromatic ring comprising mono-, di- or tri-substituted aromatic substituents activating the aromatic ring; and

W is a chlorine, bromine or iodine atom;

to yield the compound of Formula 2, where R₁, R₂, B and Z are as defined above, X is a sulfur or selenium atom, and Y is R₄C(O)O—

, in which R₄ is as defined above, wherein the compound of Formula 7 possesses an absolute configuration at the P-atom opposite to that of the diastereoisomer of Formula 2, and identical to that which is desired for the analogues of Formula 1.

15. The process according to claim 14, wherein R_x is selected from aroyl, acyl, alkoxycarbonyl, benzenesulfonic, alkyd, trialkylsilyl group and an elongated oligonucleotide chain.

17. The process according to claim 14, wherein the compound of Formula 2 is obtained by:

(i) phosphorylating with a phosphorylating reagent, a substrate of Formula 3;

wherein

R₁, R₂ and B are as defined previously;

said phosphorylating reagent comprising a compound of Formula 4;

wherein

Z and X are as defined previously,

W is a halogen;

(ii) followed by hydrolysis without isolation of the intermediate of Formula 5;

where

R₁, R₂, B, Z and X are defined previously, and Y is an oxygen atom to yield the compound of Formula 2.

18. The process according to claim 17, further comprising:

(i) separating chromatographically the compound of Formula 2 into two diastereoisomers; and

(ii) reacting the diasterosiomer with compound of Formula 7;

wherein;

R₄ is an alkyl group, perfluoroalkyl group, or an aroyl substituent containing six to fifteen carbon atoms and having an aromatic ring comprising mono-, di- or tri-substituted aromatic substituents activating the aromatic ring, and W is a halogen.

19. The process according to claim 14, wherein the activator is an organic base amine.

20. The process according to claim 14, wherein the aprotic organic solvent is tetrahydrofuran.

23. The process according to claim 14, wherein X is an oxygen, sulfur or selenium atom, and Y of Formula 2 is R₄C(O)O—

.

16. A process for the synthesis of modified P-chiral nucleotide analogues of Formula 1 in the form of a pure diastereoisomer possessing a preselected configuration at the P-atom:

wherein;

R₁ is a protecting group;

R₂ is selected from hydrogen, protected hydroxyl group, vinyl, halogen, nitrile, azide, protected amine group, chloroalkyl, perfluoroalkyl, perfluoroalkoxyl, alkoxyalkyl, vinyl, ethynyl, OQ₁, SQ₁, NHQ₁, where Q₁ stands for alkyl, aryl, alkenyl or alkynyl;

B is a purine or pyrimidine base;

Z is selected from alkyl, aryl, alkenyl, alkynyl, vinyl, ethynyl, aminomethyl or aminoethyl substituents;

R_x is a protecting group;

Y is an XR₃ substituent, where X is an oxygen, sulfur or selenium atom and R₃ is an acyl group of formula COR₄, in which R₄ is an alkyl group, perfluoroalkyl group, or an aryl substituent containing six to fifteen carbon atoms and having an aromatic ring comprising mono-, di- or tri-substituted aromatic substituents activating the aromatic ring;

said process comprising the steps of;

(a) hydrolyzing a diastereoisomer of the compound of Formula 2;

 

wherein;

R₁, R₂ and B are as defined above;

Y is an oxygen;

X is a sulfur or selenium atom, wherein the compound of Formula 2 possesses an absolute configuration at the P-atom opposite to that desired for the analogues of Formula 1, said hydrolyzing being done in the presence of an activator that inverts the absolute configuration of the P-atom resulting in a product of Formula 2,

(b) reacting the product of step (a) with a compound of Formula 7;

 

wherein

R₄ is an alkyl, perfluoroalkyl, or a aroyl substituent containing six to fifteen carbon atoms, and having an aromatic ring comprising mono-, di- or tri-substituted aromatic substituents activating the aromatic ring; and

W is a chlorine;

bromine or iodine atom, to yield the compound of Formula 2, where R₁, R₂, B and Z are as defined above, X is a sulfur or selenium atom, and

Y is R₄C(O)O—

, in which R₄ is as defined above, wherein the compound of Formula 7 possesses an absolute configuration at the P-atom opposite to that of the diastereoisomer of Formula 2, and identical to that which is desired for the analogues of Formula 1; and

(c) reacting the product with compound of Formula 6;

wherein

B, R₂ and R_x are as defined previously, under anhydrous conditions, in an aprotic organic solvent, in the presence of an activating reagent, to yield the compound of Formula 1 and isolating the compound of Formula 1.

21. The process according to claim 16, wherein the activating reagent is a lithium salt.

22. A process for the synthesis of modified P-chiral nucleotide analogues of Formula 1 in the form of a pure diastereoisomer possessing a preselected configuration at the P-atom:

wherein;

R₁ is a protecting group;

R₂ is selected from hydrogen, protected hydroxyl group, vinyl, halogen, nitrile, azide, protected amine group, chloroalkyl, perfluoroalkyl, perfluoroalkoxyl, alkoxyalkyl, vinyl, ethynyl, OQ₁, SQ₁, NHQ₁, where Q₁ stands for alkyl, aryl, alkenyl or alkynyl;

Y is an oxygen;

X is a sulfur or selenium atom wherein the compound of Formula 2 possesses an absolute configuration at the P-atom opposite to that desired for the analogues of Formula 1, comprising the steps of;

(a) hydrolizing one of two diastereoisomers of formula 2 in the presence of an activator that inverts the absolute configuration of the P-atom resulting in a product of Formula 2

(b) reacting the product of step (a) with a compound of Formula 7;

wherein

R₄ is an alkyl, perfluoroalkyl, or a aroyl substituent containing six to fifteen carbon atoms, and having an aromatic ring comprising mono-, di- or tri-substituted aromatic substituents activating the aromatic ring; and

W is a chlorine, bromine or iodine atom,

to yield the compound of Formula 2, where R₁, R₂, B and Z are as defined above, X is a sulfur or selenium atom, and Y is R₄C(O)O—

, in which R₄ is as defined above, wherein the compound of Formula 7 possesses an absolute configuration at the P-atom opposite to that of the diastereoisomer of Formula 2, and identical to that which is desired for the analogues of Formula 1; and

(c) providing the following compound of Formula 6;

wherein

B, R₂ and R_x are as defined previously;

(d) reacting the compound of Formula 6 with the compound of Formula 2 under anhydrous conditions, in an aprotic organic solvent, in the presence of an activating reagent, to yield compound of Formula 1 of desired absolute configuration at the P-atom; and

(e) isolating the compound of Formula 1.