Preparation of phosphorothioate oligomers

• US 6,031,092 A
• Filed: 10/15/1997
• Issued: 02/29/2000
• Est. Priority Date: 10/20/1995
• Status: Expired due to Term

• Current Assignee: McGill University
• Original Assignee: McGill University
• Inventors: Xin, Zhili, Jin, Yi, Marsault, Eric, Just, George
• Primary Examiner(s): Kunz, Gary L.
• Application Number:
  US08/951,132
• Time in Patent Office: 867 Days
• Field of Search: 536/25.3, 536/25.33, 536/25.34
• US Class Current: 536/25.34
• CPC Class Codes: C07F 9/1411 with hydroxyalkyl compounds with further substituents on alkyl
• CPC Class Codes: C07F 9/65846 the phosphorus atom being part of a six-membered ring which may be condensed with another ring system
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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
• CPC Class Codes: C07H 9/04 Cyclic acetals
• CPC Class Codes: C07H 9/06 the hetero ring containing nitrogen as ring hetero atoms

Abstract

Methods and intermediates for the preparation of oligomers containing diastereomerically enriched phosphorothioate linkages are disclosed.

Claims

1. A method for the preparation of an internucleotide linkage comprising the steps of:

selecting a nucleoside phosphoramidite;

reacting said nucleoside phosphoramidite with a nucleoside in the presence of a catalyst, said nucleoside having a free 5'"'"'-hydroxyl group;

thereby forming said internucleotide linkage;

said catalyst having the formula;

##STR95## wherein;

R¹² and R¹³ are independently hydrogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, an ester group, or R¹² and R¹³ together with the carbon atoms to which they are attached, form a substituted phenyl ring where said substituents are electron withdrawing; and

R¹⁴ is hydrogen, halogen, cyano, nitro, thio, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, norbornyl, substituted norbornyl, aryl, substituted aryl wherein said substituents are electron withdrawing, or has the formula;

##STR96## wherein L is a protecting group;

provided that R₁₂, R₁₃ and R₁₄ are not simultaneously H;

further provided that where one R₁₂, R₁₃ or R₁₄ is nitro, then at least one of R₁₂, R₁₃ and R₁₄ cannot be H; and

further provided that at least one R₁₂, R₁₃ and R₁₄ is electron withdrawing.

2. The method of claim 1 wherein said internucleotide linkage is a phosphite linkage.

3. The method of claim 1 further comprising the step of oxidizing said internucleotide linkage to form a phosphodiester linkage, a phosphorothioate linkage, or a phosphorodithioate linkage.

4. The method of claim 1 wherein said catalyst has the structure:

##STR97## wherein;

R¹² and R¹³ are independently hydrogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, where said substituents are electron withdrawing; and

R¹⁴ is hydrogen, halogen, cyano, nitro, thio, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, or substituted aryl wherein said substituents are electron withdrawing.

5. The method of claim 1 wherein R¹², R¹³ and R¹⁴ are independently hydrogen, or cyano.

6. The method of claim 1 wherein R¹² and R¹³ are each cyano, and R¹⁴ is hydrogen.

7. The method of claim 1 wherein R¹² and R¹³ are each cyano, and R¹⁴ is halogen.

8. The method of claim 7 wherein R¹⁴ is bromine.

9. The method of claim 2 wherein said catalyst has the structure:

##STR98## wherein;

R¹² and R¹³ are independently hydrogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, where said substituents are electron withdrawing; and

R¹⁴ is hydrogen, halogen, cyano, nitro, thio, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, or substituted aryl wherein said substituents are electron withdrawing.

10. The method of claim 3 wherein R¹², R¹³ and R¹⁴ are independently hydrogen, or cyano.

11. The method of claim 10 wherein R¹² and R¹³ are each cyano, and R¹⁴ is hydrogen.

12. The method of claim 10 wherein R¹² and R¹³ are each cyano, and R¹⁴ is halogen.

13. The method of claim 12 wherein R¹⁴ is bromine.

14. A method for the preparation of an activated nucleoside phosphoramidite comprising the steps of:

selecting a nucleoside having a free hydroxyl group;

reacting said hydroxyl group of said nucleoside with a halogenated trivalent phosphorus compound in the presence of a catalyst;

thereby forming said activated nucleoside phosphoramidite;

said catalyst having the formula;

##STR99## wherein;

R¹² and R¹³ are independently hydrogen, halogen, cyano, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, where said substituents are electron withdrawing; and

R¹⁴ is hydrogen, halogen, cyano, thio, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, or substituted aryl wherein said substituents are electron withdrawing.

15. The method of claim 14 wherein said free hydroxyl group is a 3'"'"' or 5'"'"' hydroxyl group.

16. The method of claim 14 wherein said halogenated trivalent phosphorus compound is a chlorinated trivalent phosphorus compound.

17. A method for the preparation of an activated nucleoside phosphoramidite comprising the steps of:

selecting a nucleoside having a free hydroxyl group;

reacting said hydroxyl group of said nucleoside with a trivalent phosphorus compound in the presence of a catalyst, where said trivalent phosphorus compound includes at least one hetero atom selected from nitrogen and oxygen covalently bonded to a phosphorus atom;

thereby forming said activated nucleoside phsophoramidite;

said catalyst having the formula;

##STR100## wherein;

R¹² and R¹³ are independently hydrogen, halogen, cyano, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, where said substituents are electron withdrawing; and

R¹⁴ is hydrogen, halogen, cyano, thio, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, or substituted aryl wherein said substituents are electron withdrawing.

18. The method of claim 17 wherein said free hydroxyl group is a 3'"'"' hydroxyl group or a 5'"'"' hydroxyl group.

19. The method of claim 17 wherein said trivalent phosphorus compound is a halogenated trivalent phosphorus compound.

20. The method of claim 17 said trivalent phosphorus compound is a chlorinated phosphorus compound.

21. The method of claim 17 wherein said trivalent phosphorus compound includes at least one nitrogen hetero atom covalently bonded to a phosphorus atom.

22. The method of claim 21 wherein said nitrogen atom is further substituted with an alkyl or aralkyl group having up to 15 carbon atoms.

23. The method of claim 22 wherein said nitrogen atom is further substituted with an alkyl group.

24. The method of claim 23 wherein said alkyl group is isopropyl or t-butyl.

25. The method of claim 17 wherein said trivalent phosphorus compound includes one nitrogen and one oxygen atom covalently bonded to a phosphorus atom.

26. The method of claim 25 wherein said trivalent phosphorus compound further includes a halogen atom covalently bonded to said phosphorus atom.

27. The method of claim 25 wherein each of said nitrogen atom and said oxygen atoms are further substituted with at least one carbon atom containing species.