Preparation of chiral phosphorothioate oligomers

• US 5,734,041 A
• Filed: 10/20/1995
• Issued: 03/31/1998
• 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:
  US08/546,198
• Time in Patent Office: 893 Days
• Field of Search: 536/26.31, 536/26.3
• US Class Current: 536/25.31
• 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 synthesizing an oligomer having a phosphorothioate linkage comprising the steps of:

reacting a first synthon of Formula I;

##STR94## wherein;

Q is independently O or S;

R¹ is a hydroxyl protecting group;

R² is a chiral auxiliary of formula --C(R⁸)R³ --C(R¹⁶)R⁵ --CHR⁶ --NHR⁷ ;

R³ is hydrogen, alkyl, cyanomethyl, monohalomethyl, dihalomethyl, trihalomethyl, --CH₂ Si(R⁴)₃, or --CH₂ --SO_k R⁴ where k is 0, 1 or 2;

R⁴ is independently alkyl, aryl, aralkyl or alkaryl having up to 15 carbon atoms;

R⁵ is H, --CN, --Si(R⁴)₃, SO_k R⁴ or halogen;

or R⁸ and R¹⁶ are each H, and R³ and R⁵, together, form one of the structures ##STR95## wherein;

R¹⁰ and R¹¹ are H, alkyl having from 1 to about 10 carbons, --CH₂ C(═

O)OR²², --CH₂ CN, --CH₂ Si(CH₃)₃, or o- or p-C₆ H₄ -R²¹ ;

R²¹ is hydrogen, --O--C(═

O)CH₃, alkoxy having from 1 to about 10 carbons, --NO₂, or --N(R²²)₂ ;

R²² is independently H or alkyl having from one to about 10 carbon atoms;

p is 1 or 2;

Z¹ and Z² are independently halogen, CN, --Si(CH₃)₃, and --C(═

O)OR²² ;

R³⁰ is hydrogen, --O--C(═

O)CH₃, alkoxy having from 1 to about 10 carbons, or --O--Si(R₄)₃ ;

R⁶ is H, alkyl or aralkyl having up to 15 carbon atoms;

or R⁵ and R⁶, together with the atoms to which they are attached, form a 5 or 6 membered ring;

R⁷ is alkyl or aralkyl having up to 15 carbon atoms;

or R⁶ and R⁷ together form one of the structures ##STR96## wherein V, T, and Z are independently CH or N;

R⁸ is H or methyl;

R¹⁶ is H, alkyl or aralkyl having up to 15 carbon atoms;

B is a nucleobase; and

n is an integer from 0 to 50;

with a second synthon of Formula II;

##STR97## wherein;

R⁹ is a hydroxyl protecting group or a linker connected to a solid support; and

m is an integer from 0 to 50;

for a time and under reaction conditions effective to form a third synthon of Formula III;

##STR98## and contacting said third synthon with a sulfurizing agent to form an oligomer of Formula IV;

##STR99## wherein D is said phosphorothioate linkage having the formula ##STR100##

2. The method of claim 1 wherein said phosphozothioate linkage is diastereomerically enriched.

3. The method of claim 1 wherein about 75% of said phosphorothioate linkage is in a single stereoisomeric form.

4. The method of claim 1 wherein about 85% of said phosphorothioate linkage is in a single stereoisomeric form.

5. The method of claim 1 wherein about 95% of said phosphorothioate linkage is in a single stereoisomeric form.

6. The method of claim 1 wherein said phosphorothioate linkage is in a single stereoisomeric form, substantially free of other stereoisomeric forms.

7. The method of claim 1 wherein n is 0.

8. The method of claim 1 wherein said first synthon is in a single stereoisomeric form, substantially free of other stereoisomeric forms.

9. The method of claim 1 further comprising removing said R¹ group from said phosphorothioate.

10. The method of claim 1 further comprising removing said chiral auxiliaries.

11. The method of claim 1 wherein said compound of Formula IV contains a plurality of said phosphorothioate linkages.

12. The method of claim 1 wherein said first and second synthons are reacted at a temperature of from about -20°

C. to about 40°

C.

13. The method of claim 1 wherein said first and second synthons are reacted at a temperature of from about -15°

C. to about 0°

C.

14. The method of claim 1 wherein said first synthon is formed by reacting a compound of Formula V:

##STR101## with an azaphospholane of Formula VIa;

##STR102## wherein R³ -R⁸ are as defined above; and

X is halogen, dialkylamino, imidazole, triazole or substituted phenoxy wherein said substituents are electron withdrawing.

15. The method of claim 14 wherein said electron withdrawing substituents are halogen or nitro.

16. The method of claim 14 wherein said azaphospholane is produced by reacting a reagent of formula HO--C(R⁸)R³ --C(R¹⁶)R⁵ --CHR⁶ --NHR⁷ and a phosphorus trihalide, phosphorus tri(dialkylamide), phosphorus triphenoxide, or phosphorus tri(imidazolides).

17. The method of claim 1 wherein said first synthon is formed by reacting a compound of Formula VII:

##STR103## and a γ

-amino alcohol of formula HO--C(R⁸)R³ --C(R¹⁶)R⁵ --CHR⁶ --NHR⁷ ;

wherein X and R¹ -R⁸ and R¹⁶ are as defined above.

18. The method of claim 17 wherein X is chlorine.

19. The method of claim 17 wherein said reaction is stereoselective.

20. The method of claim 18 wherein said first synthon is in a single stereoisomeric form, substantially free of other stereoisomeric forms.

21. The method of claim 1 wherein said reaction is performed in the presence of a catalyst.

22. The method of claim 21 wherein said catalyst has one of the Formulas VIII or IX:

##STR104## wherein;

R¹² and R¹³ are independently hydrogen, halogen, cyano, nitro, alkyl having from one to 10 carbons, group, or R¹² and R¹³ together with the carbon atoms to which they are attached, form a substituted or unsubstituted 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;

##STR105## wherein L is protecting group.

23. The method of claim 21 wherein R¹⁴ is halogen or nitro, and wherein R¹² and R¹³ are each halogen or each cyano.

24. The method of claim 22 wherein R¹⁴ is bromine, and R¹² and R¹³ are each cyano.

25. The method of claim 22 wherein R¹⁴, R¹² and R¹³ are each bromo.

26. The method of claim 21 wherein R¹⁴ has one of the formulas:

##STR106## wherein R¹⁵ is H, methyl, trialkylsilyl or acetyl.

27. The method of claim 1 wherein R³ is cyanomethyl or --CH₂ --SO_k R⁴ where k is 0, 1 or 2.

28. The method of claim 27 wherein R⁷ is lower alkyl or aralkyl.

29. The method of claim 1 wherein said first synthon has one of the Formulas Xa, XIa, XIIa, XIIIa or XXa:

##STR107## wherein W has the formula;

##STR108## and R¹ -R¹⁶, V, T and Z are as defined above.

30. The method of claim 29 wherein said first synthon has the Formula Xb or Xc:

##STR109##

31. The method of claim 29 wherein said first synthon has the Formula XIVa:

##STR110## wherein p, Z¹ and Z² are as defined above.

32. The method of claim 31 wherein said first synthon has the Formula XVa or XVIa:

##STR111## wherein Y, R⁷, Z¹, Z², and p are as defined above.

33. The method of claim 29 wherein said first synthon has the Formula XVIIa or XVIIIa:

##STR112## wherein W, R⁷, R¹⁰ and R¹¹ are as defined above.

34. The method of claim 1 further comprising removing said R⁹ groups.

35. The method of claim 1 further comprising removing said R² groups.

36. The oligomer produced by the method of claim 1.