Enhanced triple-helix and double-helix formation with oligomers containing modified purines

• US 5,594,121 A
• Filed: 06/07/1995
• Issued: 01/14/1997
• Est. Priority Date: 11/07/1991
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: Gilead Sciences Inc.
• Inventors: Matteucci, Mark, Froehler, Brian
• Primary Examiner(s): Leguyader, John L.
• Assistant Examiner(s): LARSON, THOMAS GEORGE
• Application Number:
  US08/479,248
• Time in Patent Office: 587 Days
• Field of Search: 536/23.1, 536/24.1, 536/24.3, 536/25.3, 536/25.6, 536/24.5, 536/27.2, 536/27.21, 536/27.6, 536/27.7, 536/27.81, 519/44, 435/6, 435/91.2, 435/91.1
• US Class Current: 536/23.5
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• 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: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays

Abstract

Novel oligomers are disclosed which have enhanced ability with respect to forming duplexes or triplexes compared with oligomers containing only conventional bases. The oligomers contain 7-deaza-7-substituted purines or related analogs. The oligomers of the invention are capable of (i) forming triplexes with various target sequences such as virus or oncogene sequences by coupling into the major groove of a target DNA duplex at physiological pH or (ii) forming duplexes by binding to single-stranded DNA or to RNA encoded by target genes. The oligomers of the invention can be constructed to have any desired sequence, provided the sequence normally includes one or more bases that is replaced with the analogs of the invention. Compositions of the invention can be used for diagnostic purposes in order to detect viruses or disease conditions.

Claims

1. An oligomer comprising from 2 to 30 nucleomonomers wherein at least one of said nucleomonomers comprises a base having the formula (1) or (2):

##STR11## wherein Pr is hydrogen or a monovalent or divalent protecting group;

W is N or CH;

R² is aryl, heteroaryl or 1-alkynylheteroaryl;

R⁸ is OH, SH, NPr, NHPr or NH₂ ;

R⁹ is H, OH, SH, NPr, NHPr or NH₂ ;

R¹⁰ is independently H, OH, CN, F, Cl, Br, I, alkyl (C1-12), alkenyl (C2-12), alkynyl (C2-12), aryl (C6-9), heteroaryl (C4-9), or both R¹⁰, taken together with the carbon atoms to which they are linked at positions 11 and 12, form

(a) a 5 or 6 membered carbocyclic ring or,

(b) a 5 or 6 membered heterocyclic ring comprising 1-3 N, O or S ring atoms, provided that no 2 adjacent ring atoms are O--O, S--S, O--S or S--O,

and wherein any unsaturated C atom of the carbocyclic or heterocyclic ring is substituted by R¹¹ and any saturated carbons contain 2 R¹¹ substituents, wherein;

R¹¹ is independently H, alkyl (C1-4), alkenyl (C2-4), alkynyl (C2-4), OR¹², SR¹², or N(R¹²)₂ or halogen provided that there are no more than four halogens per 5 or 6 member ring;

R¹² is independently H, or alkyl (C1-4);

and salts thereof.

2. The oligomer of claim 1 wherein at least one base has the formula (2) and R¹⁰ at position 11 is H and R¹⁰ at position 12 is methyl, R¹⁰ at position 12 is H or R¹⁰ at position 11 is methyl, or R¹⁰ at position 11 is H and R¹⁰ at position 12 is 1-propynyl.

3. The oligomer of claim 1 wherein adjacent nucleomonomers are linked by linkages or substitute linkages and the oligomer contains at least one substitute linkage.

4. The oligomer of claim 3 wherein the substitute linkage is selected from the group consisting of phosphoramidate, phosphorothioate, methylphosphonate, riboacetal, 3'"'"'-N-methylhydroxylamine, thionomethylphosphonate, phosphorodithioate, 2'"'"'-thioformacetal, formacetal, and 3'"'"'-thioformacetal.

5. The oligomer of claim 4 wherein the substitute linkage is a phosphorothioate, formacetal or 3'"'"'-thioformacetal linkage.

6. The oligomer of claim 1 wherein at least one base has the formula (1) and R² is phenyl, 2-s-triazinyl, 2-, 3- or 4-pyridyl, 2-, 4- or 5-pyrimidinyl, 2- or 3-thienyl, 2-, 4- or 5-thiazolyl, 2- or 4-imidazolyl, 2-, 4- or 5-oxazoyl, 2- or 3-furanyl, 2- or 3-pyrrolyl, phenyl-ethynyl, 2-, 3- or 4-pyridine-ethyl, 2-, 4- or 5-pyrimidine-ethynyl, 2-, or 3-thiophene-ethynyl, 2-, 4- or 5-thiazole-ethynyl, 2- or 4-imidazole-ethynyl, s-triazine-ethynyl, 2- or 3-furan-ethynyl, 2-, 4- or 5-oxazole-ethynyl or 2- or 3-pyrrol-ethynyl.

7. The oligomer of claim 1 having from 8 to 30 nucleomonomers and uniform polarity.

8. The oligomer of claim 7 having 2 to 12 substitute linkages or nucleomonomers at the 5'"'"'-end and at the 3'"'"'-end which comprise nuclease stable domains, and 3 to 26 substitute linkages or nucleomonomers which comprise at least one RNase H competent domain consisting of 3 or more contiguous substitute linkages selected from the group consisting of phosphorothioate and phosphorodithioate linkages disposed between the nuclease stable domains.

9. The oligomer of claim 1 comprising 2, 3, 4, 5 or 6 nucleomonomers.

10. A method of synthesizing the oligomer of claim 1, comprising the steps of:

synthesizing a protected nucleomonomer synthon having a protecting group and a base and further having a coupling group capable of coupling to a nucleomonomer or oligomer;

coupling the nucleomonomer synthon to an acceptor nucleomonomer or oligomer;

removing the protecting group; and

repeating the cycle until said oligomer is synthesized.

11. The method of claim 10 wherein the coupling group is a phosphoramidite or a hydrogen phosphonate coupling group.

12. The oligomer of claim 1 wherein at least one base has the formula (2) and both R¹⁰ are taken together with the carbon atoms to which they are linked at positions 11 and 12 to form a 6-membered ring, said base having the formula (23) or (25) ##STR12## wherein each Q is independently N or CR¹¹.

13. The oligomer of claim 12 wherein adjacent nucleomonomers are linked by linkages or substitute linkages and at least one nucleomonomer linkage is a substitute linkage.

14. The oligomer of claim 12 wherein adjacent nucleomonomers are linked by linkages or substitute linkages and at least one nucleomonomer linkage is a phosphorothioate linkage.

15. The oligomer of claim 12 wherein adjacent nucleomonomers are linked by substitute linkages and all nucleomonomer substitute linkages are phosphorothioate linkages.

16. The oligomer of claim 13 wherein the substitute linkage is selected from the group consisting of phosphoramidite, phosphorothioate, methylphosphonate, riboacetal, 3'"'"'-N-methylhydroxylamine, thionomethylphosphonate, phosphorodithioate, 2'"'"'-thioformacetal, formacetal, and 3'"'"'-thioformacetal.

17. The oligomer of claim 12 complexed with a cationic lipid.

18. The oligomer of claim 12 having from 8 to 30 nucleomonomers and uniform polarity.

19. The oligomer of claim 18 having 2 to 12 substitute linkages or nucleomonomers at the 5'"'"'-end and at the 3'"'"'-end which comprise nuclease stable domains, and 3 to 26 substitute linkages or nucleomonomers which comprise at least one RNase H competent domain consisting of 3 or more contiguous substitute linkages selected from the group consisting of phosphorothioate and phosphorodithioate linkages disposed between the nuclease stable domains.

20. An oligomer of the formula (5);

##STR13## wherein each R¹ is independently H, PO₃^-2, or a blocking group;

each R³ is independently selected from the group consisting of H, OH, F, NH₂, OCH₃, OC₂ H₅, OC₃ H₇, SCH₃, SC₂ H₅, SC₃ H₇, OC₃ H₅, and SC₃ H₅ ;

each X¹ is independently selected from the group consisting of --P(S)(O)--, --P(S)(S)--, --P(O)(O)--, --P(CH₃)(O)-- and --P(CH₃)(S)--;

n is an integer from 0 to 98; and

B is a base, provided that at least one B is of formula (1) or (2);

##STR14## wherein Pr is H or a monovalent or divalent protecting group;

W is N or CH;

R² is aryl, heteroaryl or 1-alkynylheteroaryl;

R⁸ is OH, SH, a NPr or NH₂ ;

R⁹ is H, OH, SH, NPr, NHPr or NH₂ ;

R¹⁰ is independently H, OH, CN, halogen (F, Cl, Br, I), alkyl (C1-12), alkenyl (C2-12), alkynyl (C2-12), aryl (C6-9), heteroaryl (C4-9), or both R¹⁰, taken together with the carbon atoms to which they are linked at positions 11 and 12, form

(a) a 5 or 6 membered carbocyclic ring or,

(b) a 5 or 6 membered heterocyclic ring comprising 1-3 N, O or S ring atoms, provided that no 2 adjacent ring atoms are O--O, S--S, O--S or S--O,

and wherein any unsaturated C atom of the carbocyclic or heterocyclic ring is substituted by R¹¹ and any saturated carbons contain 2 R¹¹ substituents, wherein;

R¹¹ is independently H, alkyl (C1-4), alkenyl (C2-4), alkynyl (C2-4), OR¹², SR¹², or N(R¹²)₂ or halogen provided that there are no more than four halogens per 5 or 6 member ring; and

R¹² is independently H, or alkyl (C1-4).

21. The oligomer of claim 20 wherein at least one R¹ is H, PO₃^-2, DMT, MMT, H-phosphonate, methyl phosphoramidite, methylphosphoramidite, β

-cyanoethylphosphoramidite or alkylphosphoramidite.

22. The oligomer of claim 20 wherein each R³ is independently H, OH, F or O-allyl.

23. The oligomer of claim 20 wherein at least one R³ is O-methyl, O-ethyl or O-propyl.

24. The oligomer of claim 20 wherein at least one base has the formula (2) and R¹⁰ at position 11 is H and R¹⁰ at position 12 is methyl, R¹⁰ at position 12 is H or R¹⁰ at position 11 is methyl, or R¹⁰ at position 11 is H and R¹⁰ at position 12 is 1-propynyl.

25. The oligomer of claim 20 wherein at least one base has the formula (2) and both R¹⁰ are taken together with the carbon atoms to which they are linked at positions 11 and 12 to form a 6-membered ring, said base having the formula (23) or (25) ##STR15## wherein each Q is independently N or CR¹¹.

26. The oligomer of claim 20 having a covalent link between the 5'"'"'-nucleomonomer and the 3'"'"' nucleomonomer whereby a circular oligomer is formed.

27. The oligomer of claim 20 conjugated to a solid support or label.

28. The oligomer of claim 20 which is a dimer, trimer, tetramer, pentamer or hexamer.

29. The oligomer of claim 20 complexed with a cationic lipid.

30. A method of synthesizing the oligomer of claim 20, comprising the steps of:

synthesizing a protected nucleomonomer synthon having a protecting group and a base and further having a coupling group capable of coupling to a nucleomonomer or oligomer;

coupling the nucleomonomer synthon to an acceptor nucleomonomer or oligomer;

removing the protecting group; and

repeating the cycle until said oligomer is synthesized.

31. The method of claim 30 wherein the coupling group is a phosphoramidite or a hydrogen phosphonate coupling group.

32. A nucleomonomer having the structural formula (3) or (4):

##STR16## wherein each R¹ is independently H or a blocking group;

R² is aryl, heteroaryl or 1-alkynylheteroaryl;

R³ is selected from the group consisting of H, OH, F, NH₂, OCH₃, OC₂ H₅, OC₃ H₇, SCH₃, SC₂ H₅, SC₃ H₇, OC₃ H₅, and SC₃ H₅ ;

R⁸ is OH, SH, NPr, NHPr or NH₂ ;

R⁹ is H, OH, SH, NPr, NHPr or NH₂ ;

R¹⁰ is independently H, OH, CN, halogen (F, Cl, Br, I), alkyl (C1-2), alkenyl (C2-12), alkynyl (C2-12), aryl (C6-9), heteroaryl (C4-9), or both R¹⁰, taken together with the carbon atoms to which they are linked at positions 11 and 12, form

(a) a 5 or 6 membered carbocyclic ring or,

(b) a 5 or 6 membered heterocyclic ring comprising 1-3 N, O or S ring atoms, provided that no 2 adjacent ring atoms are O--O, S--S, O--S or S--O,

and wherein any unsaturated C atom of the carbocyclic or heterocyclic ring is substituted by R¹¹ and any saturated carbons contain 2 R¹¹ substituents, wherein;

R¹¹ is independently H, alkyl (C1-4), alkenyl (C2-4), alkynyl (C2-4), OR¹², SR¹², or N(R¹²)₂ or halogen provided that there are no more than four halogens per 5 or 6 member ring;

R¹² is independently H, or alkyl (C1-4);

W is CH or N; and

Pr is H or a monovalent or divalent protecting group.

33. The nucleomonomer of claim 32 wherein R³ is hydrogen, hydroxy fluorine, O-methyl or O-allyl.

34. The nucleomonomer of claim 32 wherein R¹ is a blocking group selected from the group consisting of DMT, MMT, FMOC, phenoxyacetyl, a silyl ether, hydrogen phosphonate, methylphosphonamidite, methylphosphoramidite and β

-cyanoethylphosphoramidite.

35. The nucleomonomer of claim 32 wherein R¹ at the 3'"'"' position is selected from the group consisting of hydrogen phosphonate, N,N-diisopropylamino-β

-cyanoethoxyphosphine, N,N-diisopropyl-aminomethoxyphosphine, N,N-diethylamino-β

-cyanoethoxyphosphine, N,N-morpholino-β

-cyanoethoxyphosphine, N,N-morpholino-methoxyphosphine, N,N-diisopropylaminomethyl-phosphonamidite, N,N-diethylaminomethylphosphonamidite, bis-morpholino-phosphine, N,N-dimethylamino-β

-cyanoethyl-mercaptophosphine, 2-chlorophenyl phosphate, 4-chlorophenyl phosphate, 2,4-dichlorophenyl phosphate, 2,4-dibromophenyl phosphate, 2-chlorophenyl thiophosphate, 4-chlorophenyl thiophosphate, 2,4-dichlorophenyl thiophosphate, and 2,4-dibromophenyl thiophosphate.

36. The nucleomonomer of claim 32 having a structure of formula (4) wherein each R¹⁰ is independently H, OH, CN, halogen, alkyl (C1-9), 1-alkenyl (C2-8), 1-alkynyl (C2-8), aryl (C6-7), or heteroaryl (C4-6).

37. The nucleomonomer of claim 32 of the formula:

##STR17## wherein Q is independently N or CR¹¹.

38. The nucleomonomer of claim 37 wherein R³ is hydrogen, hydroxy fluorine, or O-allyl.

39. The nucleomonomer of claim 37 wherein both R¹ are hydrogen and R³ is hydroxy.

40. The nucleomonomer of claim 38 wherein both R¹ are hydrogen and R³ is hydroxy.

41. The nucleomonomer of claim 37 wherein R¹ is a blocking group selected from the group consisting of DMT, MMT, FMOC, phenoxyacetyl, a silyl ether, hydrogen phosphonate, methylphosphonamidite, methylphosphoramidite and β

-cyanoethylphosphoramidite.

42. The nucleomonomer of claim 37 wherein R¹ at the 3'"'"' position is selected from the group consisting of hydrogen phosphonate, N,N-diisopropylamino-β

-cyanoethoxyphosphine, N,N-diisopropyl-aminomethoxyphosphine, N,N-diethylamino-β

-cyanoethoxyphosphine, N,N-morpholino-β

-cyanoethoxyphosphine, N,N-morpholino-methoxyphosphine, N,N-diisopropylaminomethyl-phosphonamidite, N,N-diethylamino-methylphosphonamidite, bis-morpholino-phosphine, N,N-dimethylamino-β

-cyanoethyl-mercaptophosphine, 2-chlorophenyl phosphate, 4-chlorophenyl phosphate, 2,4-dichlorophenyl phosphate, 2,4-dibromophenyl phosphate, 2-chlorophenyl thiophosphate, 4-chlorophenyl thiophosphate, 2,4-dichlorophenyl thiophosphate, and 2,4-dibromophenyl thiophosphate.

43. The nucleomonomer of claim 37 wherein both R¹ are hydrogen and R³ is hydrogen.

44. The nucleomonomer of claim 32 wherein both R¹ are hydrogen and R³ is hydrogen.

45. An oligomer comprising from 2 to 30 nucleomonomers wherein at least one of said nucleomonomers comprises a base having the formula:

##STR18## wherein Pr is H or a monovalent or divalent protecting group;

R² is 1-propynyl;

R⁸ is OH, SH, NPr, NHPr or NH₂ ;

R⁹ is H, OH, SH, NPr, NHPr or NH₂ ; and

W is N or CH and salts thereof.

46. The oligomer of claim 45 wherein adjacent nucleomonomers are linked by linkages or substitute linkages and the oligomer contains at least one substitute linkage.

47. The oligomer of claim 45 having from 8 to 30 nucleomonomers and uniform polarity.

48. The oligomer of claim 45 wherein the substitute linkage is selected from the group consisting of phosphoramidate, phosphorothioate, methylphosphonate, riboacetal, 3'"'"'-N-methylhydroxylamine, thionomethylphosphonate, phosphorodithioate, 2'"'"'-thioformacetal, formacetal, and 3'"'"'-thioformacetal.

49. The oligomer of claim 48 wherein the substitute linkage is selected from the group consisting of phosphorothioate and formacetal.

50. The oligomer of claim 45 wherein R⁸ is OH and R⁹ is NH₂ or NHPr.

51. The oligomer of claim 45 complexed with a cationic lipid.

52. An oligomer of the formula (5):

##STR19## wherein each R¹ is independently H, PO₃^-2, or a blocking group;

each R³ is independently selected from the group consisting of H, OH, F, NH₂, OCH₃, OC₂ H₅, OC₃ H₇, SCH₃, SC₂ H₅, SC₃ H₇, OC₃ H₅, and SC₃ H₅ ;

each X¹ is independently selected from the group consisting of --P(S)(O)--, --P(S)(S)--, --P(O)(O)--, --P(CH₃)(O)-- and --P(CH₃)(S)--;

n is an integer from 0 to 98; and

B is a base, provided that at least one B is of formula;

##STR20## wherein R² is 1-propynyl;

R⁸ is OH or SH;

R⁹ is NPr, NHPr or NH₂ ;

Pr is H or a monovalent or divalent protecting group; and

W is N or CH.

53. The oligomer of claim 52 wherein at least one R¹ is H, PO₃^-2, DMT, MMT, H-phosphonate, methyl phosphonamidite, methylphosphoramidite, β

-cyanoethylphosphoramidite or alkylphosphoramidite.

54. The oligomer of claim 53 wherein each X¹ is --P(S)(O)--.

55. The oligomer of claim 54 complexed with a cationic lipid.

56. The oligomer of claim 52 wherein each R³ is independently H, OH, F or O-allyl.

57. The oligomer of claim 52 wherein at least one R³ is O-methyl, O-ethyl or O-propyl.

58. The oligomer of claim 52 complexed with a cationic lipid.