Granted Patent B2

US 7,999,098 · App. 11/638,062 · Granted Aug 16, 2011

Abstract

Claims (105)

1. A compound that has formula (I):

P 1 —S 1 —B 1 -M-X

or a derivative thereof, wherein:

P 1 is a triphosphate group;

S 1 is a ribose or a dideoxyribose;

B 1 is a nucleobase;

X is a protected or unprotected hydrazino group, a protected or unprotected oxyamino group, or a carbonyl derivative; and

M is a divalent group comprising any combination of any of the following groups, which are combined in any order: arylene, heteroarylene, cycloalkylene, C(R 1 ) 2 , —C(R 1 )═C(R 1 )—, >C═C(R 2 )(R 3 ), >C(R 2 )(R 3 ), —C≡C—, O, S(A) a , P(D) b (R 1 ), P(D) b (ER 1 ), N(R 1 ), >N + (R 2 )(R 3 ) and C(E); where a is 0, 1 or 2; b is 0, 1, 2 or 3; A is O or NR 1 ; D is S or O; and E is S, O or NR 1 ;

wherein:

each R 1 is a monovalent group independently selected from hydrogen and M 1 -R 4 ;

each M 1 is a divalent group each independently comprising any combination of the following groups, which groups are combined in any order: a direct link, arylene, heteroarylene, cycloalkylene, C(R 5 ) 2 , —C(R 5 )═C(R 5 )—, >C═C(R 2 )(R 3 ), >C(R 2 )(R 3 ), —C≡C—, O, S(A) a , P(D) b (R 5 ), P(D) b (ER 5 ), N(R 5 ), N(COR 5 ), >N + (R 2 )(R 3 ) and C(E); where a is 0, 1 or 2; b is 0, 1, 2 or 3; A is O or NR 5 ; D is S or O; and E is S, O or NR 5 ;

R 4 and R 5 are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR 6 R 7 R 8 , alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR 9 R 10 ;

R 9 and R 10 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R 2 and R 3 are selected from (i) or (ii) as follows:

(i) R 2 and R 3 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl; or

(ii) R 2 and R 3 together form alkylene, alkenylene or cycloalkylene;

R 6 , R 7 and R 8 are each independently a monovalent group selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR 9 R 10 ; and

each R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 is unsubstituted or substituted with one or more substituents each independently selected from Z, wherein Z is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, S(O) h R 20 , NR 20 R 21 , COOR 20 , COR 20 , CONR 20 R 21 , OC(O)NR 20 R 21 , N(R 20 )C(O)R 21 , alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido; h is 0, 1 or 2; and R 20 and R 21 are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino.

2. The compound of claim 1 , wherein X is a protected hydrazino group.

3. The compound of claim 1 , wherein X is a carbonyl group.

4. The compound of claim 3 , wherein X is an aldehyde or ketone group.

5. The compound of claim 1 , wherein X is a protected oxyamino group.

6. The compound of claim 1 , wherein the protecting group is a salt, an amine protecting group, or a hydrazine protecting group.

7. The compound of claim 1 , wherein M has 1-50 of the following groups, which can be combined in any order: arylene, heteroarylene, C(R 5 ) 2 , O, S(A) a , N(R 5 ), N(COR 5 ) and C(E); where a is 0, 1 or 2; A is O or NR 5 ; and E is S, O or NR 5 .

8. The compound of claim 1 , wherein M has 1-50 of the following groups, which can be combined in any order: heteroarylene, C(R 5 ) 2 , N(R 5 ) and C(E); where E is S, O or NR 5 .

9. The compound of claim 1 , wherein M has 1-50 of the following groups, which can be combined in any order: arylene, C(R 5 ) 2 , N(R 5 ) and C(E); where E is S, O or NR 5 .

10. The compound of claim 1 , wherein M is a chain of 1-2000 repeating monomer units selected from ethylene oxide, propylene oxide, methacrylamide, or ethylene glycol.

11. The compound of claim 1 , wherein M has a cleavable linkage selected from a disulfide, an ester, an enzyme specific peptide, a photocleavable linkage, or an acid labile group.

12. The compound of claim 1 , wherein X is —C(O)R 30 , —Y—N(R 31 )—Y 1 —N(R 32 )—Y 2 or —O—N(R 30 )—Y 2 ;

where R 30 , R 31 and R 32 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl or cycloalkyl; Y and Y 1 are selected as in (i) or (ii) as follows:

(i) Y is a direct link, and Y 1 is a direct link, C(O)N(R 35 ), N(R 35 )C(O)N(R 36 ), C(S)N(R 35 ), N(R 35 )C(S)N(R 36 ) or C(O)N(R 35 )N(R 36 )C(O)N(R 37 ); or

(ii) Y is C(O) or OC(O), and Y 1 is a direct link;

where R 35 , R 36 and R 37 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl and cylcoalkyl; and

Y 2 is a salt of the hydrazino or oxyamino group, or any amino or hydrazino protecting group;

where R 30 , R 31 , R 32 , R 35 , R 36 , R 37 and Y 2 are unsubstituted or substituted with one or more substituents each independently selected from Z, wherein Z is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, S(O) h R 20 , NR 20 R 21 , COOR 20 , COR 20 , CONR 20 R 21 , OC(O)NR 20 R 21 , N(R 20 )C(O)R 21 , alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido; h is 0, 1 or 2; and R 20 and R 21 are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino.

13. The compound of claim 12 , wherein Y 2 is selected from monomethoxytrityl (MMT), dimethoxytrityl (DMT), 9-fluorenylmethoxy-carbonyl (FMOC), acetyl, trifluoroacetyl (TFA), benzoyl, or a lower aliphatic hydrazone or oxime.

14. The compound of claim 1 , wherein X has the formula:

—Y—N(R 31 )—Y 1 —N(R 32 )—Y 2 ,

wherein:

Y and Y 1 are selected as in (i) or (ii) as follows:

(i) Y is a direct link, and Y 1 is a direct link, C(O)N(R 35 ), N(R 35 )C(O)N(R 36 ), C(S)N(R 35 ), N(R 35 )C(S)N(R 36 ) or C(O)N(R 35 )N(R 36 )C(O)N(R 37 ); or

(ii) Y is C(O) or OC(O), and Y 1 is a direct link;

where R 35 , R 36 and R 37 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl and cylcoalkyl;

Y 2 is a salt, or any amino or hydrazino protecting group; and

R 31 and R 32 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl or cycloalkyl;

where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 31 , R 32 , R 35 , R 36 , R 37 and Y 2 are unsubstituted or substituted with one or more substituents each independently selected from Z.

15. The compound of claim 14 , wherein X has the formula:

—Y—NH—Y 1 —NH—Y 2 ;

wherein Y and Y 1 are selected from (i) or (ii) as follows:

(i) Y is a direct link, and Y 1 is a direct link, C(O)NH, NHC(O)NH, C(S)NH, NHC(S)NH or C(O)NHNHC(O)NH; or

(ii) Y is C(O) or OC(O), and Y 1 is a direct link.

16. The compound of claim 1 , wherein M has 2,5-pyridylene attached to X; and 1-9 of the following groups, which can be combined in any order: CH 2 , NH and C(O).

17. The compound of claim 1 , wherein M has the formula:

18. The compound of claim 1 that has any of the formulae:

19. The compound of claim 1 , wherein B 1 is a cytosine.

20. The compound of claim 1 that has the formula:

21. A compound that has formula (II):

or a derivative thereof, wherein:

O 1 and O 2 are each independently an oligonucleotide or an analogs thereof;

P 2 is a phosphodiester group;

S 1 is a ribose, a 2′-deoxyribose, or a 2′,3′-dideoxyribose;

B 1 is a nucleobase;

X is a protected or unprotected hydrazino group, a protected or unprotected oxyamino group, or a carbonyl derivative; and

M is a divalent group having any combination of any of the following groups, which can be combined in any order: arylene, heteroarylene, cycloalkylene, C(R 1 ) 2 , —C(R 1 )═C(R 1 )—, >C═C(R 2 )(R 3 ), >C(R 2 )(R 3 ), —C≡C—, O, S(A) a, P(D) b (R 1 ), P(D) b (ER 1 ), N(R 1 ), >N + (R 2 )(R 3 ) and C(E); where a is 0, 1 or 2; b is 0, 1, 2 or 3; A is O or NR 1 ; D is S or O; and E is S, O or NR 1 ;

wherein:

P 2 is located at the 5′-position of S 1 ;

each R 1 is a monovalent group independently selected from hydrogen and M 1 -R 4 ;

each M 1 is a divalent group independently having any combination of the following groups, which groups can be combined in any order: a direct link, arylene, heteroarylene, cycloalkylene, C(R 5 ) 2 , —C(R 5 )═C(R 5 )—, >C═C(R 2 )(R 3 ), >C(R 2 )(R 3 ), —C≡C—, O, S(A) a , P(D) b (R 5 ), P(D) b (ER 5 ), N(R 5 ), N(COR 5 ), >N + (R 2 )(R 3 ) and C(E); where a is 0, 1 or 2; b is 0, 1, 2 or 3; A is O or NR 5 ; D is S or O; and E is S, O or NR 5 ;

R 4 and R 5 are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR 6 R 7 R 8 , alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR 9 R 10 ;

R 9 and R 10 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R 2 and R 3 are selected from (i) or (ii) as follows:

(i) R 2 and R 3 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl; or

(ii) R 2 and R 3 together form alkylene, alkenylene or cycloalkylene;

R 6 , R 7 and R 8 are each independently a monovalent group selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR 9 R 10 ; and

R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are unsubstituted or substituted with one or more substituents each independently selected from Z, wherein Z is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, S(O) h R 20 , NR 20 R 21 , COOR 20 , COR 20 , CONR 20 R 21 , OC(O)NR 20 R 21 , N(R 20 )C(O)R 21 , alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido; h is 0, 1 or 2; and R 20 and R 21 are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino.

22. The compound of claim 21 that is immobilized on a surface.

23. The compound of claim 21 that is conjugated to a second component.

24. A method for formation of an oligonucleotide conjugate, comprising the step of:

reacting the compound of claim 21 with a second component to form an oligonucleotide conjugate;

wherein the compound and the second component comprise complementary groups.

25. An oligonucleotide conjugate prepared by the method of claim 24 .

26. The conjugate of claim 23 , wherein the second component is selected from the group consisting of a fluorescein, a rhodamine and a cyanine dye.

27. The compound of claim 1 , wherein M has the formula:

28. The compound of claim 1 , wherein M has the formula:

29. The compound of claim 1 , wherein M has 1-10 of the following groups, which can be combined in any order: arylene, C(R 1 ) 2 , —C≡C—, N(R 1 ) and C(E); where E is S, O or NR 1 .

30. The compound of claim 1 , wherein M has 1-10 of the following groups, which can be combined in any order: C(R 1 ) 2 , —C≡C—, N(R 1 ) and C(E); where E is O or NR 1 .

31. A composition, comprising nucleoside triphosphates, wherein at least one of the nucleoside triphosphates is a compound of claim 1 .

32. A method of synthesizing a nucleic acid using the composition of claim 31 .

33. A method synthesis of a modified oligonucleotide, comprising contacting a composition having at least one nucleoside triphosphate of claim 1 with an enzyme for oligonucleotide synthesis under conditions where oligonucleotide synthesis occurs.

34. The compound of claim 1 , comprising the formula:

35. The compound of claim 1 , comprising the formula:

where each R is OH or each R is H.

36. The compound of claim 1 , comprising the formula:

where each R is OH or each R is H.

37. The compound of claim 1 , comprising the formula:

where each R is OH or each R is H.

38. The compound of claim 21 , comprising the formula:

wherein:

(i) R 40 is selected from the group consisting of an oligonucleotide, H and OH; and

(ii) R 41 is selected from the group consisting of H and OH.

39. The method of claim 24 , wherein the formation of the oligonucleotide conjugate results in the formation of a hydrazone or oxime covalent linkage.

40. The method of claim 24 , wherein the reaction to form the oligonucleotide conjugate is performed under aqueous conditions.

41. The method of claim 24 , wherein the second component comprises a biopolymer, a polymer, and/or a compound useful in an assay, kit, diagnostic array, and the like.

42. The oligonucleotide conjugate of claim 25 , wherein the oligonucleotide conjugate comprises a hydrazone or oxime covalent linkage.

43. The oligonucleotide conjugate of claim 25 , wherein the second component comprises a biopolymer, a polymer, and/or a compound useful in an assay, kit, diagnostic array, and the like.

Assignments (18)