Triphosphate oligonucleotide modification reagents and uses thereof

• US 7,999,098 B2
• Filed: 12/11/2006
• Issued: 08/16/2011
• Est. Priority Date: 08/01/2000
• Status: Active Grant

• Current Assignee: Vector Laboratories
• Original Assignee: Solulink Biosciences Incorporated
• Inventors: Schwartz, David A., Hogrefe, Richard I.
• Primary Examiner(s): LEWIS, PATRICK T
• Application Number:
  US11/638,062
• Publication Number:
  US 20070179288A1
• Time in Patent Office: 1,709 Days
• Field of Search: None
• US Class Current: 536/25.3
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• 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 21/04 with deoxyribosyl as saccharide radical

Abstract

Hydrazino, oxyamino and carbonyl-based monomers and methods for incorporation into oligonucleotides during enzymatic synthesis are provided. Modified oligonucleotides are provided that incorporate the monomers provided herein. Immobilized oligonucleotides and oligonucleotide conjugates that contain covalent hydrazone or oxime linkages are provided. Methods for preparation of surface bound oligonucleotides are provided. Methods for the preparation of oligonucleotide conjugates are also provided.

Claims

1. A compound that has formula (I):

P¹—

S¹—

B¹-M-X

or a derivative thereof, wherein;

P¹ is a triphosphate group;

S¹ is a ribose or a dideoxyribose;

B¹ 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(R¹)₂, —

C(R¹)═

C(R¹)—

, >

C═

C(R²)(R³), >

C(R²)(R³), —

C≡

C—

, O, S(A)_a, P(D)_b(R¹), P(D)_b(ER¹), N(R¹), >

N⁺(R²)(R³) and C(E);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

A is O or NR¹;

D is S or O; and

E is S, O or NR¹;

wherein;

each R¹ is a monovalent group independently selected from hydrogen and M¹-R⁴;

each M¹ 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(R⁵)₂, —

C(R⁵)═

C(R⁵)—

, >

C═

C(R²)(R³), >

C(R²)(R³), —

C≡

C—

, O, S(A)_a, P(D)_b(R⁵), P(D)_b(ER⁵), N(R⁵), N(COR⁵), >

N⁺(R²)(R³) and C(E);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

A is O or NR⁵;

D is S or O; and

E is S, O or NR⁵;

R⁴ and R⁵ are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR⁶R⁷R⁸, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR⁹R¹⁰;

R⁹ and R¹⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R² and R³ are selected from (i) or (ii) as follows;

(i) R² and R³ are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or

(ii) R² and R³ together form alkylene, alkenylene or cycloalkylene;

R⁶, R⁷ and R⁸ 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 NR⁹R¹⁰; and

each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ 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)_hR²⁰, NR²⁰R²¹, COOR²⁰, COR²⁰, CONR²⁰R²¹, OC(O)NR²⁰R²¹, N(R²⁰)C(O)R²¹, 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

R²⁰and R²¹ 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(R⁵)₂, O, S(A)_a, N(R⁵), N(COR⁵) and C(E);

where a is 0, 1 or 2;

A is O or NR⁵; and

E is S, O or NR⁵.

8. The compound of claim 1, wherein M has 1-50 of the following groups, which can be combined in any order:

heteroarylene, C(R⁵)₂, N(R⁵) and C(E);

where E is S, O or NR⁵.

9. The compound of claim 1, wherein M has 1-50 of the following groups, which can be combined in any order:

arylene, C(R⁵)₂, N(R⁵) and C(E);

where E is S, O or NR⁵.

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)R³⁰, —

Y—

N(R³¹)—

Y¹—

N(R³²)—

Y² or —

O—

N(R³⁰)—

Y²;

where R³⁰, R³¹ and R³² are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl or cycloalkyl;

Y and Y¹ are selected as in (i) or (ii) as follows;

(i) Y is a direct link, and Y¹ is a direct link, C(O)N(R³⁵), N(R³⁵)C(O)N(R³⁶), C(S)N(R³⁵), N(R³⁵)C(S)N(R³⁶) or C(O)N(R³⁵)N(R³⁶)C(O)N(R³⁷);

or

(ii) Y is C(O) or OC(O), and Y¹ is a direct link;

where R³⁵, R³⁶ and R³⁷ 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

Y² is a salt of the hydrazino or oxyamino group, or any amino or hydrazino protecting group;

where R³⁰, R³¹, R³², R³⁵, R³⁶, R³⁷ and Y² 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)_hR²⁰, NR²⁰R²¹, COOR²⁰, COR²⁰, CONR²⁰R²¹, OC(O)NR²⁰R²¹, N(R²⁰)C(O)R²¹, 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

R²⁰ and R²¹ 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 Y² 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(R³¹)—

Y¹—

N(R³²)—

Y²,

wherein;

Y and Y¹ are selected as in (i) or (ii) as follows;

(i) Y is a direct link, and Y¹ is a direct link, C(O)N(R³⁵), N(R³⁵)C(O)N(R³⁶), C(S)N(R³⁵), N(R³⁵)C(S)N(R³⁶) or C(O)N(R³⁵)N(R³⁶)C(O)N(R³⁷);

or

(ii) Y is C(O) or OC(O), and Y¹ is a direct link;

where R³⁵, R³⁶ and R³⁷ are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl and cylcoalkyl;

Y² is a salt, or any amino or hydrazino protecting group; and

R³¹ and R³² are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl or cycloalkyl;

where R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R³¹, R³², R³⁵, R³⁶, R³⁷ and Y² 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—

Y¹—

NH—

Y²;

wherein Y and Y¹ are selected from (i) or (ii) as follows;

(i) Y is a direct link, and Y¹ 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 Y¹ 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;

CH₂, 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 B¹ is a cytosine.

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

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(R¹)₂, —

C≡

C—

, N(R¹) and C(E);

where E is S, O or NR¹.

30. The compound of claim 1, wherein M has 1-10 of the following groups, which can be combined in any order:

C(R¹)₂, —

C≡

C—

, N(R¹) and C(E);

where E is O or NR¹.

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:

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

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

21. A compound that has formula (II):

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.

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

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.