Reagents useful for synthesizing rhodamine-labeled oligonucleotides

• US 9,040,674 B2
• Filed: 04/02/2007
• Issued: 05/26/2015
• Est. Priority Date: 03/31/2006
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Benson, Scott C., Upadhya, Krishna G., Cassel, Jonathan M., Zou, Ruiming N., Kenney, Paul M.
• Primary Examiner(s): Lau, Jonathan S
• Application Number:
  US11/695,548
• Publication Number:
  US 20080071070A1
• Time in Patent Office: 2,976 Days
• Field of Search: 536/23.1, 536/25.32
• US Class Current: 536/23.1
• CPC Class Codes: C07D 311/96 spiro-condensed with carbocyclic rings or ring systems
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/242 of hydroxyaryl compounds
• CPC Class Codes: C07F 9/650952 having the nitrogen atoms in the positions 1 and 4
• CPC Class Codes: C07F 9/65312 having the two nitrogen atoms in positions 1 and 2
• CPC Class Codes: C07F 9/65515 the oxygen atom being part of a five-membered ring
• CPC Class Codes: C07F 9/6561 containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes

Abstract

The present disclosure provides reagents that can be used to label synthetic oligonucleotides with rhodamine dyes or dye networks that contain rhodamine dyes.

Claims

1. An oligonucleotide comprising a label moiety produced by reacting an oligonucleotide attached to a solid support with a reagent having a structure of formula (VII):

LM-L-PEP 

 

(VII)

wherein PEP is a phosphate ester precursor group, L is an optional linker linking the label moiety to the phosphate ester precursor group and LM comprises an N-protected NH-rhodamine moiety selected from the group consisting

of structures having formulae (IIIa), (IIIb) and (IIIc);

2. The oligonucleotide of claim 1, wherein the N-protected NH-rhodamine moiety is selected from the group consisting of structures having formulae (IIIa.1), (IIIa.2), (IIIb.1), (IIIb.2), (IIIc.1) and (IIIc.2):

3. The oligonucleotide of claim 1, wherein the label moiety is linked to the 3′

- or 5′

-hydroxyl of the oligonucleotide.

4. The oligonucleotide of claim 1 in which the label moiety is linked to a nucleobase of the oligonucleotide.

5. The oligonucleotide of claim 1, wherein the oligonucleotide is further labeled with a donor and/or acceptor moiety for the N-protected NH-rhodamine moiety.

6. The oligonucleotide of claim 1, wherein the oligonucleotide is further labeled with a quencher moiety.

7. The oligonucleotide of claim 1, wherein the oligonucleotide is further labeled with a minor groove binding moiety.

8. The oligonucleotide of claim 1, wherein the N-protected NH-rhodamine moiety has at least one applicable feature selected from the group consisting of:

(i) each of R⁴ and R⁷ is chloro;

(ii) each of R^1′

and R^8′

is hydrogen;

(iii) R^1′

and R^2′

or R^7′

and R^8′

are taken together to form a benzo group;

(iv) each of R^2′

and R^7′

selected from the group consisting of hydrogen and a lower alkyl;

(v) R′

is R^3′

and R″

is R^6′

; and

(vi) R′

is R^3′

, R″

is R^6′

, and R^3′

and R^6′

are taken together with a substituent group on an adjacent carbon atom to form a moiety selected from the group consisting of —

CH₂CH₂—

, —

CH₂CH₂CH₂—

, —

C(CH₃)₂CH═

C(CH₃)—

, —

C(CH₃)₂CH═

CH—

, —

CH₂—

C(CH₃)₂— and

9. The oligonucleotide of claim 1, wherein R¹⁰ is selected from the group consisting of methyl and trifluoromethyl.

10. The oligonucleotide of claim 1, wherein the label moiety further comprises a donor moiety or an acceptor moiety for the N-protected NH-rhodamine moiety.

11. The oligonucleotide of claim 10, wherein the label moiety further comprises a donor moiety for the N-protected NH-rhodamine moiety.

12. The oligonucleotide of claim 11, wherein the donor moiety comprises an N-protected NH-rhodamine moiety or an O-protected fluorescein moiety.

13. The oligonucleotide of claim 12, wherein the 2′

-, 2″

-, 4′

-, 5′

-, 7′

-, 7″

-, 5- or 6-position of the donor moiety is linked to the 2′

-, 2″

-, 4′

-, 5′

-, 7′

-, 7″

-, 5- or 6-position of the N-protected NH-rhodamine moiety.

14. The oligonucleotide of claim 13, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a head-to-head orientation.

15. The oligonucleotide of claim 13, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a head-to-tail orientation.

16. The oligonucleotide of claim 13, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a tail-to-tail orientation.

17. The oligonucleotide of claim 13, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a side-to-side orientation.

18. The oligonucleotide of claim 13, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a side-to-head orientation.

19. The oligonucleotide of claim 13, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a side-to-tail orientation.

20. The oligonucleotide of claim 13, wherein the label moiety has structural formula (VI):

A-Z¹-Sp-Z²-D 

 

(VI)

wherein A represents the N-protected NH-rhodamine moiety, D represents the donor moiety, Z¹ and Z², which may be the same or different, represent portions of linkages provided by linking moieties comprising a functional group F^z, and Sp represents a spacing moiety.

21. The oligonucleotide of claim 20, wherein A is selected from the group consisting of moieties having structural formulae A.1, A.2, A.3, A.4, A.5 and A.6, and D is selected from the group consisting of moieties having structural formulae D.1, D.2, D.3, D.4, D.5 and D.6, or A is selected from the group consisting of moieties having structural formulae A.7, A.8, A.9, A.10, A.11 and A.12, and D is selected from the group consisting of moieties having structural formulae D.7, D.8, D.9, D.10, D.11 and D.12:

22. The oligonucleotide of claim 21, wherein the moieties having the structural formulae A.1-A.12 and/or D.1-D.12 further include at least one applicable feature selected from the group consisting of:

(i) each of Y^1a, Y^2a and Y^3a is independently selected from the group consisting of —

C(O)— and

—

S(O)₂—

;

(ii) each of Y^1b, Y^2b and Y^3b is —

NH—

;

(iii) each of R⁴ and R⁷ is chloro;

(iv) each of R^1′

and R^8′

is hydrogen;

(v) R^1′

and R^2′

or R^7′

and R^8′

are taken together to form a benzo group;

(vi) each of R^2′

and R^7′

is selected from the group consisting of hydrogen and a lower alkyl;

(vii) R′

is R^3′

and R″

is R^6′

; and

(viii) R′

is R^3′

, R″

is R^6′

, and R^3′

and R^6′

are taken together with a substituent group on an adjacent carbon atom to form a group selected from —

CH₂CH₂—

, —

CH₂CH₂CH₂—

, —

C(CH₃)₂CH═

C(CH₃)—

, —

C(CH₃)₂CH═

CH—

, —

CH₂—

C(CH₃)₂— and

23. The oligonucleotide of claim 21, wherein:

(i) each of Y^1a, Y^2a and Y^3a is NH—

;

(ii) each of Y^1b, Y^2b and Y^3b is independently selected from the group consisting of —

C(O)— and

—

S(O)₂—

;

(iii) Z¹ is selected from the group consisting of —

C(O)— and

—

S(O)₂—

;

(iv) Z² is —

NH—

; and

(v) Sp is selected from the group consisting of —

(CH₂)_a—

[(Ar)_b—

(CH₂)_a]_c—

, —

(CH₂)_a—

[C≡

C—

(CH₂)_a]_c—

, —

(CH₂)_a—

[C≡

C—

(Ar)_b]_c—

(CH₂)_a—

, —

(CH₂)_d—

NH—

C(O)—

[(CH₂)_a—

(Ar)—

(CH₂)_a—

C(O)—

NH]_c—

(CH₂)_d—

, and —

[CH₂(CH₂)_eO]_f—

CH₂(CH₂)—

, wherein;

each a is independently an integer having the value between 0 and 4;

each b is independently an integer having the value between 1 and 2;

each c is independently an integer having the value between 1 and 5;

each d is independently an integer having the value between 1 and 10;

each e is independently an integer having the value between 1 and 4;

each f is independently an integer having the value between 1 and 10; and

each Ar is independently an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene, arylene or heteroarylene group.

24. The oligonucleotide of claim 21, wherein in the structures D.1-D.12, R^1′

and R^2′

and/or R^7′

and R^8′

are taken together with the carbon atoms to which they are bonded to form a benzo bridge.

25. The oligonucleotide of claim 21, wherein each of E¹ and E² is —

OR^9b.

26. The oligonucleotide of claim 25, wherein R^9b is a group of the formula —

C(O)R^10b, wherein R^10b is a lower alkyl.

27. The oligonucleotide of claim 26, wherein R^10b is tert-butyl.

28. The oligonucleotide of claim 21, wherein R⁹ is a group of the formula —

C(O)R¹⁰, wherein R¹⁰ is selected from the group consisting of hydrogen, a lower alkyl, —

CX₃, —

CHX₂, —

CH₂X, —

CH₂—

OR^d and phenyl optionally mono-substituted with a lower alkyl, —

X, —

OR^d, cyano or nitro group, wherein R^d is selected from the group consisting of a lower alkyl, phenyl and pyridyl, and each X is a halo group.

29. The oligonucleotide of claim 28, wherein R¹⁰ is selected from the group consisting of methyl and trifluoromethyl.

30. The oligonucleotide of claim 1, wherein the L of the reagent is selected from -Z-(CH₂)_3-6—

O—

, -Z-(CH₂)_a—

[(Ar)_b—

(CH₂)_a]_c—

O—

, -Z-(CH₂)_a—

[C≡

C—

(CH₂)_a]_c—

O—

, -Z-(CH₂)_a—

[C≡

C—

(Ar)_b]_c—

(CH₂)_a—

O—

, -Z-(CH₂)_d—

NH—

C(O)—

[(CH₂)_a—

(Ar)—

(CH₂)_a—

C(O)—

NH]_c—

(CH₂)_d—

O—

, and -Z-[CH₂(CH₂)_eO]_f—

CH₂(CH₂)_eO—

, wherein;

each Z represents, independently of the others, a portion of a linkage contributed by a functional group F^z;

each a represents, independently of the others, an integer ranging from 0 to 4;

each b represents, independently of the others, an integer ranging from 1 to 2;

each c represents, independently of the others, an integer ranging from 1 to 5;

each d represents, independently of the others, an integer ranging from 1 to 10;

each e represents, independently of the others, an integer ranging from 1 to 4;

each f represents, independently of the others, an integer ranging from 1 to 10; and

each Ar represents, independently of the others, an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene, arylene or heteroarylene group.

31. The oligonucleotide of claim 30, wherein each Ar of L, independently of the others, is a group derived cyclohexane, piperazine, benzene, napthalene, phenol, furan, pyridine, piperidine, imidazole, pyrrolidine or oxadizole.

32. The oligonucleotide of claim 1, wherein the reagent further comprises a suitably protected synthesis handle comprising a reactive group selected from the group consisting of amino, hydroxyl, thiol and aldehyde and a protecting group configured a) to be removed to provide the reactive group for attachment of additional moieties during the course of synthesis of the oligonucleotide or b) to be stable during the course of synthesis of the oligonucleotide and be removed following synthesis of the oligonucleotide to provide the reactive group for attachment of additional moieties.

33. The oligonucleotide of claim 32, wherein the reagent is a compound according to structural formula (VIII):

R^eO-L-LM-L-PEP 

 

(VIII)

wherein R^e represents an acid-labile protecting group, each L represents, independently of the other, an optional linker, LM represents the label moiety and PEP represents the phosphate ester precursor group.

34. The oligonucleotide of claim 32, wherein the reagent is a compound according to structural formula (IX):

35. The oligonucleotide of claim 34, wherein the reagent is a compound according to structural formula (IX.1):

36. The oligonucleotide of claim 35, wherein Sp¹, Sp² and Sp³ are each, independently of one another, selected from an alkylene chain containing from 1 to 10 carbon atoms, —

(CH₂)_a—

[(Ar)_b—

(CH₂)_a]_c—

, —

(CH₂)_a—

[C≡

C—

(CH₂)_a]_c—

, —

(CH₂)_a—

[C≡

C—

(Ar)_b]_c—

(CH₂)_a—

, —

(CH₂)_d—

NH—

C(O)—

[(CH₂)_a—

(Ar)—

(CH₂)_a—

C(O)—

NH]_c—

(CH₂)_d— and

—

[CH₂(CH₂)_eO]_f—

CH₂(CH₂)—

, where each a represents, independently of the others, an integer ranging from 0 to 4;

each b represents, independently of the others, an integer ranging from 1 to 2;

each c represents, independently of the others, an integer ranging from 1 to 5;

each d represents, independently of the others, an integer ranging from 1 to 10;

each e represents, independently of the others, an integer ranging from 1 to 4;

each f represents, independently of the others, an integer ranging from 1 to 10; and

Ar represents, independently of the others, an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene, arylene or heteroarylene group.

37. The oligonucleotide of claim 32, wherein the reagent is a compound according to structural formula (IX.2), (IX.3), (IX.4) or (IX.5):

38. The oligonucleotide of claim 37, wherein B of the reagent is selected from A^iBu, A^Pac, C^Ac, G^iPr-Pac, T and U.

39. The oligonucleotide of claim 37, wherein L² of the reagent is selected from —

C≡

C—

CH₂—

NH—

, —

C≡

C—

C(O)—

, —

CH═

CH—

NH—

, —

CH═

CH—

C(O)—

, —

C≡

C—

CH₂—

NH—

C(O)—

(CH₂)_1-6—

NH—

, —

CH═

CH—

C(O)—

NH—

(CH₂)_1-6—

NH—

C(O)—

, —

C═

CH—

CH₂—

O—

CH₂CH₂—

[O—

CH₂CH₂]_0-6—

NH—

, —

C≡

C—

C≡

C—

CH₂—

O—

CH₂CH₂—

[O—

CH₂CH₂]_0-6—

NH—

, —

C≡

C—

(Ar)_1-2—

C≡

C—

CH₂—

O—

CH₂CH₂—

[O—

CH₂CH₂]_0-6—

NH—

, —

C≡

C—

(Ar)_1-2—

O—

CH₂CH₂—

[O—

CH₂CH₂]_0-6—

NH— and

—

C≡

C—

(Ar)_1-2—

O—

CH₂CH₂—

[O—

CH₂CH₂]_0-6—

NH—

, where Ar represents, independently of the others, an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene, arylene or heteroarylene group.

40. The oligonucleotide of claim 1, wherein the phosphate ester precursor group comprises a phosphoramidite group and an H-phosphonate group.

41. The oligonucleotide of claim 40, wherein the phosphate ester precursor group comprises a phosphoramidite of the formula (P.1):

42. The oligonucleotide of claim 40, wherein R²⁰ is beta-cyanoethyl and R²¹ and R²² are each isopropyl.

43. The oligonucleotide of claim 32, wherein the synthesis handle has the formula —

OR^e, where R^e is an acid-labile protecting group.

44. The oligonucleotide of claim 43 wherein the acid-labile protecting group is selected from the group consisting of triphenylmethyl (trityl), 4-monomethoxytrityl, 4,4′

-dimethoxytrityl, 4,4′

,4″

-trimethoxytrityl, bis (p-anisyl)phenylmethyl, naphthyldiphenylmethyl, p-(p′

-bromophenacyloxy)phenyldiphenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl and 9-(9-phenyl-10-oxo)anthryl.