Linking reagents for nucleotide probes

• US 5,585,481 A
• Filed: 01/14/1994
• Issued: 12/17/1996
• Est. Priority Date: 09/21/1987
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Bhatt, Ram S., Reynolds, Mark A., Arnold, Lyle J. Jr.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/182,666
• Time in Patent Office: 1,068 Days
• Field of Search: 536/25.33, 536/25.34, 536/26.1, 536/26.11, 536/26.13, 536/26.14, 536/26.2, 536/26.21, 536/26.22, 536/26.23, 536/26.26, 536/26.3, 536/26.5 , 536/26.6, 536/26.7, 536/26.74, 536/26.8, 536/26.9, 558/70, 558/199, 558/202, 562/8, 562/10, 562/23, 525/155
• US Class Current: 536/25.33
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• 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

Abstract

A versatile reagent with a non-nucleotide monomeric unit having a ligand, and first and second coupling groups which are linked to the non-nucleotide monomeric unit. The ligand can be either a chemical moiety, such as a label or intercalator, or a linking arm which can be linked to such a moiety. Such reagent permits preparation of versatile nucleotide/non-nucleotide polymers, having any desired sequence of nucleotide and non-nucleotide monomeric units, each of the latter of which bear a desired ligand. These polymers can for example, be used as probes which can exhibit enhanced sensitivity and/or which are capable of detecting a genus of nucleotides each species of which has a common target nucleotide sequence of interest bridged by different sequences not of interest.

Claims

1. A linking reagent having the formula:

space="preserve" listing-type="equation">D--X.sup.1 --R.sup.2 (X.sup.3 --M).sub.n --Z

wherein;

Z is a reactive phosphorus-containing group attached to the end of R², wherein said reactive phosphorus-containing group is able to couple to, or is activatable for coupling to, an OH group of a first nucleotide;

R² comprises a chain of atoms which is 2-20 atoms in length joined to X¹ and Z, wherein R² is stable to DNA synthesis and deprotection conditions;

X^l is either O, S, NH or NNH;

D is a protecting group which may be removed to permit X¹ to be coupled to the phosphorus group of a second nucleotide;

each X³ is independently a linker-arm joined at a first end to R² and joined at a second end to M, wherein each X³ is stable to DNA synthesis and deprotection conditions;

each M is independently either a label stable to DNA synthesis and deprotection conditions or a protecting group which can be removed to permit X³ to be coupled to a label; and

n is a positive integer.

2. The linking reagent of claim 1, wherein Z has the formula:

##STR3## wherein, X² is either a halogen or a substituted amino;

R³ is selected from the group consisting of alkyl, alkoxy and phenoxy;

X⁴ is selected from the group consisting of halogen, amino and O^- ; and

R⁵ is selected from the group consisting of alkyl, alkoxy, aryloxy and H, provided that R⁵ may be H only if X⁴ is O^-.

3. The linking reagent of claim 2, wherein Z has the formula:

##STR4##

4. The linking reagent of claim 3, wherein X² is either Cl or a secondary amino;

and

R³ is selected from the group consisting of chlorophenoxy, methoxy, ethoxy and beta-cyanoethoxy.

5. The linking reagent of claim 4, wherein X² is selected from the group consisting of diisopropylamino, dimethyl and morpholino.

6. The linking reagent of claim 2, wherein Z has the formula:

##STR5##

7. The linking reagent of claim 6, wherein X⁴ is selected from the group consisting of Cl, secondary amino and O^- ;

and

R⁵ is selected from the group methoxy, ethoxy, monochlorophenoxy, beta-cyanoethoxy and H, provided that R⁵ may be H only if X⁴ is O^-.

8. The linking reagent of claim 2, wherein X¹ is O.

9. The linking reagent of any one of claims 1-8, wherein R² consists essentially of a hydrocarbon chain optionally substituted by one or more heteroatoms, each independently selected from the group consisting of oxygen, nitrogen and sulfur;

and

each X³ is independently either NH, O, S, NNH, or a chain 1-25 atoms in length ending in either NH, O, S or NNH, wherein each of said X³ chain of atoms consists essentially of a hydrocarbon chain optionally substituted by one or more heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur.

10. The linking reagent claim 9, wherein R² is acyclic.

11. The linking reagent of claim 10, wherein R² in an acyclic hydrocarbon chain.

12. The linking reagent of claim 11, wherein said hydrocarbon chain has a length of 2 to 10 carbon atoms.

13. The linking reagent of claim 12, wherein said hydrocarbon chain has a length of 2 to 3 carbon atoms.

14. The linking reagent of claim 13, wherein n is 1.

15. The linking reagent of any one of claims 1-8, wherein R² is an acyclic hydrocarbon chain.

16. The linking reagent of claim 15, wherein said hydrocarbon chain has a length of 2 to 10 carbon atoms.

17. The linking reagent of claim 16, wherein said hydrocarbon chain has a length of 2 to 3 carbon atoms.

18. The linking reagent of claim 17, wherein n is 1.

19. The linking reagent of claim 2, wherein each X³ is linked to R² by a carbon and to M by a nitrogen and each M is independently trifluoroacetyl or 9-trifluorenylmethoxycarbonyl.

20. The linking reagent of claim 2, or 8, wherein D is triphenylmethyl or dimethoxytriphenylmethyl.

21. The linking reagent of claim 2, wherein M is said label stable to DNA synthesis and deprotection conditions.

22. The linking reagent of claim 2, wherein M is said protecting group which may be removed to permit X³ to be coupled to said label.

23. A linking reagent composition having the formula:

space="preserve" listing-type="equation">Q--X.sup.1 --R.sup.2 (X.sup.3 --M).sub.n --T

wherein;

T is a DNA synthesis support attached to the end of R² through a cleavable ester linkage,

R² comprises a chain of atoms which is 2-20 atoms in length joining X¹ to T, wherein each R² is stable to DNA synthesis and deprotection conditions,

X¹ is O, S, NH or NNH,

Q is a nucleotide or a protecting group which may be removed to permit X¹ to be coupled to the phosphorus group of a nucleotide,

each X³ is independently a linker-arm joined at a first end to R² and joined at a second end to M, wherein each X³ is stable to DNA synthesis and deprotection conditions,

each M is independently a label stable to DNA synthesis and deprotection conditions or a protecting group which can be removed to permit X³ to be coupled to a label, and

each n is a positive integer.

24. The composition of claim 23, wherein Q is said protecting group which may be removed to permit X¹ to be coupled to the phosphorus group of said nucleotide.

25. The composition of claim 24, wherein X¹ is 0.

26. The composition of claim 23, wherein R² consists essentially of a hydrocarbon chain optionally substituted by one or more heteroatoms each independently selected from the group consisting of oxygen, nitrogen and sulfur;

and

each X³ is independently either NH, O, S, NNH, or a chain of 1-25 atoms in length ending in NH, O, S or NNH, wherein each of said X³ chain of atoms consists essentially of a hydrocarbon chain optionally substituted by one or more heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur.

27. The composition of claim 26, wherein X¹ is 0.

28. The composition of any one of claims 23-27, wherein R² is acyclic.

29. The linking reagent of claim 28, wherein R² in an acyclic hydrocarbon chain.

30. The composition of claim 29, wherein said hydrocarbon chain has a length of 2 to 10 carbon atoms.

31. The composition of claim 30, wherein said hydrocarbon chain has a length of 2 to 3 carbon atoms.

32. The composition of claim 31, wherein each n is 1.

33. A linking reagent having the formula:

##STR6## wherein;

Z is a reactive phosphorus-containing group, OH or a cleavable ester attached to a DNA synthesis support, wherein said reactive phosphorus-containing group is able to couple, to or is activatable for coupling to, an OH group of a first nucleotide;

D is a protecting group which may be removed to permit said non-nucleotide linking reagent to be coupled to a second nucleotide;

i is either 0, 1, 2 or 3, and j is either 0, 1, 2 or 3, provided that i+j is at least 1;

L¹ is a first linker-arm;

L² is either H or a second linker-arm;

wherein said first linker-arm, and said second linker-arm each independently have the formula;

space="preserve" listing-type="equation">(CH.sub.2).sub.k --NH--(CO--(CH.sub.2).sub.q NH).sub.r --M

wherein each M is independently H, fluorenylmethoxycarbonyl, trifluoroacetyl or a label stable to DNA synthesis and deprotection conditions,

each k is independently an integer between 0 and 4, inclusive,

each q is independently an integer between 1 and 11, inclusive,

each r is independently either 0, 1, or 2,

provided that each k+1+(2+q)r in said first linker-arm, and in said second linker-arm are independently an integer between 1 and 25, inclusive.

34. The linking reagent of claim 33, wherein Z has the formula:

##STR7## wherein X² is a halogen or a substituted amino;

R³ is selected from the group consisting of alkyl, alkoxy and phenoxy;

X⁴ is selected from the group consisting of halogen, amino and O^- ; and

R⁵ is selected from the group consisting of alkyl, alkoxy, aryloxy and H, provided that R⁵ may be H only if X⁴ is O^-.

35. The linking reagent of claim 34, wherein Z has the formula:

##STR8##

36. The linking reagent of claim 35, wherein X² is Cl or a secondary amino;

and

R³ is selected from the group consisting of chlorophenoxy, methoxy, ethoxy and beta-cyanoethoxy.

37. The linking reagent of claim 36, wherein X² is selected from the group consisting of diisopropylamino, dimethylamino and morpholino.

38. The linking reagent of claim 34, wherein Z has the formula:

##STR9##

39. The linking reagent of claim 38, wherein X⁴ is selected from the group consisting of Cl, secondary amino and O^- ;

and

R⁵ is selected from the group methoxy, ethoxy, monochlorophenoxy, beta-cyanoethoxy and H, provided that R⁵ may be H only if X⁴ is O^-.

40. The linking reagent of claim 33 or 34, wherein j is 0.

41. The linking reagent of claim 33 or 34, wherein L² is H.

42. The linking reagent of claim 33 or 34, wherein L² is H and j is 0.

43. The linking reagent of claim 33 or 34, wherein i is 1.

44. The linking reagent of claim 33 or 34, wherein i is 1 and j is 1.

45. The linking reagent of claim 33 or 34, wherein each r is 0.

46. The linking reagent of claim 33 or 34, wherein each r is 0 and X⁵ is H.

47. The linking reagent of claim 33 or 34, wherein each q is an integer between 1 and 6, inclusively.

48. The linking reagent of claim 42, wherein each q is an integer between 1 and 6, inclusively.

49. The linking reagent of claim 33 or 34, wherein each k is independently an integer between 1 and 3, inclusively.

50. The linking reagent of claim 44, wherein each q is an integer between 1 and 6, inclusively.