Methods for making nucleotide polymers using novel linking reagents

• US 5,656,744 A
• Filed: 06/07/1995
• Issued: 08/12/1997
• 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:
  US08/490,109
• Time in Patent Office: 797 Days
• Field of Search: 536/25.1, 536/25.3, 536/25.32, 536/25.33, 536/25.34, 536/25.4
• US Class Current: 536/25.3
• 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 method for making a substituted nucleotide polymer, said method comprising:

(a) coupling, under DNA synthesis conditions, the reactive phosphorus group of a linking reagent to a first nucleotide or a chain of nucleotides protected so as to be stable to DNA synthesis and deprotection conditions, said 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 able to couple to, or activatable for coupling to, an OH group of said 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;

X¹ is 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 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;

(b) removing D under conditions which permit X¹ to be coupled to the activated phosphorus group of a second nucleotide or to a second said linking reagent which may have the same or different D, X¹, R², X³, M and Z groups, and n as said linking reagent.

2. The method 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 =O--.

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

##STR4## wherein X² is either Cl or a secondary amino; and

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

4. The method of claim 3 wherein X² is selected from the group consisting of diisopropylamino, dimethylamino and morpholino.

5. The method of claim 2 wherein Z has the formula:

##STR5## wherein X⁴ is selected from the group consisting of Cl, secondary amino, and O--; and

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

6. The method of claim 1, 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.

7. The method of claim 2, 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.

8. The method of claim 4, 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.

9. The method of claim 5, 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 method of claim 6, wherein X¹ is O.

11. The method of claim 7, wherein X¹ is O.

12. The method of claim 8, wherein X¹ is O.

13. The method of claim 9, wherein X¹ is O.

14. The method of claim 10, wherein n is 1.

15. The method of claim 11, wherein n is 1.

16. The method of claim 12, wherein n is 1.

17. The method of claim 13, wherein n is 1.

18. The method of claim 1 further comprising

reacting X¹ from step (b) with the activated phosphorus group of a second nucleotide or of a second said linking reagent which may have the same or different D, X¹, R², X³, M and Z groups, and n as said linking reagent.

19. The method of claim 1 further comprising

removing M under conditions which permit X³ to be coupled to H, a label or a solid-phase support.

20. The method of claim 19 further comprising

coupling X³ to a label or a solid-phase support.

21. The method of claim 20 wherein X³ is coupled to a label selected from the group consisting of biotin, fluorescein and acridinium ester molecules.

26. A method of claim 1, wherein the linking reagent has the formula:

##STR6## wherein;

Z is either a reactive phosphorus-containing group, 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 linking reagent to be coupled to a second nucleotide;

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

L¹ is a first linker-arm;

L² is 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 1 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.

27. The method of claim 26, wherein Z is said reactive phosphorus-containing group.

28. The method of claim 27, wherein Z has the formula:

##STR7## 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 =O^--.

29. The method of claim 28, wherein Z has the formula:

##STR8## wherein X² is either Cl or a secondary amino; and

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

30. The method of claim 29, wherein X² is selected from the group consisting of diisopropylamino, dimethylamino or morpholino.

31. The method of claim 26, wherein j is 0 or 1.

32. The method of claim 31, wherein L² is H.

33. The method of claim 30, wherein L² is H and j is 0 or 1.

34. The method of claim 26, wherein i is 1.

35. The method of claim 30, wherein i is 1 and j is 1.

36. The method of claim 26, wherein each r is 0.

37. The method of claim 30, wherein L² is H and r is 0.

38. The method of claim 26, wherein each q is an integer between 1 and 6, inclusively.

39. The method of claim 30, wherein each q is an integer between 1 and 6, inclusively.

40. The method of claim 26, wherein each k is independently an integer between 1 and 3, inclusively.

22. A method for making a substituted nucleotide polymer, said method comprising:

coupling, under DNA synthesis conditions, an activated phosphorus group of a nucleotide, to a linking reagent attached through a cleavable ester linkage to a DNA synthesis support and stable to DNA synthesis and deprotection conditions having the formula;

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

wherein;

T is a DNA synthesis support;

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

X¹ is O, S, NH or NNH;

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

n is a positive integer;

wherein said conditions allow coupling of said activated phosphorus group of said nucleotide to said X¹ of said linking reagent.

23. The method of claim 22, 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 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.

24. The method of claim 23, wherein X¹ is O.

25. The method of claim 24, wherein n is 1.

41. A method of claim 22, wherein the linking reagent has the formula:

##STR9## wherein;

T comprises a cleavable group attached to a DNA synthesis support and stable to DNA synthesis conditions;

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

L¹ is a first linker-arm;

L² is 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 1 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.

42. The method of claim 41, wherein j is 0 or 1.

43. The method of claim 41, wherein L² is H.

44. The method of claim 41, wherein L² is H and j is 0 or 1.

45. The method of claim 41, wherein i is 1.

46. The method of claim 41, wherein i is 1 and j is 1.

47. The method of claim 41, wherein each r is 0.

48. The method of claim 41, wherein L² is H and r is 0.

49. The method of claim 41, wherein each q is an integer between 1 and 6, inclusively.

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

51. The method of claim 41, wherein each k is independently an integer between 1 and 3, inclusively.