Photocleavable agents and conjugates for the detection and isolation of biomolecules

• US 6,057,096 A
• Filed: 06/07/1995
• Issued: 05/02/2000
• Est. Priority Date: 05/11/1994
• Status: Expired due to Term

• Current Assignee: Boston University
• Original Assignee: Trustees of Boston University
• Inventors: Olejnik, Jerzy, Rothschild, Kenneth J., Sonar, Sanjay M.
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Riley, Jezia
• Application Number:
  US08/479,389
• Time in Patent Office: 1,791 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/24.3, 536/24.33, 536/25.32
• US Class Current: 435/6.13
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 41/0042 Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: C07C 205/45 the carbon skeleton being further substituted by at least one doubly—bound oxygen atom, not being part of a —CHO group
• CPC Class Codes: C07C 205/57 having nitro groups and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
• CPC Class Codes: C07D 207/404 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. succinimide
• CPC Class Codes: C07D 311/16 substituted in position 7
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• 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: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals

Abstract

This invention relates to agents and conjugates that can be used to detect and isolate target components from complex mixtures such as nucleic acids from biological samples, cells from bodily fluids, and nascent proteins from translation reactions. Agents comprise a detectable moiety bound to a photoreactive moiety. Conjugates comprise agents coupled to substrates by covalent bounds which can be selectively cleaved with the administration of electromagnetic radiation. Targets substances labeled with detectable molecules can be easily identified and separated from a heterologous mixture of substances. Exposure of the conjugate to radiation releases the target in a functional form and completely unaltered. Using photocleavable molecular precursors as the conjugates, label can be incorporated into macromolecules, the nascent macromolecules isolated and the label completely removed. The invention also relates to targets isolated with these conjugates which may be useful as pharmaceutical agents or compositions that can be administered to humans and other mammals. Useful compositions include biological agents such as nucleic acids, proteins, lipids and cytokines. Conjugates can also be used to monitor the pathway and half-life of pharmaceutical composition in vivo and for diagnostic, therapeutic and prophylactic purposes. The invention also relates to kits comprised of agents and conjugates that can be used for the detection of diseases, disorders and nearly any individual substance in a complex background of substances.

Claims

1. A method, comprising the steps of:

a) conjugating a bioreactive agent to one or more oligonucleotide primers with a covalent bond that is selectively cleavable with electromagnetic radiation, wherein the conjugated bioreactive agent has a chemical structure selected from the group consisting of;

##STR7## wherein SUB comprises the oligonucleotide primer;

R₁ and R₂ are selected from the group consisting of hydrogen, alkyls, substituted alkyls, aryls, substituted aryls, --CF₃, --NO₂, and --COOH, and may be the same or different;

A is a divalent functional group selected from the group consisting of --O--, --S-- and --N--;

Y comprises one or more polyatomic groups which may be the same or different;

V comprises one or more monoatomic groups which may be the same or different;

Q comprises a spacer moiety;

m₁ and m₂ are integers between 1-5 which can be the same or different; and

D comprises a selectively detectable moiety which is distinct from R₁ and R₂, wherein D is not --NO₂ ;

b) amplifying a nucleic acid sequence with said conjugated oligonucleotide primers; and

c) treating said amplified sequences with electromagnetic radiation.

2. The method of claim 1, wherein said nucleic acid sequence is selected from the group consisting of nucleic acid sequences found in biological samples, bacterial DNA and eukaryotic DNA.

3. The method of claim 1, wherein Y is selected from the group consisting of NO₂, N(R₃)₂ and OR₃, wherein R₃ is selected from the group consisting of hydrogen, alkyls, substituted alkyls, aryls and substituted aryls.

4. The method of claim 1, wherein V is a halide.

5. The method of claim 1, wherein said detectable moiety is an antigen selected from the group consisting of nucleic acids, proteins, and polypeptides.

6. The method of claim 1, wherein said detectable moiety is selected from the group consisting of biotin, coumarin, dansyl, rhodamines, fluoresceins, dinitrophenyl, and combinations thereof.

7. The method of claim 1, wherein said detectable moiety is magnetic.

8. The method of claim 1, wherein said detectable moiety has a net electric charge.

9. The method of claim 1, wherein said spacer moiety comprises a branched or straight chain hydrocarbon.

10. The method of claim 1, wherein Q is represented by the formula:

##STR8## wherein W and W'"'"' are each selected from the group consisting of --C(O)--, --C(O)--NH--, --HN--C(O)--, --NH--, --O--, --S-- and --CH₂ --, and may be the same or different; and

n1 and n2 are integers from 0-10 which can be the same or different and if either n1 or n2 is zero, then W and W'"'"' are optional.

60. The method of claim 8, wherein the application of an electric field to said detectable moiety induces a response that is detectable by spectrometry.

11. A method, comprising the steps of:

a) conjugating an oligonucleotide with a bioreactive agent comprising a chemical structure selected from the group consisting of;

##STR9## wherein G is an O-ester selected from the group consisting of cyanomethyl, o and p nitrophenyl, 2,4-dinitrophenyl, 2,4,5-trichlorophenyl, pentachlorophenyl, pentafluorophenyl, N-hydroxyphthalimidyl, N-hydroxysuccinimidyl, 1-hydroxypiperidinyl, 5-chloro-8-hydroxy-quinolyl, 1-hydroxybenzotriazolyl, 3,4-dihydro-4-oxobenzotriazin-3-yl, 2,3-dihydro-2,5-diphenyl-3-oxo-thiophen-1,1-dioxide-4-yl, 1,2-benzisoxasolyl, 2-hydroxypyridyl, and combinations thereof;

A is a divalent functional group selected from the group consisting of --O--, --S-- and --N--;

Y comprises one or more polyatomic groups which may be the same or different;

V comprises one or more monoatomic groups which may be the same or different;

Q comprises a spacer moiety;

m₁ and m₂ are integers from 0-5 and may be the same or different; and

D comprises a selectively detectable moiety;

b) amplifying a nucleic acid sequence with said conjugated oligonucleotide; and

c) treating said amplified sequences with electromagnetic radiation.

12. The method of claim 11, wherein said nucleic acid sequence is selected from the group consisting of nucleotide sequences found in biological samples, bacterial DNA and eukaryotic DNA.

13. The method of claim 11, wherein Y is selected from the group consisting of NO₂, N(R₃)₂ and OR₃, wherein R₃ is selected from the group consisting of hydrogen, alkyls, substituted alkyls, aryls and substituted aryls.

14. The method of claim 11, wherein V is a halide.

15. The method of claim 11, wherein said detectable moiety is an antigen selected from the group consisting of nucleic acids, proteins, and polypeptides.

16. The method of claim 11, wherein said detectable moiety is selected from the group consisting of biotin, coumarin, dansyl, rhodamines, fluoresceins, dinitrophenyl, and combinations thereof.

17. The method of claim 11, wherein said detectable moiety is magnetic.

18. The method of claim 11, wherein said detectable moiety has a net electric charge.

19. The method of claim 11, wherein said spacer moiety comprises a branched or straight chain hydrocarbon.

20. The method of claim 11, wherein Q is represented by the formula:

##STR10## wherein W and W'"'"' are each selected from the group consisting of --C(O)--, --C(O)--NH--, --HN--C(O)--, --NH--, --O--, --S-- and --CH₂ --, and may be the same or different; and

n1 and n2 are integers from 0-10 which can be the same or different and if either n1 or n2 is zero, then W and W'"'"' are optional.

61. The method of claim 18, wherein the application of an electric field to said detectable moiety induces a response that is detectable by spectrometry.

21. A method, comprising the steps of:

a) conjugating an oligonucleotide with a bioreactive agent comprising a chemical structure selected from the group consisting of;

##STR11## wherein G is an N-amide selected from the group consisting of imidazolyl, benzimidazolyl, benzisooxasolyl, 3,5-dioxo-4-methyl-1, 2,4-oxadiazolidinyl, and combinations thereof;

A is a divalent functional group selected from the group consisting of --O, --S-- and --N--;

Y comprises one or more polyatomic groups which may be the same or different;

V comprises one or more monoatomic groups which may be the same or different;

Q comprises a spacer moiety;

m₁ and m₂ are integers from 0-5 and may be the same or different; and

D comprises a selectively detectable moiety;

b) amplifying a nucleic acid sequence with said conjugated oligonucleotide; and

c) treating said amplified sequences with electromagnetic radiation.

22. The method of claim 21, wherein said nucleotide sequence is selected from the group consisting of nucleotide sequences found in biological samples, bacterial DNA and eukaryotic DNA.

23. The method of claim 21, wherein Y is selected from the group consisting of NO₂, N(R₃)₂ and OR₃, wherein R₃ is selected from the group consisting of hydrogen, alkyls, substituted alkyls, aryls and substituted aryls.

24. The method of claim 21, wherein V is a halide.

25. The method of claim 21, wherein said detectable moiety is an antigen selected from the group consisting of nucleic acids, proteins, and polypeptides.

26. The method of claim 21, wherein said detectable moiety is selected from the group consisting of biotin, coumarin, dansyl, rhodamines, fluoresceins, dinitrophenyl, and combinations thereof.

27. The method of claim 21, wherein said detectable moiety is magnetic.

28. The method of claim 21, wherein said detectable moiety has a net electric charge.

29. The method of claim 21, wherein said spacer moiety comprises a branched or straight chain hydrocarbon.

30. The method of claim 21, wherein Q is represented by the formula:

##STR12## wherein W and W'"'"' are each selected from the group consisting of --C(O)--, --C(O)--NH--, --HN--C(O)--, --NH--, --O--, --S-- and --CH₂ --, and may be the same or different; and

n1 and n2 are integers from 0-10 which can be the same or different and if either n1 or n2 is zero, then W and W'"'"' are optional.

62. The method of claim 28, wherein the application of an electric field to said detectable moiety induces a response that is detectable by spectrometry.

31. A method, comprising the steps of:

a) conjugating a bioreactive agent to one or more oligonucleotide primers with a covalent bond that is selectively cleavable with electromagnetic radiation, wherein the conjugated bioreactive agent has a chemical structure selected from the group consisting of;

##STR13## wherein SUB comprises the oligonucleotide primer;

R₁ and R₂ are selected from the group consisting of hydrogen, alkyls, substituted alkyls, aryls and substituted aryls, --CF₃, --NO₂, and --COOH, and may be the same or different;

A is a divalent functional group selected from the group consisting of --O--, --S-- and --N--;

Ym₁ wherein Y comprises one or more polyatomic groups which may be the same or different and m₁ is an integer from 1-5;

Vm₂ wherein V comprises one or more monoatoms and m₂ is an integer between 1-5;

Q is a spacer moiety; and

D comprises a detectable moiety which is distinct from R₁ and R₂, wherein D is not --NO₂ ;

b) producing nucleic acid with said conjugated oligonucleotide primers; and

c) treating said produced nucleic acid with electromagnetic radiation.

32. The method of claim 31, wherein said nucleic acid is selected from the group consisting of nucleic acid found in biological samples, bacterial DNA and eukaryotic DNA.

33. The method of claim 31, wherein Y is selected from the group consisting of NO₂, N(R₃)₂ and OR₃, wherein R₃ is selected from the group consisting of hydrogen, alkyls, substituted alkyls, aryls and substituted aryls.

34. The method of claim 31, wherein V is a halide.

35. The method of claim 31, wherein said detectable moiety is an antigen selected from the group consisting of nucleic acids, proteins, and polypeptides.

36. The method of claim 31, wherein said detectable moiety is selected from the group consisting of biotin, coumarin, dansyl, rhodamines, fluoresceins, dinitrophenyl, and combinations thereof.

37. The method of claim 31, wherein said detectable moiety is magnetic.

38. The method of claim 31, wherein said detectable moiety has a net electric charge.

39. The method of claim 31, wherein said spacer moiety comprises a branched or straight chain hydrocarbon.

40. The method of claim 31, wherein Q is represented by the formula:

##STR14## wherein W and W'"'"' are each selected from the group consisting of --C(O)--, --C(O)--NH--, --HN--C(O)--, --NH--, --O--, --S-- and --CH₂ --, and may be the same or different; and

n1 and n2 are integers from 0-10 which can be the same or different and if either n1 or n2 is zero, then W and W'"'"' are optional.

63. The method of claim 38, wherein the application of an electric field to said detectable moiety induces a response that is detectable by spectrometry.

41. A method, comprising the steps of:

a) conjugating a bioreactive agent to one or more oligonucleotide primers with a covalent bond that is selectively cleavable with electromagnetic radiation, wherein the conjugated bioreactive agent has a chemical structure selected from the group consisting of;

##STR15## wherein SUB comprises the oligonucleotide primer;

R₁ and R₂ are selected from the group consisting of hydrogen, alkyls, substituted alkyls, aryls and substituted aryls, --CF₃, --NO₂, and --COOH, and may be the same or different;

A is a divalent functional group selected from the group consisting of --O--, --S-- and --N--;

Ym₁ wherein Y comprises one or more polyatomic groups which may be the same or different and m₁ is an integer from 1-5;

Vm₂ wherein V comprises one or more monoatoms and m₂ is an integer between 1-5;

Q is a spacer moiety; and

may be the same or different; and

D comprises a detectable moiety which is distinct from R₁ and R₂, wherein D is not --NO₂ ;

b) amplifying a nucleic acid sequence with said conjugated oligonucleotide primers; and

c) treating said amplified nucleic acid with electromagnetic radiation.

42. The method of claim 41, wherein said nucleic acid sequence is selected from the group consisting of nucleic acid sequences found in biological samples, bacterial DNA and eukaryotic DNA.

43. The method of claim 41, wherein Y is selected from the group consisting of NO₂, N(R₃)₂ and OR₃, wherein R₃ is selected from the group consisting of hydrogen, alkyls, substituted alkyls, aryls and substituted aryls.

44. The method of claim 41, wherein V is a halide.

45. The method of claim 41, wherein said detectable moiety is an antigen selected from the group consisting of nucleic acids, proteins, and polypeptides.

46. The method of claim 41, wherein said detectable moiety is selected from the group consisting of biotin, coumarin, dansyl, rhodamines, fluoresceins, dinitrophenyl, and combinations thereof.

47. The method of claim 41, wherein said detectable moiety is magnetic.

48. The method of claim 41, wherein said detectable moiety has a net electric charge.

49. The method of claim 41, wherein said spacer moiety comprises a branched or straight chain hydrocarbon.

50. The method of claim 41, wherein Q is represented by the formula:

##STR16## wherein W and W'"'"' are each selected from the group consisting of --C(O)--, --C(O)--NH--, --HN--C(O)--, --NH--, --O--, --S-- and --CH₂ --, and may be the same or different; and

n1 and n2 are integers from 0-10 which can be the same or different and if either n1 or n2 is zero, then W and W'"'"' are optional.

64. The method of claim 48, wherein the application of an electric field to said detectable moiety induces a response that is detectable by spectrometry.

51. A method, comprising the steps of:

a) conjugating an oligonucleotide with a bioreactive agent comprising a chemical structure selected from the group consisting of;

##STR17## wherein X is selected from the group consisting of a halogen, N₂, CH₂ -halogen, --OH, --NHNH₂, --OP(OR₃)N(R₄)R₅, --N═

C═

O, --N═

C═

S, --S--S--R, NC₂ H₄, --NC₄ H₂ O₂ and --OCO--G wherein G is selected from the group consisting of a halogen, N₃, O-esters and N-amides;

R₁, R₂, R₃, R₄ and R₅ are selected from the group consisting of hydrogen, alkyls, substituted alkyls, aryls and substituted aryls, --CF₃, --NO₂, --COOH and --COOR, and may be the same or different;

A is a divalent functional group selected from the group consisting of --O, --S-- and --NR₁ --;

Y comprises one or more polyatomic groups which may be the same or different;

V comprises one or more monoatomic groups which may be the same or different;

Q is represented by the formula;

##STR18## wherein W and W'"'"' are each selected from the group consisting of --C(O)--, --C(O)--NH--, --HN--C(O)--, --NH--, --O--, --S-- and --CH₂ --, and may be the same or different; and

n1 and n2 are integers from 0-10 which can be the same or different and if either n1 or n2 is zero, then W and W'"'"' are optional;

m1 and m2 are integers from 0-5 and may be the same or different; and

D comprises a selectively detectable moiety which is distinct from R₁ -R₅ ;

b) amplifying a nucleic acid sequence with said conjugated oligonucleotide; and

c) treating said amplified sequences with electromagnetic radiation.

52. The method of claim 51, wherein said nucleic acid sequence is selected from the group consisting of nucleic acid sequences found in biological samples, bacterial DNA and eukaryotic DNA.

53. The method of claim 51, wherein Y is selected from the group consisting of NO₂, N(R₃)₂ and OR₃, wherein R₃ is selected from the group consisting of hydrogen, alkyls, substituted alkyls, aryls and substituted aryls.

54. The method of claim 51, wherein V is a halide.

55. The method of claim 51, wherein said detectable moiety is an antigen selected from the group consisting of nucleic acids, proteins, and polypeptides.

56. The method of claim 51, wherein said detectable moiety is selected from the group consisting of biotin, coumarin, dansyl, rhodamines, fluoresceins, dinitrophenyl, and combinations thereof.

57. The method of claim 51, wherein said detectable moiety is magnetic.

58. The method of claim 51, wherein said detectable moiety has a net electric charge.

59. The method of claim 57, wherein the application of an electric field to said detectable moiety induces a response that is detectable by spectrometry.