Photocleavable agents and conjugates for the detection and isolation of biomolecules

• US 5,986,076 A
• Filed: 11/22/1994
• Issued: 11/16/1999
• Est. Priority Date: 05/11/1994
• Status: Expired due to Term

• Current Assignee: Trustees of 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:
  US08/345,807
• Time in Patent Office: 1,820 Days
• Field of Search: 435/6, 536/22.1, 536/23.1, 536/25.32, 424/130.1, 424/184.1, 514/2, 514/44, 530/300, 530/350
• US Class Current: 536/22.1
• 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 conjugate comprising a chemical structure selected from the group consisting of:

##STR7## wherein SUB comprises a substrate;

R₁ and R₂ are selected from the group consisting of hydrogen, alkyls, substituted alkyls, aryls, 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 --;

Ym₁ wherein Y comprises one or more polyatomic groups selected from the group consisting of nitro groups, sulfoxide groups, alkyl groups, alkoxyl groups, wherein said polyatomic groups may be the same or different and m₁ is an integer between 1-5;

Vm₂ wherein V comprises one or more monoatomic groups selected from the group consisting of chloride, fluoride, iodide, bromide, and hydrogen wherein said monoatomic groups may be the same or different and m₂ is an integer between 1-5;

Q comprises a spacer moiety; and

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

2. The conjugate of claim 1 wherein Q comprises a branched or unbranched hydrocarbon spacer moiety.

3. The conjugate of claim 1 wherein Q is represented by the formula:

##STR8## wherein W and W'"'"' are each selected from the group consisting of --CO--, --CO--NH--, --HN--CO--, --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.

4. The conjugate of claim 1 wherein the detectable moiety is selected from the group consisting of biotin, coumarin, dansyl, rhodamines, fluoresceins, dinitrophenyl.

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

6. The conjugate of claim 1 which has the formula:

##STR9##

7. The conjugate of claim 1 which has the formula:

8. The conjugate of claim 1 which has the formula:

9. The conjugate of claim 1 which has the formula:

10. The conjugate of claim 1 which has the formula:

11. The conjugate of claim 1 which has the formula:

12. The conjugate of claim 1 which has the formula:

13. The conjugate of claim 1 wherein the substrate is a pharmaceutical composition.

14. The pharmaceutical composition of claim 13 and a pharmaceutically acceptable carrier selected from the group consisting of water, oils, lipids, saccharides, polysaccharides, glycerols, collagens and combinations thereof.

15. A bioreactive agent for forming a detectable photocleavable conjugate comprising the chemical structure:

wherein X comprises --OP(OR₃)N(R₄)R₅ or --A--(C═

O)--G;

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 --;

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 comprises a spacer moiety; and

D comprises a detectable moiety which is distinct form R₁ -R₅, wherein D is not --NO₂.

16. The agent of claim 15 wherein X further comprises a nucleotide selected from the group consisting of NTP, dNTP;

and ddNTP.

17. The agent claim of 15 wherein X further comprises an oligonucleotide.

18. The agent claim of 17 wherein the detectable moiety is an antigen selected from the group consisting of nucleic acids, proteins, and polypeptides.

19. A conjugate comprising the chemical structure selected from the group consisting of:

##STR10## wherein SUB comprises a substrate;

R₁ and R₂ 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 --;

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 form R₁ -R₅, wherein D is not --NO₂.

20. The conjugate of claim 19 wherein the detectable moiety is magnetic.

21. The conjugate of claim 19 wherein the detectable moiety has a net electric charge.

22. The conjugate of claim 19 wherein the application of an electric field induces a response that is detectable by spectrometry.

23. The conjugate of claim 19 wherein the substrate is an NTP-phosphoramidite or a dNTP-phosphoramidite.

24. A bioreactive agent for forming a detectable photocleavable conjugate comprising a chemical structure selected from the group consisting of:

##STR11## wherein G is the adduct product of forming an ester linkage to one of the moiety selected from the group consisting of N-hydroxyphthalimidyl, N-hydroxysuccinimidyl, 1-hydroxypiperidinyl, 5-chloro-8-hydroxy-quinolyl, 1-hydroxybenzotriazolyl, 2-hydroxypyridyl;

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

Ym₁ wherein Y comprises one or more polyatomic groups selected from the group consisting of nitro groups, sulfoxide groups, alkyl groups, alkoxyl groups wherein said polyatomic groups may be the same or different and m₁ is an integer between 1-5;

Vm₂ wherein V comprises one or more monoatomic groups selected from the group consisting of chloride, fluoride, iodide, bromide, and hydrogen wherein said monoatomic groups may be the same of different and m₂ is an integer between 1-5;

Q comprises a spacer moiety; and

D comprises a selectively detectable moiety.

25. The agent of claim 24 wherein Q comprises a branched or unbranched hydrocarbon spacer moiety.

26. The agent of claim 24 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.

27. The agent of claim 24 wherein the monoatomic group is a halide.

28. The agent of claim 24 wherein the detectable moiety is selected from the group consisting of moieties of biotin, coumarin, dansyl, rhodamines, fluoresceins, dinitrophenyl.

29. The agent of claim 24 wherein the detectable moiety is selected from the group consisting of antibodies, antibody fragments and antigens.

30. A bioreactive agent for forming a detectable photocleavable conjugate comprising a chemical structure selected from the group consisting of:

##STR13## wherein G is the adduct product of forming an amide linkage to one of the moiety selected from the group consisting of imidazolyl, benzimidazolyl, benzisooxasolyl, 3,5-dioxo-4-methyl-1, 2,4-oxadiazolidinyl;

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

Ym₁ wherein Y comprises one or more polyatomic groups selected from the group consisting of nitro groups, sulfoxide groups, alkyl groups, alkoxyl groups wherein said polyatomic groups may be the same or different and m₁ is an integer between 1-5;

Vm₂ wherein V comprises one or more monoatomic groups selected from the group consisting of chloride, fluoride, iodide, bromide, and hydrogen wherein said monoatomic groups may be the same of different and m₂ is an integer between 1-5;

Q comprises a spacer moiety; and

D comprises a selectively detectable moiety.

31. The agent of claim 30 wherein Q comprises a branched or unbranched hydrocarbon spacer moiety.

32. The agent of claim 30 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.

33. The agent of claim 30 wherein the monoatomic group is a halide.

34. The agent of claim 30 wherein the detectable moiety is selected from the group consisting of moieties of biotin, coumarin, dansyl, rhodamines, fluoresceins, dinitrophenyl.

35. The agent of claim 30 wherein the detectable moiety is selected from the group consisting of antibodies, antibody fragments and antigens.

36. A bioreactive agent for forming a detectable photocleavable conjugate comprising a chemical structure selected from the group consisting of:

##STR15## wherein X is selected from the group consisting of a halogen, N₂, CH₂ -halogen, --OH, --NHNH₂, --OP(OR₃)N(R₄)R₅, --N═

C═

, --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 --;

Ym₁ wherein Y comprises one or more polyatomic groups selected from the group consisting of nitro groups, sulfoxide groups, alkyl groups, alkoxyl groups wherein said polyatomic groups may be the same or different and m₁ is an integer between 1-5;

Vm₂ wherein V comprises one or more monoatomic groups selected from the group consisting of chloride, fluoride, iodide, bromide, and hydrogen wherein said monoatomic groups may be the same of different and m₂ is an integer between 1-5;

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; and

D comprises a selectively detectable moiety which is distinct from R₁ -R₅, wherein D is not --NO₂.

37. The agent of claim 36 wherein the monoatomic group is a halide.

38. The agent of claim 36 wherein the detectable moiety is selected from the group consisting of moieties of biotin, coumarin, dansyl, rhodamines, fluoresceins, dinitrophenyl.

39. The agent of claim 36 wherein the detectable moiety is selected from the group consisting of antibodies, antibody fragments and antigens.

40. A bioreactive agent for forming a detectable photocleavable conjugate comprising a chemical structure which has the formula:

##STR17## wherein R₁ and R₂ 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;

Q comprises a spacer moiety; and

D comprises a selectively detectable moiety which is distinct from R₁ and R₂.

41. The agent of claim 40 wherein said Q comprises a branched or unbranched hydrocarbon spacer moiety.

42. The agent of claim 40 wherein said 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.

43. A bioreactive agent for forming a detectable photocleavable conjugate comprising the chemical structure which has the formula:

##STR19## wherein R₁ and R₂ 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;

Q comprises a spacer moiety; and

D comprises a selectively detectable moiety which is distinct from R₁ and R₂.

44. The agent of claim 43 wherein said Q comprises a branched or unbranched hydrocarbon spacer moiety.

45. The agent of claim 43 wherein said Q is represented by the formula:

##STR20## 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.

46. A bioreactive agent for forming a detectable photocleavable conjugate comprising the chemical structure which has the formula:

##STR21## wherein R₁ and R₂ 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;

Q comprises a spacer moiety;

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

D comprises a selectively detectable moiety which is distinct from R₁ and R₂.

47. The agent of claim 46 wherein said Q comprises a branched or unbranched hydrocarbon spacer moiety.

48. The agent of claim 46 wherein said Q is represented by the formula:

##STR22## 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.

49. The agent of claim 46, wherein Y is selected from the group consisting of N(R₃)₂ and OR₃, wherein R₃ is selected from the group consisting of hydrogen, alkyls, substituted alkyls, aryls and substituted aryls.

50. The agent of claim 46, wherein Y is selected from the group consisting of:

--CH═

CH--Ar and --N═

N--Ar.