Bioreactive agents

• US 8,288,098 B2
• Filed: 10/27/2011
• Issued: 10/16/2012
• Est. Priority Date: 05/11/1994
• Status: Expired due to Fees

• Current Assignee: Ambergen Inc., Trustees of Boston University
• Original Assignee: Ambergen Inc.
• Inventors: Rothschild, Kenneth J., Sonar, Sanjay M., Olejnik, Jerzy
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/283,209
• Publication Number:
  US 20120088241A1
• Time in Patent Office: 355 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/26.6
• US Class Current: 435/6.1
• 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: 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 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

This invention relates to agents and conjugates 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.

Claims

1. A method, comprising, a) providing a conjugate comprising a bioreactive agent bound to a substrate, wherein said substrate comprises a nucleotide, said bioreactive agent comprises a cleavable moiety and a detectable moiety, wherein said cleavable moiety is positioned between said detectable moiety and said substrate;

b) contacting said conjugate with a mixture to create a coupled conjugate;

c) removing uncoupled conjugates; and

d) treating said coupled conjugate so as to release said detectable moiety.

2. The method of claim 1, wherein said mixture is a biological sample.

3. The method of claim 1, wherein said mixture is a proteinaceous mixture.

4. The method of claim 1, wherein said mixture comprises nucleic acid strands.

5. The method of claim 4, wherein said coupled conjugate is coupled to at least one nucleic acid strand.

6. The method of claim 5, wherein said coupled conjugate is created by incorporating said conjugate into said at least one nucleic acid strand by enzymatic techniques.

7. The method of claim 1, wherein said cleavable moiety is a photocleavable moiety.

8. The method of claim 7, wherein said photocleavable moiety is a photocleavable biotin.

9. The method of claim 8, wherein said treating of step c) comprises treating with electromagnetic radiation.

10. The method of claim 1, wherein said bioreactive agent is bound to said substrate via a moiety selected from the group consisting of amines, hydroxyls, imidazoles, aldehydes, carboxylic acids, esters and thiols.

11. The method of claim 1, wherein said detectable moiety is fluorescent.

12. The method of claim 1, wherein said bioreactive agent is attached to the nucleotide through the base moiety.

13. The method of claim 1, wherein said nucleotide is a derivative of dATP, dCTP, dTTP or dGTP.

14. The method of claim 1, wherein said nucleotide is a derivative of ATP, CTP, UTP or GTP.