Hydrazine-based and carbonyl-based bifunctional crosslinking reagents

• US 6,800,728 B2
• Filed: 03/22/2001
• Issued: 10/05/2004
• Est. Priority Date: 03/22/2000
• Status: Expired due to Term

• Current Assignee: Vector Laboratories
• Original Assignee: Solulink Biosciences Incorporated
• Inventors: Schwartz, David A.
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US09/815,978
• Publication Number:
  US 20030013857A1
• Time in Patent Office: 1,293 Days
• Field of Search: 424/179.1, 424/180.1, 424/181.1, 435/174, 435/175, 435/176, 435/177, 435/178, 435/179, 435/180, 435/181, 435/182, 514/2, 514/8, 514/12, 514/21, 514/44, 530/375, 530/391.1, 530/391.3, 530/391.5, 530/391.7, 530/391.9, 530/404, 530/405, 530/408, 530/409, 530/810, 530/811, 530/812, 530/813, 530/814, 530/815, 530/816, 536/23.1, 548/542, 564/17, 564/18, 564/34, 564/37
• US Class Current: 530/345
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: A61K 47/643 Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• CPC Class Codes: A61K 47/6801 Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
• CPC Class Codes: C07D 207/452 with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 213/82 in position 3
• CPC Class Codes: C07D 401/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07F 7/1804 Compounds having Si-O-C linkages Si-O-acyl linkages C07F7/1896
• 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: C08G 65/329 with organic compounds
• CPC Class Codes: C08G 65/336 containing silicon
• CPC Class Codes: C08L 2203/02 for biomedical use
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: Y10S 530/81 Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier

Abstract

Reagents and methods are provided for crosslinking and immobilizing biomolecules, drugs and synthetic polymers. The reagents possess (i) a thiol or amino reactive group; and (ii) a hydrazino or oxyamino moiety. Conjugates and immobilized biomolecules are also provided.

Claims

1. A compound of formula 1

2. The compound according to claim 1, wherein X is a halide or trifluoroacetate.

3. The compound according to claim 1, wherein B is an amino reactive moiety which is a succinimidyl ester, a hydroxybenzotriazolyl ester or a pentafluorophenol ester.

4. A conjugate comprising the compound according to claim 1, bound to a biological molecule.

5. The conjugate according to claim 4, wherein the biological molecule is a protein, a glycoprotein, or a peptide.

6. The conjugate according to claim 4, wherein the biological molecule is a polynucleotide, an oligonucleotide, an RNA or a DNA.

7. The conjugate according to claim 5, wherein the protein is an antibody.

8. A method of immobilizing a biological molecule, comprising:

(a) preparing the conjugate according to claim 4; and

(b) applying the conjugate to a surface wherein the surface has at least one carbonyl moiety for a time and under conditions such that the hydrazine moiety of the conjugate reacts with the at least one carbonyl moiety of the surface forming a hydrazone bond to the surface.

9. A method of immobilizing a biological molecule, comprising:

(a) applying the compound according to claim 1 to a surface comprising at least one amine moiety; and

(b) applying a biological molecule having at least one carbonyl moiety for a time and under conditions such that the hydrazine moiety of the surface reacts with the at least one carbonyl moiety of the biological molecule forming a hydrazone bond to the surface.

10. A method of crosslinking a first biological molecule to a second biological molecule, comprising:

(a) preparing the conjugate of the first biological molecule according to claim 4; and

(b) mixing the conjugate with the second biological molecule wherein the second biological molecule has at least one carbonyl moiety for a time and under conditions such that the hydrazine moiety of the conjugate reacts with the at least one carbonyl moiety of the second biological molecule forming a hydrazone bond crosslinking the first biological molecule to the second biological molecule.

11. The method according to claim 10 wherein the first biological molecule is a protein, a glycoprotein, or a peptide.

12. The method according to claim 10, wherein the first biological molecule is a polynucleotide, an oligonucleotide, an RNA or a DNA.

13. The method according to claim 11, wherein the protein is an antibody.

14. The compound according to claim 1 of the formula: