N-ARYL-CARBAMIC ACID ESTER-DERIVED AND VALERIC ACID ESTER-DERIVED CROSS-LINKERS AND CONJUGATES, AND METHODS FOR THEIR SYNTHESIS AND USE

• US 20050181416A1
• Filed: 03/14/2005
• Published: 08/18/2005
• Est. Priority Date: 02/12/2004
• Status: Active Grant

• Current Assignee: Biosite Diagnostics Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Biosite Diagnostics Incorporated (Abbott Laboratories Incorporated)
• Inventors: Buechler, Kenneth F., Banaszczyk, Mariusz G., Noar, Joseph Barry
• Granted Patent: US 6,967,107 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom

Abstract

The present invention describes carbamic acid ester-derived and valeric acid ester-derived polyfunctional cross-linker molecules, and methods for their synthesis and use. The inclusion of polymeric moieties such as poly(alkylene oxide) in the cross-linkers of the present invention can provide advantageous solubility properties in aqueous environments. Such cross-linkers may be used to form conjugates for use in a variety of assay formats.

Claims

1. A method of conjugating a first species selected from the group consisting of a protein, a polypeptide, an antibody, a nucleic acid, a small molecule, an aptamer, a carbohydrate, and a peptidomimetic to a second species selected from the group consisting of a detectable label, a solid phase, a protein, a polypeptide, an antibody, a nucleic acid, a small molecule, an aptamer, a carbohydrate, and a peptidomimetic, the method comprising:

contacting said first and second species with a crosslinker of the formula;

wherein X is (alkylene oxide)_n;

n is between about 40 to about 450; and

Y and Z are independently arylene or heteroarylene units having 5 or 6 ring atoms, optionally substituted with from 1 to 4 substituents independently selected from the group consisting of C_1-6 alkyl straight or branched chain, halogen, trihalomethyl, C_1-6 alkoxy, —

NO₂, —

NH₂, —

OH, —

COOR′

, where R′

is H or lower alkyl, —

CH₂OH, —

CONH₂, and a linkage to a poly(alkylene oxide) moiety, wherein z is optionally present;

under conditions selected to conjugate said first species to said second species, wherein one of the first or second species becomes covalently bound to said crosslinker through a moiety reactive with an n-hydroxysuccinimide functional group, and the other of the first or second species becomes covalently bound to said crosslinker through a moiety reactive with a maleimide functional group.

2. A method according to claim 1, wherein the crosslinker has the formula:

3. A method according to claim 1, wherein the (alkylene oxide)_n group is a copolymer of two or more units selected from the group consisting of methylene oxide, ethylene oxide, propylene oxide, isopropylene oxide, and butylene oxide.

4. A method according to claim 1, wherein the (alkylene oxide)_n group is a homopolymer of units selected from the group consisting of methylene oxide, ethylene oxide, propylene oxide, isopropylene oxide, and butylene oxide.

5. A method according to claim 1, wherein the crosslinker has the formula:

6. A method according to claim 5, wherein n is between 60 and 100.

7. A method according to claim 5, wherein n is between 70 and 90.

8. A method according to claim 5, wherein n averages about 77.

9. A method according to claim 1, wherein the conjugating step comprises (i) covalent binding of the crosslinker to the first or second species through a moiety reactive with an N-hydroxysuccinimide functional group to form a covalently bound crosslinker intermediate;

(ii) removal of unreacted crosslinker from the covalently bound crosslinker intermediate; and

(iii) covalent binding of the crosslinker to the other of the first or second species through a moiety reactive with a maleimide functional group.

10. A method according to claim 1, wherein the first or second species is an antibody.

11. A method according to claim 1, wherein the second species is a latex particle.

12. A method according to claim 1, wherein the second species is a solid phase modified to provide linkage sites for the crosslinker.

13. A method according to claim 12, wherein the second species is a latex particle, and the linkage sites are provided by an intermediary protein.

14. A method according to claim 1, wherein the first species is an antibody, and the second species is a solid phase.

15. A method according to claim 14, wherein the second species is a latex particle.

16. A method according to claim 15, wherein the latex particle is covalently bound to the crosslinker through a moiety reactive with an N-hydroxysuccinimide functional group provided on an intermediary protein, and wherein the antibody is covalently bound through a moiety reactive with a maleimide functional group provided by cystenylation of the antibody.