Carbohydrate-directed cross-linking reagents

• US 6,582,928 B1
• Filed: 03/02/2000
• Issued: 06/24/2003
• Est. Priority Date: 08/05/1992
• Status: Expired due to Fees

• Current Assignee: California Institute of Technology
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Chamow, Steven M., Ashkenazi, Avi J., Kogan, Timothy P.
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US09/517,876
• Time in Patent Office: 1,209 Days
• Field of Search: 548/546, 548/547, 548/548, 548/549, 548/545, 530/351, 530/391.1, 530/391.3, 530/391.5, 530/391.7, 530/391.9, 530/408, 530/409, 530/812, 424/450, 436/503, 436/546, 436/811, 436/829, 435/7.21, 435/960, 435/974
• US Class Current: 435/7.21
• CPC Class Codes: C07D 207/452 with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
• CPC Class Codes: C07K 1/1072 by covalent attachment of residues or functional groups
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: G01N 2474/20 Immunohistochemistry assay
• CPC Class Codes: Y10S 435/974 Aids related test
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation
• CPC Class Codes: Y10S 530/812 Peptides or proteins is immobilized on, or in, an organic carrier

Abstract

The invention provides heterobifunctional cross-linking reagents and methods of using the cross-linking reagents. The cross-linking reagents of the invention combine a nucleophilic hydrazide residue with an electrophilic maleimide residue allowing coupling of aldehydes to free thiols. In the methods of the invention, human immunodeficiency virus (HIV) infected cells can be detected using conjugates that include CD4 molecules conjugated to detectable markers via the disclosed cross-linking reagents.

Claims

1. A conjugate comprising an aldehyde moiety connected to a thiol group via a heterobifunctional compound of formula (I)

wherein (I) is linked to the aldehyde moiety via the hydrazide group and (I) is linked to the thiol group via the maleimide group, wherein X is a hydrocarbon of 6 to about 25 carbon atoms, or a salt thereof.

2. The conjugate of claim 1 wherein said heterobifunctional compound connects a carbohydrate-containing compound via the aldehyde moiety to a compound having a thiol group or a compound to which a thiol group can be attached.

3. The conjugate of claim 2 wherein said carbohydrate-containing compound is a glycophospholipid.

4. The conjugate of claim 2 wherein said carbohydrate-containing compound is a glycoprotein.

5. The conjugate of claim 2 wherein said compound having a thiol group or a group convertible to a thiol group is a toxin.

6. The conjugate of claim 5 wherein said toxin is in a liposome.

7. The conjugate of claim 4 wherein said glycoprotein is a cell surface receptor.

8. The conjugate of claim 7 wherein said cell surface receptor is a cytokine, a hormone or a growth factor receptor.

9. The conjugate of claim 7 wherein said cell surface receptor is member of the immunoglobulin gene superfamily.

10. The conjugate of claim 9 wherein said cell surface receptor is CD4.

11. The conjugate of claim 10 wherein said compound having a thiol group or to which a thiol group can be attached is an anti-human immunodeficiency virus (anti-HIV) agent.

12. The conjugate of claim 11 wherein said anti-HIV agent is in a liposome.

13. The conjugate of claim 10 wherein said compound having a thiol group or to which a thiol group can be attached is a detectable marker.

14. A method for detecting human immunodeficiency virus (HIV) infected cells, comprising:

(a) contacting the cells to be tested with a conjugate of claim 1, the conjugate comprising a CD4 molecule chemically linked to a detectable marker via the heterobifunctional compound of claim 1; and

(b) detecting the signal of said marker.

15. The method of claim 14 comprising additionally isolating said HIV-infected cells.

16. The conjugate of claim 1, wherein X comprises an aliphatic chain and an alicyclic or aromatic moiety.

17. The conjugate of claim 1, wherein X is interrupted by one or more heteroatoms selected from the group consisting of sulfur, oxygen, and nitrogen.