Thiol-selective water-soluble polymer derivatives
The present invention provides water-soluble, polymer derivatives having a thiol-selective terminus suitable for selective coupling to thiol groups, such as those contained in the cysteine residues of proteins.
1. A method for preparing a thiol-selective derivative of a water-soluble polymer comprising the steps of:
(i) providing an electrophilically-activated polymer, POLY-L 0,1 -E, wherein:
POLY is a water-soluble polymer segment,
L is an optional linker,
E is an electrophile, and
(ii) reacting the POLY-L 0,1 -E with a symmetrical disulfide reagent, (NU-Y-S-) 2 , wherein:
NU is a nucleophile, and
Y is alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, aryl, or substituted aryl, comprising from about 2 to about 10 carbon atoms,
under conditions effective to promote reaction between E and NU to thereby form POLY-L 0,1 -X-Y-S-S-Y-X-L 0,1 -POLY, wherein X is a group resulting from the reaction between E and NU.
2. The method of claim 1 , further comprising the step of (iii) reducing the disulfide bond in POLY-L 0,1 -X-Y-S-S-Y-X-L 0,1 -POLY to form POLY-L 0,1 -X-Y-SH.
3. The method of claim 1 , wherein L 1 is C 1 -C 10 alkylene or C 1 -C 10 substituted alkylene.
4. The method of claim 3 , wherein L 1 is—(CH 2 ) 1-5 —.
5. The method of claim 1 , wherein E is a carboxylic acid or an activated carboxylic acid derivative.
6. The method of claim 1 , wherein E is a carboxylic acid, activated carboxylic acid ester, acid halide, or active anhydride.
7. The method of claim 6 , wherein E is an N-hydroxysuccinimidyl ester.
8. The method of claim 1 , wherein NU is amino, hydroxy, imino, or thiol.
9. The method of claim 1 , wherein NU is —NH 2 .
10. The method of claim 1 , wherein X is an amide, carbamate, carbonate ester, ether, or thioester.
11. The method of claim 1 , wherein POLY is a polyalkylene oxide.
12. The method of claim 11 , wherein POLY is a polyethylene glycol having the structure H 3 CO—(CH 2 CH 2 O) n CH 2 CH 2 — wherein n ranges from 10 to about 4,000.
13. The method of claim 11 , wherein POLY is end-capped.
14. The method of claim 12 , wherein L is absent or —CH 2 —, and E is N-hydroxysuccinimidyl ester or 1-hydroxybenzotriazolyl carbonate.
15. The method of claim 14 , wherein the symmetrical disulfide reagent is cystamine, where NU is primary amino and Y is —(CH 2 ) 2 —.
16. A method for preparing a polymer-protein conjugate comprising the steps of:
(i) providing an electrophilically-activated polymer, POLY-L 0,1 -E, wherein:
POLY is a water-soluble polymer segment,
L is an optional linker, and
E is an electrophile,
(ii) reacting the POLY-L 0,1 -E with a symmetrical disulfide reagent, (NU-Y-S-) 2 , wherein:
NU is a nucleophile, and
Y is selected from the group consisting of C 2 -C 10 alkylene, C 2 -C 10 substituted alkylene, arylene, and substituted arylene,
under conditions effective to promote reaction between E and NU to thereby form POLY-L 0,1 -X-Y-S-S-Y-X-L 0,1 -POLY wherein X is a group resulting from the reaction between E and NU,
(iii) reducing the disulfide bond in POLY-L 0,1 -X-Y-S-S-Y-X-L 0,1 -POLY to form POLY-L 0,1 -X-Y-SH, and
(iv) reacting POLY-L 0,1 -X-Y-SH with a thiol or protected thiol group of a protein to form a protein conjugate, POLY-L 0,1 -X-Y-S-S-protein.
17. The method of claim 16 , wherein the protein is a therapeutic protein.