Thiol-selective water-soluble polymer derivatives
View Patent ↗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, said method comprising:
reacting a water-soluble polymer comprising a water-soluble polymer segment (“POLY”) having one terminus activated with an electrophile (“—E”) with a molecule of the structure “NU—Y—S, comprising a nucleophile (“—NU”) and a thiol-selective moiety (“—S”) selected from the group consisting of thiol, protected thiol, symmetrical disulfide, maleimide, and iodoacetamide, and a linear group (“—Y—”) interposed between NU and S, under conditions effective to promote reaction between said electrophile and said nucleophile to form a polymer product comprising a thiol-selective terminus selected from the group consisting of thiol, protected thiol, disulfide, maleimide, and iodoacetamide (“POLY—S”),
wherein the molecular mass of the water-soluble polymer segment is from 1,000 Daltons to 50,000 Daltons.
2. The method of claim 1 , wherein said thiol-selective moiety is a disulfide, and said method further comprises the step of reducing the disulfide bond in said POLY-S to form a polymer having a terminal thiol (POLY-SH).
3. The method of claim 1 , wherein said water-soluble polymer segment is selected from the group consisting of poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), and poly(oxyethylated polyol).
4. The method of claim 1 , wherein the POLY-S product from step (ii) comprises greater than about 95% by weight mono-functionally substituted POLY-S.
5. The method of claim 1 , wherein the POLY-S product from step (ii) comprises less than about 5% di-functionally-substituted POLY-S.
6. The method of claim 3 , wherein the water-soluble polymer segment is a polyalkylene oxide.
7. The method of claim 6 , wherein said-water soluble polymer segment is a polyethylene glycol (PEG).
8. The method of claim 7 , wherein said polyethylene glycol is end-capped.
9. The method of claim 1 , wherein said electrophile is a carboxylic acid or a carboxylic acid derivative.
10. The method of claim 1 , wherein said electrophile is selected from the group consisting of carboxylic acid ester, carbonate ester, carbonic acid, acid halide, and anhydride.
11. The method of claim 1 , further comprising the step of purifying said water-soluble polymer prior to said reacting.
12. The method of claim 9 , further comprising the step of purifying said water-soluble polymer prior to said reacting.
13. The method of claim 11 , wherein said purifying step comprises a chromatographic or a chemical separation.
14. The method of claim 12 , wherein said purifying step comprises ion exchange chromatography.
15. The method of claim 6 , wherein said water-soluble polymer contains less than about 5% polyalkylene oxide dial.
16. The method of claim 1 , wherein said nucleophile is selected from the group consisting of primary amino, secondary amino, hydroxy, imino, thiol, and thioester.
17. The method of claim 16 , wherein said nucleophile is primary or secondary amino.
18. The method of claim 7 , wherein said polymer comprises an end-capping group selected from the group consisting of alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy, aryloxy, and substituted aryloxy.
19. The method of claim 7 , wherein said water-soluble polymer is an N-hydroxysuccinimidyl propionate or an N-hydroxysuccinimidyl butanoate of polyethylene glycol.
20. The method of claim 1 , wherein said molecule is cystamine or cysteamine.
21. The method of claim 1 , wherein said thiol selective moiety is thiol, and said method further comprises the step of reacting said POLY-S thiol with a thiol or protected thiol group of a protein to form a disulfide-linked polymer-protein conjugate (“POLY-S—S-protein”).
22. The method of claim 2 , further comprising the step of reacting said POLY-SH with a thiol or protected thiol group of a protein to form a disulfide-linked polymer-protein conjugate (“POLY-S—S-protein”).
23. The method of claim 7 , wherein said polyethylene glycol has a structure selected from the group consisting of linear, branched, forked, and multi-armed.
24. The method of claim 7 , wherein said polyethylene glycol comprises a hydrolyzable linkage.
25. The method of claim 1 , wherein said molecule is a symmetrical disulfide reagent comprising a nucleophile (-NU), and said reacting step results in formation of a symmetrical water-soluble polymer having a central disulfide bond.
26. A water-soluble polymer having a thiol-selective terminus (POLY-S) produced by a method comprising:
reacting a water-soluble polymer comprising a water-soluble polymer segment (“POLY”) having one terminus activated with an electrophile (“-E”) with a molecule of the structure “NU-Y—S, comprising a nucleophile (”-NU″) and a thiol-selective moiety (“—S”) selected from the group consisting of thiol, protected thiol, disulfide, maleimide, and iodoacetamide, and a linear group (“—Y—”) interposed between NU and S, under conditions effective to promote reaction between said electrophile and said nucleophile to form a polymer product comprising a thiol-selective terminus selected from the group consisting of thiol, protected thiol, disulfide, maleimide, and iodoacetamide (“POLY-S”),
wherein the molecular mass of the water-soluble polymer segment is from 1,000 Daltons to 50,000 Daltons.
27. An activated polymer comprising the structure:
POLY-L-C(O)G-Y—S—W,
wherein POLY is a water-soluble polymer segment,
L is a linker,
G is a heteroatom selected from the group consisting of O, NH, and S,
Y is selected from the group consisting of C 2 -C 10 alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, aryl, and substituted aryl,
S is a sulfur atom, and
W is H or a protecting group,
wherein the molecular mass of the water-soluble polymer segment is from 1,000 Daltons to 50,000 Daltons.
28. The activated polymer of claim 27 , wherein POLY is a polyalkylene glycol.
29. The activated polymer of claim 27 , wherein POLY is a polyethylene glycol, L is —CH 2 —, G is N(H), and Y is —(CH 2 ) 2 —.
30. The activated polymer of claim 27 , wherein L is an aliphatic linker comprising from one to 10 carbon atoms.
31. The activated polymer of claim 27 , wherein L is a linker selected from the group consisting of (CH 2 ) 2, 3, 4 and 5 .
32. The activated polymer of claim 28 , wherein said polyethylene glycol has a structure selected from the group consisting of linear, branched, forked, and multi-armed.
33. The activated polymer of claim 28 , comprising the structure:
W—S—Y-G-(O)C-L-POLY-L-C(O)G-Y—S—W.
34. An activated polymer comprising the structure:
POLY-L-C(O)G-Y-Q,
wherein POLY is a water-soluble polymer segment,
L is a linker,
G is a heteroatom selected from the group consisting of O, NH, and S,
Y is selected from the group consisting of C 2 -C 10 alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, aryl, and substituted aryl, and
Q is selected from the group consisting of thiol, protected thiol, maleimide, and iodoacetamide,
wherein the molecular mass of the water-soluble polymer segment is from 1,000 Daltons to 50,000 Daltons.
35. A composition comprising the activated polymer of any one of claims 27 to 34 .
36. The composition of claim 35 , comprising greater than about 95% by weight monofunctional product.
37. A polymer-conjugate comprising the structure:
POLY-L-C(O)G-Y—S—S-Drug
wherein POLY is a water-soluble polymer segment,
L is a linker,
G is a heteroatom selected from the group consisting of O, NH, and S, and
Y is selected from the group consisting of C 2 -C 10 alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, aryl, and substituted aryl,
wherein the molecular mass of the water-soluble polymer segment is from 1,000 Daltons to 50,000 Daltons.
38. The polymer conjugate of claim 37 , wherein said drug is selected from the group consisting of proteins, peptides, and small molecules.
39. A composition comprising the polymer conjugate of claim 37 .
40. The composition of claim 39 further comprising a pharmaceutical excipient.
41. A method for delivering a drug to a subject in need thereof, said method comprising administering the polymer-conjugate of claim 37 .