US2004132965A1PendingUtilityA1
Chemical reagents for formation of disulfide bonds in peptides
Est. expiryMay 26, 2020(expired)· nominal 20-yr term from priority
C40B 40/10C07K 1/1133B01J 2219/00277B01J 2219/00725C07K 1/006C07K 1/107
45
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Claims
Abstract
This invention pertains to the discovery of a class of reagents that effectively form intramolecular disulfide bonds in peptides. Intermolecular disulfide linkage formation is low or essentially non-existent. In addition, preferred reagents of this invention are relatively mild and do not oxidize “vulnerable” residues in the subject peptide(s). In addition the reagents and reaction products are safe and essentially non-toxic. One particularly preferred reagent is [Pt(en) 2 Cl 2 ] 2+ where en is ethylenediamine.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A method of forming an intramolecular disulfide bond in a peptide, said method comprising:
contacting a peptide comprising at least two sulfhydryl (SH) groups with a compound having the formula: wherein R 1 and R 2 are independently selected from the group consisting of Cl—, Br—, I—, OH—, and RCO 2 — where R is an alkyl or substituted alkyl; Pt is Pt(IV); X 1 , X 2 , X 3 , and X 4 are ligands independently selected from the group consisting of a nitrogen ligand, an oxygen ligand, a phosphorous ligand, and a sulfur ligand; i, k, m, and p are independently 0 or 1; L 1 , L 2 , L 3 , and L 4 when present, are independently selected linkers.
2 . The method of claim 1 , wherein said ligand is a nitrogen ligand selected from the group consisting of cyanide, ammonia, and an amine nitrogen.
3 . The method of claim 1 , wherein X 1 , X 2 , X 3 , and X 4 are the same.
4 . The method of claim 1 , wherein R 1 and R 2 are the same.
5 . The method of claim 1 , wherein L 1 and L 2 are the same.
6 . The method of claim 1 , wherein
i, k, m, and p are zero; X 1 , X 2 , X 3 , and X 4 are cyanide or ammonia.
7 . The method of claim 6 , wherein R 1 and R 2 are the same.
8 . The method of claim 1 , wherein
i and k are 1 m and p are 0; and L 1 i and L 2 k are linkers selected from the group consisting of alkyl linkers, substituted alkyl linkers, and aromatic linkers.
9 . The method of claim 8 , wherein L 1 i and L 2 k are alkyl linkers comprising from 1 to 4 carbons.
10 . The method of claim 9 , wherein R 1 and R 2 are independently selected from the group consisting of Cl—, Br—, and I—.
11 . The method of claim 9 , wherein -X 1 -L 1 i-X 4 - and -X 2 -L 2 k -X 3 - are both:
—NH 2 CH 2 CH 2 NH 2 —.
12 . The The method of claim 1 , wherein
i, k, m, and p are 1; and X 1 -L 3 -X 2 -L 2 -X 3 -L 4 -X 4 -L 1 is H 2 NCH 2 CH 2 NHCH 2 CH 2 NHCH 2 CH 2 NH 2 .
13 . The method of claim 1 , wherein said peptide ranges in length from about 2 amino acids to about 60 amino acids.
14 . The method of claim 1 , wherein two amino acids that form a disulfide bond are adjacent amino acids.
15 . The method of claim 1 , wherein two amino acids that form a disulfide bond are separated by no more than about 58 amino acids.
16 . The method of claim 11 , wherein said peptide contains a methionine.
17 . The method of claim 1 , wherein said contacting is at a pH ranging from about pH 0 to about pH 9.
18 . The method of claim 1 , wherein said peptide is a chemically synthesized peptide.
19 . The method of claim 1 , wherein said peptide is a chemically synthesized peptide bearing a protecting group.
20 . The method of claim 19 , wherein said protecting group is an Fmoc or a tBoc.
21 . The method of claim 1 , wherein said peptide is attached to a substrate.
22 . The method of claim 1 , wherein X 1 , X 2 , X 3 , and X 4 are not cyanide.
23 . A solution comprising:
a peptide comprising at least two sulfhydryl groups; and a compound having the formula: wherein R 1 and R 2 are independently selected from the group consisting of Cl—, Br—, I—, OH—, and RCO 2 — where R is an alkyl or substituted alkyl; Pt is Pt(IV); X 1 , X 2 , X 3 , and X 4 are ligands independently selected from the group consisting of a nitrogen ligand, an oxygen ligand, a phosphorous ligand, and a sulfur ligand; i, k, m, and p are independently 0 or 1; L 1 , L 2 , L 3 , and L when present, are independently selected linkers.
24 . The solution of claim 23 , wherein said ligand is a nitrogen ligand selected from the group consisting of cyanide, ammonia, and an amine nitrogen.
25 . The solution of claim 23 , wherein X 1 , X 2 , X 3 , and X 4 are the same.
26 . The solution of claim 23 , wherein R 1 and R 2 are the same.
27 . The solution of claim 23 , wherein L 1 and L 2 are the same.
28 . The solution of claim 23 , wherein
i, k, m, and p are zero; X 1 , X 2 , X 3 , and X 4 are cyanide or ammonia.
29 . The solution of claim 28 , wherein R 1 and R 2 are the same.
30 . The solution of claim 23 , wherein
i and k are 1; m and p are zero; and L 1 and L 2 are linkers selected from the group consisting of alkyl linkers, substituted alkyl linkers, and aromatic linkers.
31 . The method of claim 30 , wherein L 1 i and L 2 k are alkyl linkers comprising from 1 to 4 carbons.
32 . The method of claim 31 , wherein R 1 and R 2 are independently selected from the group consisting of Cl—, Br—, and I—.
33 . The method of claim 31 , wherein -X 1 -L 1 i-X 1 - and -X 2 -L 2 k -X 3 - are both:
—NH 2 CH 2 CH 2 NH 2 —.
34 . The The method of claim 1 , wherein
i, k, m, and p are 1; and X 1 -L 3 -X 2 -L 2 -X 3 -L 4 -X 4 -L 1 is H 2 NCH 2 CH 2 NHCH 2 CH 2 NHCH 2 CH 2 NH 2 .
35 . The solution of claim 30 , wherein L 1 and L 2 are alkyl linkers comprising from 1 to 4 carbons.
36 . The solution of claim 35 , wherein R 1 and R 2 are the same and are selected from the group consisting of Cl—, Br—, and I—.
37 . The solution of claim 35 , wherein -X 1 -L 1 -X 4 - and -X 2 -L 2 -X 3 - are both:
—NH 2 CH 2 CH 2 NH 2 —.
38 . The solution of claim 23 , wherein said peptide ranges in length from about 2 amino acids to about 100 amino acids.
39 . The solution of claim 23 , wherein two amino acids that form a disulfide bond are adjacent amino acids.
40 . The solution of claim 23 , wherein two amino acids that form a disulfide bond are separated by no more than 60 amino acids.
41 . The solution of claim 23 , wherein said peptide contains a methionine.
42 . The solution of claim 23 , wherein said solution has a pH ranging from about pH 1 to about pH 9.
43 . The solution of claim 23 , wherein said peptide is a chemically synthesized peptide.
44 . The solution of claim 23 , wherein said peptide is a chemically synthesized peptide bearing a protecting group.
45 . The solution of claim 23 , wherein said protecting group is an Fmoc or a tBoc.
46 . The solution of claim 23 , wherein said peptide is attached to a substrate.
47 . A peptide synthesizer for the synthesis of a peptide having an intramolecular disulfide linkage, said peptide synthesizer comprising:
a plurality of vials, said vials containing amino acids derivatized for chemical peptide synthesis wherein at least one of said vials comprises an amino acid that, when fully deprotected, bears a sulfhydryl (SH) group; and a vial comprising a reagent having the formula: herein R 1 and R 2 are independently selected from the group consisting of Cl—, Br—, I—, OH—, and RCO 2 — where R is an alkyl or substituted alkyl; Pt is Pt(IV); X 1 , X 2 , X 3 , and X 4 are independently selected from the group consisting of a nitrogen ligand, an oxygen ligand, a phosphorous ligand, and a sulfur ligand; i, k, m, and p are independently 0 or 1; L 1 , L 2 , L 3 , and L 4 when present, are independently selected linkers.
48 . The peptide synthesizer of claim 47 , wherein said nitrogen ligand is selected from the group consisting of cyanide, ammonia, and an amine nitrogen.
49 . A method of chemically synthesizing a peptide comprising a disulfide linkage, said method comprising:
chemically coupling a plurality of amino acids to form a peptide comprising at least two sulfhydryl (SH) groups; and contacting said peptide with a reagent having the formula: wherein R 1 and R 2 are independently selected from the group consisting of Cl—, Br—, I—, OH—, and RCO 2 — where R is an alkyl or substituted alkyl; Pt is Pt(IV); X 1 , X 2 , X 3 , and X 4 are ligands independently selected from the group consisting of a nitrogen ligand, an oxygen ligand, a phosphorous ligand, and a sulfur ligand; i, k, m, and p are independently 0 or 1; L 1 , L 2 , L 3 , and L 4 when present, are independently selected linkers.
50 . The method synthesizer of claim 49 , wherein said ligand is a nitrogen ligand selected from the group consisting of cyanide, ammonia, and an amine nitrogen.
51 . The method of claim 49 , wherein said contacting is in a buffer having a pH ranging from about pH 1 to about pH 9.
52 . A kit for forming an intramolecular disulfide bond in a peptide, said kit comprising:
a container containing a reagent having the formula: wherein R 1 and R 2 are independently selected from the group consisting of Cl—, Br—, I—, OH—, and RCO 2 — where R is an alkyl or substituted alkyl; Pt is Pt(IV); X 1 , X 2 , X 3 , and X 4 are ligands independently selected from the group consisting of a nitrogen ligand, an oxygen ligand, a phosphorous ligand, and a sulfur ligand; i, k, m, and p are independently 0 or 1; L 1 , L 2 , L 3 , and L 4 when present, are independently selected linkers.
53 . The kit of claim 51 , wherein said ligand is a nitrogen ligand selected from the group consisting of cyanide, ammonia, and an amine nitrogen.
54 . The kit of claim 51 , wherein said reagent is in a buffer at a pH ranging from about pH 1 to about pH 9.Join the waitlist — get patent alerts
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