Methods for site-specific pegylation

A chemical and free aldehyde technology, applied in the preparation method of peptides, chemical instruments and methods, specific peptides, etc., can solve the problems of non-site selectivity and achieve high site selectivity, easy characterization, and small content differences.

Inactive Publication Date: 2009-07-29
益普生制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this method, the thiolactone used reacts with the N-terminus of the protein and with any amino functional group in the lysine residue, and this method is not site-selective (October 30, 2001 Granted U.S. Patent No. 6,310,180)

Method used

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  • Methods for site-specific pegylation
  • Methods for site-specific pegylation
  • Methods for site-specific pegylation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Embodiment 1) preparation H-NMeCys-Lys-Phe-NH 2

[0146]

[0147] Rink amide MBHA resin (211 mg, 0.152 mmol) (Novabiochem, San Diego, Calif.) was swollen in dichloromethane (DCM) and washed with dimethylformamide (DMF). The resin was debolcked by treatment with 25% piperidine / DMF (10 mL) solution 2 x 10 min. The resin was washed three times with DMF (10 mL). By using Fmoc-Phe-OH (Novabiochem, San Diego, Calif.) (235 mg, 0.606 mmol), 1-hydroxybenzotriazole (HOBt) (92.3 mg, 0.606 mmol) and diisopropylcarbodiene A solution of amine (DIC) (77 mg, 0.606 mmol) in N-methylpyrrolidone (NMP) (2 mL) was treated for 1 hour to couple the first amino acid to the resin. The resin was filtered and washed three times with DMF (10 mL).

[0148] The Fmoc protecting group was removed by treatment with 25% piperidine / DMF (10 mL) solution for 2 x 10 min, and the resin was washed three times with DMF (10 mL). Fmoc-Lys(Boc)-OH (Novabiochem, San Diego, Calif.) (285 mg, 0.606 mol) c...

Embodiment 2

[0152] Embodiment 2) prepare mPEG-Tmc-Lys-Phe-NH 2

[0153] mPEG has CH here 3 O(CH 2 CH 2 O) n -(CH 2 ) 2 -The structure of which n is a positive integer.

[0154]

[0155]The peptide product of Example 1 (0.5 mg, 1.22 micromol) was dissolved in 1.0 mL of pH 4 buffer (20 mmol NaOAc, 150 mmol NaCl and 1 mmol EDTA). mPEG-aldehyde (1.5 equivalents, average molecular weight 31378 Daltons, NOF Corp., Tokyo, Japan) was added to the resulting solution. According to the reverse phase analytical HPLC system (Vydac C 18 5 μ peptide / protein column, 4.6 x 250 mm), the reaction was approximately 90% complete after 27 hours at room temperature. Load the reaction mixture onto 5 mL Zeba TM Desalting spin columns (Pierce Biotechnology, Rockford, IL). A white foam (36.7 mg) was obtained after lyophilization.

Embodiment 3

[0156] Embodiment 3) preparation H-NMeCys (Prd-PEG)-Lys-Phe-NH 2

[0157]

[0158] The peptide product of Example 1 (0.5 mg, 1.22 micromol) was dissolved in 1.0 mL of pH 7 buffer (20 mmol NaOAc). α-(3-(3-Maleimido-1-oxopropyl)amino)propyl-ω-methoxy-polyoxyethylene (1.5 equivalents, average molecular weight 11962 Daltons, NOF Corp. ., Tokyo, Japan) and 2 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were added to the resulting solution. According to the reverse phase analytical HPLC system (Vydac C 18 5 μ peptide / protein column, 4.6 x 250 mm), the reaction was completed after 1 hour at room temperature. Load the reaction mixture onto 5 mL Zeba TM Desalting spin columns (Pierce Biotechnology, Rockford, IL). A white foam (15.1 mg) was obtained after lyophilization. Put this product in the High Trap TM Further purification was performed on a SPXL cation exchange column (GE Healthcare, Piscataway, NJ). The molecular weight distribution of the pu...

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Abstract

The present invention relates to methods for the chemo-selective pegylation of the cysteine residue having unoxidized sulfhydryl side-chain and free a-amino group in proteins, peptides and other molecules. Similar methods are provided for the chemo- selective pegylation of the homocysteine, selenocysteine, penicillamine, and N-methyl- cysteine residues.

Description

technical field [0001] The present invention relates to a method for the chemoselective pegylation of cysteine ​​residues with unoxidized sulfhydryl side chains and free alpha-amino groups in proteins, peptides and other molecules. Background technique [0002] Unlike small molecule drugs, which are usually administered by the oral route, protein and peptide based therapeutics are usually administered by injection due to their extremely low oral bioavailability. After injection, most proteins and peptides are rapidly cleaved by enzymes and cleared from the body, resulting in a short circulating half-life in vivo. This short circulating half-life is a major reason for lower potency, more frequent dosing, reduced patient compliance, and higher cost of protein and peptide therapeutics. Therefore, there is a strong need to develop methods to extend the duration of action of protein and peptide drugs. [0003] Covalent attachment of proteins or peptides to polyethylene glycol (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08G63/00C07K16/00
CPCC08G65/329C08G65/334C07K1/1077A61K47/48215C08G65/3348A61K47/60C07K17/08C07K17/00C07K5/02
Inventor 董正新J·S·埃耶昂
Owner 益普生制药股份有限公司
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