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Methods and reagents for preparing peptide/protein conjugates

A protein and compound technology, applied in the preparation method of peptide, animal/human protein, anti-animal/human immunoglobulin, etc., can solve the problem of cell toxicity

Active Publication Date: 2019-08-30
SHENZHEN INST OF RES & INNOVATION THE UNIV OF HONG KONG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some byproducts are even toxic to cells

Method used

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  • Methods and reagents for preparing peptide/protein conjugates
  • Methods and reagents for preparing peptide/protein conjugates
  • Methods and reagents for preparing peptide/protein conjugates

Examples

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preparation example Construction

[0029] The present invention relates to a preparation method of compound (1) represented by formula (1),

[0030]

[0031] wherein R' represents a substituent, and R represents H or at least one substituent.

[0032] The compound (1) is produced by reacting the compound (2) represented by the formula (2) with the compound (3) represented by the formula (3).

[0033] R'-NH 2 (2)

[0034]

[0035] In the above formula, R' can be any substituent, especially a peptide residue and / or a protein residue. The peptide residues may be synthetic or natural peptide residues. The protein residues may be natural protein residues or recombinant protein residues, or enzyme residues or antibody residues. The peptide residues may have at least one lysine residue in the sequence. R can be any substituent, preferably selected from alkyl, epoxide, amide, amine, ketone, aldehyde, phosphate, sulfate, halide, nitrile, nitro, azido , thiol group, carboxylic acid group, alkynyl group, este...

Embodiment 1

[0047] Will NH 2 CH 2 CH 2 NHBoc was dissolved in PBS buffer (pH=7.4) at a concentration of 1 mM, and compound 2 (1.2 equivalents) was added. The reaction mixture was stirred at room temperature for 1 hour. The product (%) was isolated using chromatography. 1 H NMR (CDCl 3 )δ7.30(2H,m),7.05(1H,m),5.02(1H,s),4.30(2H,s),3.84(3H,s),3.80(2H,m),3.38(2H,m ),1.28(9H,s); 13 C NMR (CDCl 3 )δ170.8,161.4,157.6,135.2,134.9,125.0,121.2,107.8,80.9,57.1,51.5,43.9,40.8,29.8.( figure 2 )

Embodiment 2

[0049] As a model study, the peptide Ac-AKLHA-OH was prepared by solid-phase peptide synthesis. A typical procedure for OPA / amine conjugation is as follows: Compound 1 is added to a solution of lysine-containing peptide in PBS buffer (pH=7.4) at a concentration of 0.01 mM. The reaction mixture was stirred at room temperature. The progress of the reaction is monitored by measuring the change in fluorescence. The fluorescence intensity increased with time and reached a maximum at 10 min ( image 3 ). The product was analyzed by LC-MS ( Figure 4 ). Ac-AKPHA-OH, ESI-MS, calculated M+H+: 546.4, measured 564; product ESI-MS, calculated M+H+: 680.3, measured 680.1.

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Abstract

The invention relates to a method and a reagent for preparing a peptide / protein conjugate. The method comprises the step of reacting a compound (2) expressed by a formula (2) with a compound (3) expressed by a formula (3) to obtain a compound (1) expressed by a formula (1), wherein R' represents a substituent, and R represents H or at least one substituent.

Description

technical field [0001] The present invention relates to methods and reagents for preparing peptide / protein conjugates. The invention also relates to methods of labeling and modifying unprotected peptides and native proteins and preparing protein conjugates with polyethylene glycol (PEG), small molecules, probes, and the like. Background technique [0002] Protein labeling provides valuable tools for studying the function of intracellular proteins such as immunostaining and immunolocalization and for developing therapeutic biologics, including polyethylene glycol-protein conjugate drugs, antibody-drug Conjugate (ADC), and glycoprotein conjugate vaccine, etc. Native protein labeling often involves conjugation chemistry that occurs under physiological conditions at the nucleophilic side chains of amino acids in proteins, with lysine and cysteine ​​being the most common labeling sites. Conjugation at lysine residues and primary amines at the N-terminus involves the use of acyl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/82C07K14/765C07K16/18C07K1/113
Inventor 李学臣童俊凌王搢铤
Owner SHENZHEN INST OF RES & INNOVATION THE UNIV OF HONG KONG