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Pyridoxal derivative for pegylation modification of N terminal of protein and preparation method and application thereof

A technology of polyethylene glycol and protein, applied in the field of chemical synthesis and modification of protein, can solve the problems of long reaction time and low yield, and achieve the goal of improving efficiency, improving transamination efficiency, and shortening reaction transamination time Effect

Inactive Publication Date: 2013-03-06
JIANGSU SINOCOMPOUND CATALYST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The first problem to be solved by the present invention is to overcome the problems of long reaction time and low yield in existing transamination methods involving pyridoxal, and provide a designed and synthesized pyridoxal derivative for improving protein N Transamination efficiency of terminal amino acids, especially for amino acids with high steric hindrance, such as leucine, isoleucine, valine, etc.

Method used

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  • Pyridoxal derivative for pegylation modification of N terminal of protein and preparation method and application thereof
  • Pyridoxal derivative for pegylation modification of N terminal of protein and preparation method and application thereof
  • Pyridoxal derivative for pegylation modification of N terminal of protein and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0038] Preparation of 4-formyl-3-hydroxy-5-(methoxymethyl)-1,2-lutidine salt of formula VIII:

[0039] (1). Dissolve pyridoxine in acetone, add a catalytic amount of p-toluenesulfonic acid, and reflux the reaction solution overnight. After cooling, spin off the solvent under vacuum conditions, redissolve in dichloromethane, and use sodium bicarbonate aqueous solution for the solution Washing, the aqueous layer was further extracted with dichloromethane, the organic phases were combined, dried over magnesium sulfate, filtered, and further separated and purified by column chromatography to obtain (2,2,8-trimethyl-4H-[1,3]diox [4,5-c]pyridin-5-yl)methanol.

[0040]

[0041] (2). Dissolve (2,2,8-trimethyl-4H-[1,3]dioxy[4,5-c]pyridin-5-yl)methanol in anhydrous tetrahydrofuran and cool to 0oC , and then sodium hydride (70% oil droplet dispersion) was added in portions. After stirring for 30 minutes, a solution of methyl iodide in tetrahydrofuran was added dropwise to the reacti...

Embodiment 2

[0054] Preparation of formula VIII 4-formyl-3-hydroxyl-5-(methoxymethyl)-1,2-lutidine salt:

[0055] (1). Preparation of (2,2,8-trimethyl-4H-[1,3]dioxy[4,5-c]pyridin-5-yl)methanol of formula II;

[0056]

[0057] Pyridoxine (4.06g, 24mmol) was dissolved in 120ml of acetone, a catalytic amount of p-toluenesulfonic acid (4.93g, 28.68mmol) was added, and the reaction solution was refluxed overnight. After cooling, spin off the solvent under vacuum, redissolve in 120ml of dichloromethane, wash the solution with aqueous sodium bicarbonate, and extract the aqueous layer with dichloromethane, combine the organic phases, dry over magnesium sulfate, filter, and use column chromatography Separation and purification gave white solid (2,2,8-trimethyl-4H-[1,3]dioxy[4,5-c]pyridin-5-yl)methanol (5.02 g, 63.3%). 1HNMR (CDCl 3 , 300MHz): 1.55(d, J=3.8Hz, 6H), 2.41(s, 3H), 4.59(d, J=5.5Hz, 2H), 4.94(s, 2H), 7.96(s, 1H).

[0058] (2). Preparation of formula III 5-(methoxymethyl)-2,2,8-trim...

Embodiment 3

[0077] N-terminal transamination of the model polypeptide leucine-glutamic acid-tryptophan-glycine-alanine (LEWGA).

[0078] To the in situ generated pyridoxal derivative of formula VIII was added 500 μL of 100 mM phosphate buffered saline. The solution was mixed well, and the solution was adjusted to pH=6.5 with 1M sodium hydroxide and 1M hydrochloric acid. Then an equal volume of 2 mM peptide (LEWGA) solution was added, the solution was remixed and the pH was adjusted to 6.5. The reaction was stirred at room temperature and followed by RP-HPLC (C18 column, UV detection at 215 nm and 280 nm) and LC-MS. The reaction time was 27 hours and the yield was 88%. LC-MS: (CH 3 ) 2 CHCH2 COCO-Glu-Trp-Gly-Ala-OH (theoretical 573.2, actual [M+H] + 574.2).

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Abstract

The invention overcomes the problems of long reaction time and low yield of the existing transamination method involving pyridoxal, and provides a designed and synthesized pyridoxal derivative for improving the transamination efficiency of N-terminal amino acid of protein, particularly the high-steric hindrance amino acids such as leucine, isoleucine, and valine. The pyridoxal derivative is characterized by having a structure shown by the formula VIII. The pyridoxal derivative disclosed by the invention improves the transamination efficiency of the N-terminal amino acid of the protein, particularly the transamination reaction of the polypeptide containing high-steric hindrance amino acids (Leu, Ile and Val) at N terminal, thereby being favorable for further pegylation modification of protein.

Description

technical field [0001] The invention relates to the field of chemical synthesis and modification of protein, and relates to a novel pyrrole aldehyde derivative used for PEGylation modification of protein N-terminus, a preparation method and application thereof. Background technique [0002] With the development of modern biotechnology, protein drugs develop rapidly. Especially with the completion of the Human Genome Project and the progress of proteomics research, more and more protein drugs will be used for disease treatment. However, these drugs have a short half-life in vivo due to rapid renal elimination and proteolysis. In order to maintain the efficacy of the drug, it is usually necessary to use high doses of protein drugs, which not only increases the cost of treatment, but also easily increases adverse side effects such as immune response. So far, the most successful means to solve the defects of protein drugs is to modify and shield the surface of proteins by cova...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D213/66C07K17/08
Inventor 赵劲张美娟刘磊李娟
Owner JIANGSU SINOCOMPOUND CATALYST
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