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Site-directed single-substituted pegylated exendin analogue and preparation method thereof

A technology of PEGylation and analogues, which is applied in the field of fixed-point monosubstituted PEGylation of Exendin analogues, which can solve the problems of unclear efficacy and toxicity, adverse effects on stability, easy oxidation of cysteine, etc. , to avoid multiple substitution side reactions, reduce production costs, and avoid side effects

Active Publication Date: 2021-06-01
SHANGHAI HUAYI BIO LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantage of this method is that, on the one hand, the change in the structure of the drug causes unclear effects on its efficacy and toxicity, and on the other hand, the introduction of cysteine ​​will have an adverse effect on its stability.
These two contradictory facts make it easy for the Fmoc protecting group to fall off when the Fmoc-protected polypeptide undergoes pegylation modification under partial alkaline conditions, which inevitably results in the generation of a considerable number of side-reactants with multiple substitutions

Method used

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  • Site-directed single-substituted pegylated exendin analogue and preparation method thereof
  • Site-directed single-substituted pegylated exendin analogue and preparation method thereof
  • Site-directed single-substituted pegylated exendin analogue and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066](1) Amino acid monomer used to synthesize

[0067]FMoc-His (TRT) -OH, DDE-His (TRT) -OH, Fmoc-ala-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Thr (TBU) -OH, FMOC- PHE-OH, FMOC-SER (TBU) -OH, FMoc-ASP (OTBU) -OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Lys (DDE) -OH, FMOC-GLN ( TRT) -OH, Fmoc-Val-OH, FMOC-ILE-OH, FMOC-TRP (Boc) -OH, Fmoc-Asn (TRT) -OH, Fmoc-Pro-OH, Fmoc-Cys (TRT) -OH

[0068]Abbreviations are: FmoC 9-fluorenyl methoxycarbonyl; BoC tert-butoxycarbonyl; TRT triphenyl group; OTBU tert-butyloxy; TBU t-butyl; DDE N- [1- (4, 4-dimethyl) Base-2,6-dioxide subunisyl)

[0069](2) reagent: N, N-diisopropylamine, diisopropyl carbon diimide (DIC), N, N-dimethylformamide (DMF), dichloromethane, hexapyridine, 1 - Hydroxybenzotriazole, RINK Amide resin, ninluol, methanol, triisopropylsilane, trifluoroacetic acid.

[0070](3) operation

[0071]A synthesis: Take 0.25 mmol size as an example, weigh 0.5 g of Rink Amide resin is placed in the reactor, and the amino acid is given 1 mmol, activated by...

Embodiment 2

[0089]Method for protecting Exendin-4 analog polyethylene glycolization

[0090]This example uses conventional amino polyethylene glycolification reagents such as (SC-PEG, SS-PEG, NHS-PEG, etc.) to protect the side chain of polyethylene glycolinated reactions on the exendin-4 analog. The amino group is combined with modification. Among them, polyethylene glycolified reagents are preferably 40 kD Y-type NHS-PEG, and preferentially protect exendin-4 analogues.

[0091](FMOC) His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- (DDE) Lys-Gln-Z-Glu-Glu-Glu-ala-Val-Lys-Leu-Phe- ILE-GLU-TRP-Leu- (DDE) Lys-asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser- (DDE) Lys or

[0092](FMOC) His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- (DDE) Lys-Gln-Z-Glu-Glu-Glu-ala-Val- (DDE) Lys-Leu -Phe-ILE-GLU-TRP-LEU- (DDE) Lys-asn-gly-gly-pro-ser-ser-gly-ala-prop-pro-pro-ser-lys.

[0093]The point protects the exendin-4 analog 2g, 40 kD Y-type NHS-PEG 26g (with polypeptide molar ratio 1.5: 1) dissolved in 400 ml DMSO, 40 ° C to ...

Embodiment 3

[0096]Polyethylene glycolified Exendin-4 analog isolation purification method

[0097]The fixed-point protection Exendin-4-analog polyethylene glycolized end reaction liquid is respectively obtained by reverse phase HPLC, ion exchange, ultrafiltration, molecular sieve and freeze-drying to obtain pure polyethylene glycolization Exendin-4 analog producers.

[0098]A, Source reverse phase HPLC purification

[0099]Mobile phase: a phase A phase 20mm Hac 5% acetonitrile; B phase 20mm Hac 50% acetonitrile

[0100]Column: GE FineLine Pilot 35

[0101]Filler: Source 30RPC 175ml

[0102]Flow speed: 30ml / min

[0103]Elution gradient: balance 2 column volume, 0-30% 5 min, 30% -100% 5 min

[0104]image 3 It is a polyethylene glycolified Exendin-4 analog source Source purified chromatogram.

[0105]B, SP cation exchange purification

[0106]Mobile phase: a phase 10mm pH 3.5CBS

[0107]B1 phase 10mm pH 3.5cbs + 0.15m NaCl

[0108]B2 phase 10mm pH 3.5CBS + 1.5M NaCl

[0109]Column: GE XK 50

[0110]Filler: SP Sepharose Fast Flow 600ml

[0...

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Abstract

The invention discloses a compound capable of point-directed mono-substituted pegylation on any amino group in the structure of an exendin analogue, its preparation method and application. The method of the present invention avoids the use of an unstable protecting group by using a more stable protecting group Dde (N-α-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)). The multi-substitution side reactions brought about can prepare high-yield mono-substituted PEGylated Exedin analogues with a low reaction molar ratio.

Description

[0001]Presidentification statement[0002]This application is a score application number 2011110078314.x, 2011, 2011110078314.x, inventive, is a appended claims for Chinese patent application for "designated single-substituted polyethylene glycooleration Exendin analogs and preparation methods thereof".Technical field[0003]The present invention relates to a fixed point monochronous polyethylene glycolification Exendin analog, which is prepared and a medical purpose.Background technique[0004]As early as in the 1960s, McKoldtar and Elrick and others found that oral glucose on insulin secretion was significantly higher than intravenous injection, which is called "intestinal promotel". Perth et al. Further study confirmed that the insulin produced by this "intestinal promotelin effect" has more than 50% of the total amount of insulin after food. In 1986, Nak et al. Found that the effect of macrophypsopropin in patients with type 2 diabetes. This suggests that an impercussulopolysis system...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/575C07K1/107C07K1/06C07K1/02
CPCC07K14/57563A61K38/00A61K47/60A61P3/10C07K1/06C07K14/575A61K38/22Y02P20/55
Inventor 岳鹏
Owner SHANGHAI HUAYI BIO LAB CO LTD
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