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Fixed point mono-substituted pegylation of Exendin analogue and preparation method thereof

A technology of PEGylation and PEGylation, which is applied in the field of fixed-point monosubstituted PEGylation of Exendin analogues, can solve the problems of easy oxidation and shedding of cysteine, and unclear effects of drug efficacy and toxicity and other problems to achieve the effect of avoiding multiple substitution reactions and reducing by-products

Inactive Publication Date: 2012-10-10
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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  • Fixed point mono-substituted pegylation of Exendin analogue and preparation method thereof
  • Fixed point mono-substituted pegylation of Exendin analogue and preparation method thereof
  • Fixed point mono-substituted pegylation of Exendin analogue and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] (1) Amino acid monomers used in the synthesis

[0065] 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

[0066] Abbreviations above are: Fmoc 9-fluorenylmethoxycarbonyl; Boc tert-butoxycarbonyl; Trt trityl; OtBu tert-butyloxy; tBu tert-butyl; Dde N-[1-(4,4-dimethyl base-2,6-dioxocyclohexylidene)

[0067] (2) Reagents: N,N-diisopropylethylamine, diisopropylcarbodiimide (DIC), N,N-dimethylformamide (DMF), dichloromethane, hexahydropyridine, 1 -Hydroxybenzotriazole, Rink Amide resin, ninhydrin, methanol, triisopropylsilane, trifluoroacetic acid.

[0068] (3) Operation

[0069] A Synthesis: Take 0.25 mmol scale as an example, weigh 0.5 g of Rink Amide resin into a reactor, weigh 1 mmol of amino acids, activate by...

Embodiment 2

[0087] Method for site-directed protection of Exendin-4 analogs PEGylation

[0088] In this example, conventional amino PEGylation modification reagents such as (SC-PEG, SS-PEG, NHS-PEG, etc.) are used to protect the only free side chain on the Exendin-4 analog that can be PEGylated. Amino group for binding modification. Among them, the PEGylation reagent is preferably 40KD Y-type NHS-PEG, and the site-directed protection Exendin-4 analog is preferably

[0089] (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

[0090] (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-Pro-Pro-Pro-Ser-Lys.

[0091] Take 2 g of site-protected Exendin-4 analog and 26 g of 40KD Y-type NHS-PEG (about 1.5:1 molar ratio to the polypeptide) and dissolve it in...

Embodiment 3

[0094] Separation and purification method of PEGylated Exendin-4 analogs

[0095] The final reaction solution of PEGylation of exendin-4 analogs with site-specific protection is respectively subjected to reverse-phase HPLC, ion exchange, ultrafiltration concentration, molecular sieves and freeze-drying to obtain pure PEGylated Exendin-4 analog raw materials.

[0096] A. SOURCE reversed-phase HPLC purification

[0097] Mobile phase: A phase A 20mM HAc 5% acetonitrile; B phase 20mM HAc 50% acetonitrile

[0098] Column: GE Fineline Pilot 35

[0099] Filler: SOURCE 30RPC 175mL

[0100] Flow rate: 30mL / min

[0101] Elution gradient: Equilibrate 2 column volumes after loading, 0-30% 5min, 30%-100% 5min

[0102] image 3 is the SOURCE purification chromatogram of the PEGylated Exendin-4 analog.

[0103] B, SP cation exchange purification

[0104] Mobile phase: Phase A 10mM pH3.5CBS

[0105] Phase B1 10mM pH3.5 CBS+0.15M NaCl

[0106] Phase B2 10mM pH3.5 CBS+1.5M NaCl

[010...

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Abstract

The invention discloses a compoundwhich is obtained by fixed point mono-substitution pegylation of any amidogen in an Exendin analogue structure, a preparation method and use thereof. The method of this invention adopts a more stable protecting group of Dde(N- alpha-1(4,4-dimethyl -2,6- dioxo cyclohexyl idene) to avoid the polysubstitution side reaction caused by using unstable protecting groups, such that high yield of mono-substituted pegylation Exendin analogue can be obtained with a low reaction molar ratio.

Description

technical field [0001] The invention relates to a site-specific mono-substituted PEGylated Exendin analog, a preparation method and medical use thereof. Background technique [0002] As early as the 1960s, McIntyre and Elric et al. found that oral glucose promoted insulin secretion significantly more than intravenous injection, an additional effect known as the "incretin effect". Further research by Poley et al. confirmed that the insulin produced by this "incretin effect" accounts for more than 50% of the total insulin after eating. In 1986, Nauk et al. found that the incretin effect was diminished in patients with type 2 diabetes. This suggests that the abnormality of the incretin system may be one of the pathogenesis of type 2 diabetes. [0003] With the development of cellular and molecular biology, the mysterious veil of incretin has been slowly unveiled. Studies have confirmed that incretin is a gut-derived hormone in the human body. After eating, these hormones ca...

Claims

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Application Information

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IPC IPC(8): C07K17/08A61K38/22A61K47/48A61P3/10
CPCA61K47/48215C07K14/57563A61K38/00A61K47/60A61P3/10C07K1/06C07K14/575A61K38/22Y02P20/55
Inventor 岳鹏
Owner SHANGHAI HUAYI BIO LAB CO LTD
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