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Fixed point monosubstituted pegylation 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 problems such as easy shedding, easy oxidation of cysteine, and adverse effects on stability, achieving The effect of reducing by-products and avoiding multiple substitution reactions

Active Publication Date: 2017-07-07
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 monosubstituted pegylation Exendin analogue and preparation method thereof
  • Fixed point monosubstituted pegylation Exendin analogue and preparation method thereof
  • Fixed point monosubstituted pegylation Exendin analogue and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] (1) Amino acid monomers used for synthesis

[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] The above abbreviations 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)

[0069] (2) Reagents: N,N-diisopropylethylamine, diisopropylcarbodiimide (DIC), N,N-dimethylformamide (DMF), dichloromethane, hexahydropyridine, 1 -Hydroxybenzotriazole, Rink Amide Resin, Ninhydrin, Methanol, Triisopropylsilane, Trifluoroacetic Acid.

[0070] (3) Operation

[0071] A Synthesis: Taking 0.25mmol scale as an example, weigh 0.5g Rink Amide resin and place it in a reactor, weigh 1mmol of amino acid, act...

Embodiment 2

[0089] Method for site-specific protection of PEGylation of Exendin-4 analogs

[0090] In this example, conventional amino pegylation 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 groups are modified. Among them, the PEGylation reagent is preferably 40KD Y-type NHS-PEG, and the Exendin-4 analogue for site-specific protection is preferably

[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-Pro-Pro-Pro-Pro-Ser-Lys.

[0093] Take 2g of Exendin-4 analogue for site-specific protection, 26g of 40KD Y-type NHS-PEG (about 1.5:1 molar ratio to polypeptide) was dissolved in 400mL...

Embodiment 3

[0096] Separation and purification method of pegylated Exendin-4 analog

[0097] Targeted protection Exendin-4 analogue PEGylation final reaction solution is respectively subjected to reverse phase HPLC, ion exchange, ultrafiltration concentration, molecular sieve and freeze-drying to obtain pure PEGylated Exendin-4 analogue API.

[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] Packing: SOURCE 30RPC 175mL

[0102] Flow rate: 30mL / min

[0103] Elution gradient: 2 column volumes equilibrated after loading, 0-30% 5min, 30%-100% 5min

[0104] image 3 is the SOURCE purification chromatogram of the pegylated Exendin-4 analogue.

[0105] B. SP cation exchange purification

[0106] Mobile phase: Phase A 10mM pH3.5CBS

[0107] Phase B1 10mM pH3.5CBS+0.15M NaCL

[0108] Phase B2 10mM pH3.5CBS+1.5M NaCL

[0109] Column: GE XK 50

...

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Abstract

The invention discloses a compound capable of monosubstituting pegylation on any amino in an Exendin analogue structure at a fixed point, a preparation method of the compound, and an application of the compound. The method uses a more stable protecting group Dde(N-a-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)), the side effect of polysubstitution, caused by the use of an unstable protecting group, is avoided, so that the high-yield monosubstituted pegylation Exedin analogue is prepared at the low reaction molar ratio.

Description

[0001] Divisional statement [0002] This application is a divisional application of the Chinese patent application filed on March 30, 2011 with the application number 201110078314.X and the title of the invention "Site-Site-Substituted PEGylated Exendin Analog and Preparation Method thereof". technical field [0003] The invention relates to a site-specific monosubstituted pegylated Exendin analogue, its preparation method and medical application. Background technique [0004] As early as the 1960s, McIntyre and Elrick et al. found that the promotion of insulin secretion by oral glucose was significantly higher than that by intravenous injection. This additional effect was called "incretin effect". Further studies by Perley and others 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 decreased in patients with type 2 diabetes. This suggests that ab...

Claims

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Application Information

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Patent Type & Authority Applications(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