SITE-DIRECTED MONO-SUBSTITUTED PEGYLATED EXENDIN ANALOG AND PREPARATION METHOD THEREFOR
The invention discloses site-specific mono-PEGylated Exendin analogs in which any amino groups can be mono-PEGylated as well as their preparation method and use. The method of the present invention adopts a more stable protective group Dde (N-α-1-(4,4-dimethyl-2,6-dioxo-cyclohexylene)) to avoid multi-PEGylated side reactions rendered by unstable protecting groups, achieving mono-PEGylated Exendin analogs at a high recovery with a low reaction molar ratio.
1 . A method for preparing PEGylated Exendin analogs, including the steps as follows:
(1) Synthesize peptide raw materials of Exendin analogues: some of Lysine residues of the peptide have been protected by Dde, wherein, Dde is N-α-1-(4,4-dimethyl-6-dioxo-cyclohexylene);
(2) The peptide materials mentioned in step (1) react with the PEG derivatives in alkaline organic solvent, allowing Lys residues without Dde protecting group to bind the polyethylene glycol group;
(3) Remove the protecting groups of the product produced from step (2), isolate and purify the product, obtaining the PEGylated Exendin analogs.
2 . The method according to claim 1 , wherein said peptide material includes only one locus' Lys residue without protected by Dde.
3 . The method of claim 1 , wherein said peptide material, N-terminus is protected by Dde or Fmoc and Fmoc is 9-fluorenyl-methoxycarbonyl.
4 . The method of claim 1 , wherein said peptide material has the following structure:
(X)His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Y1-Gln-Z-Glu-Glu-Glu-Ala-Val-Y2-Leu-Phe-Ile-Glu-Trp-Leu-Y3-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Y4, wherein, X is Fmoc or Dde; Z is Leu or Ile; Y1-Y4 is Lys or (Dde) Lys, and at least one of Y1-Y4 is Lys.
5 . The method of claim 4 , wherein Y1-Y4, only one of Y2-Y4 is Lys, the rest are (Dde) Lys.
6 . The method of claim 5 , wherein Y2 is Lys, Y1, Y3 and Y4 are (Dde) Lys.
7 . PEGylated Exedin analogue prepared according to claim 1 .
8 . A PEGylated Exedin wherein the analogs have the following sequence structure:
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Z-Glu-Glu-Glu-Ala-Val-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys,
Wherein, Z is Leu or Ile, and in said structure, and the amino group of one Lys residue is connected to polyethylene glycol.
9 . The PEGylated Exedin analogue of claim 8 , wherein Lys20 residue or Lys27 residue is connected to polyethylene glycol.
10 . A method for treating diabetes comprising administering an Exedin analogue according to claim 7 .
11 . PEGylated Exedin analogue prepared according to claim 2 .
12 . PEGylated Exedin analogue prepared according to claim 3 .
13 . PEGylated Exedin analogue prepared according to claim 4 .
14 . PEGylated Exedin analogue prepared according to claim 5 .
15 . PEGylated Exedin analogue prepared according to claim 6 .
16 . A method for treating diabetes comprising administering an Exedin analogue according to claim 8 .
17 . A method for treating diabetes comprising administering an Exedin analogue according to claim 9 .
18 . A method for treating diabetes comprising administering an Exedin analogue according to claim 11 .
19 . A method for treating diabetes comprising administering an Exedin analogue according to claim 12 .
20 . A method for treating diabetes comprising administering an Exedin analogue according to claim 13 .
21 . A method for treating diabetes comprising administering an Exedin analogue according to claim 14 .
22 . A method for treating diabetes comprising administering an Exedin analogue according to claim 15 .