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All amino acids were purchased from NovaBiochem Company. Unless otherwise specified, all other reagents were analytically pure and purchased from Sigma Company. Protein Technologies PRELUDE 6-channel polypeptide synthesizer was used. Phenomenex Luna C18 preparative column (46 mm×250 mm) was used for purification of the polypeptides. High performance liquid chromatograph was manufactured by Waters Company. MS analysis was determined using Agilent mass spectrometer. Synthetic method of polypeptide compounds of the invention is illustrated by taking the polypeptide compound 6 as an example: Structure Sequence: His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-gammaGlu-CO(CH2)14CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-As n-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 a) Main peptide chain assembly: The following polypeptide in a scale of 0.25 mmol was synthesized on a CS336X peptide synthesizer (CS Bio American Company) according to Fmoc/t-Bu strategy: Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Lys(ivDde)-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met- n(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin (1) Step 1: 0.75 g of Rink amide MBHA-LL resin (Novabiochem, loading 0.34 mmol/g) was swelled in dichloromethane (DCM) for 1 hour, and the resin was fully washed with N,N-dimethylformamide (DMF) for three times (2) Step 2: The procedure reaction was performed using Rink amide resin as carrier, the mixture of 6-chloro-benzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), organic base N,N-diisopropylethylamine (DIEPA) at a molar ratio of 1:1 as coupling agent, and N,N-dimethylformamide (DMF) as solvent, the condensation reactions were performed to successively link. Fmoc-Ser(t-Bu)-OH, Fmoc-Pro-OH (3x), Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(t-Bu)-OH (2x), Fmoc-Pro-OH, Fmoc-Gly-OH (2x), Fmoc-Thr(t-Bu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH (2x), Fmoc-Lys(ivDde)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Phe-OH, Thr(t-Bu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-D-Ser(t-Bu)-OH, Boc-His(Boc)-OH to obtain: Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Lys(ivDde)-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met- Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. Subsequently, the resin was fully washed with N,N-dimethylformamide (DMF), dichloromethane (DCM), Methanol, dichloromethane (DCM), and N,N-dimethylformamide (DMF) in sequence for three times respectively. In the reaction, 1) the amount of the first amino acid Fmoc-Ser(t-Bu)-OH and the amount of the resin was at a ratio of 1:16:1; and 2) in each of the subsequent condensation reactions, each of the amount of Fmoc protected amino acid, 6 -chloro-benzotriazole-1,1,3,3 -tetram ethyluronium hexafluorophosphate (HCTU), organic base N,N-diisopropylethylamine (DIEPA) was excess by 2-8 times, the reaction time was 1-5 hours. b) Removal of 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)-3-methyl-butyl (ivDde) and introduction of lipophilic substituent: The resin was washed twice in the solution of N,N-dimethylformamide (DMF)/dichloromethane (DCM)=1:1 (volume ratio), and added with freshly prepared 3.0% hydrazine hydrate in N,N-dimethylformamide (DMF). The reaction mixture was shaken at room temperature for 10-30 minutes, and then filtered. The hydrazine treatment step was repeated five times to obtain: Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Lys-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met- Asn(Trt)Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. Subsequently, the resin was fully washed with N,N-dimethylformamide (DMF), dichloromethane (DCM), Methanol, dichloromethane (DCM), N,N-dimethylformamide (DMF) in sequence for three times respectively. Thereto was added an N,N-dimethylformamide (DMF) mixed coupling solution (5 times excess of each) of FmocNH-PEG2-OH (Quanta BioDesign), 2-(7-azo BTA)-N,N,N?,N?-tetramethyluronium hexafluorophosphate (HATU) and diisopropylethyl amine (DIEPA), shaken for 2 hours, and filtrated. Subsequently, the resin was fully washed with N,N-dimethylformamide (DMF), dichloromethane (DCM), methanol, dichloromethane (DCM), and N,N-dimethylformamide (DMF) in sequence for three times respectively to obtain: Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Ph... |
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