Branched peg-modified glp-1 analogs and pharmaceutically acceptable salts thereof
A GLP-1, branched technology, applied in the field of branched PEG-modified GLP-1 analogs and their pharmaceutically acceptable salts, can solve the problem of inability to maintain blood sugar metabolism and endocrine regulation, loss of function, and ineffective long-term effects. First-class problems, to achieve the effect of low cost, optimized design, long-term activity
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Embodiment 1
[0025] Embodiment 1: Synthesis of GLP-1 analogue SEQIDNO.95
[0026] The sequence of SEQ ID NO.95 is shown in formula (I): HdAEGTFTSDLSKQNleEEEAVRLFIEWLKQGGPSSGAPPPC-NH 2
[0027] (I)
[0028] According to the structural characteristics, it can be synthesized by relatively mature solid-phase synthesis technology.
[0029] 1.1 Amino acid derivatives used in the synthesis
[0030] Fmoc-His(Trt)-OH, Fmoc-D-Ala-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ser (tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Nle-OH, Fmoc-Ala-OH , Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH.
[0031] The above amino acid derivatives were purchased from Jill Biochemical.
[0032] 1.2 Resins and other reagents
[0033] Resin: RinkAmide-AM resin (Gill Biochemical).
[0034]Other reagents: N, N'-diisopropylcarbodiimide (DIC), hydroxybenzotriazole (HOBT), dimethylformamide (DMF), dichlorom...
Embodiment 2
[0040] Embodiment 2: the synthesis of maleimide-based active polyethylene glycol
[0041] This example is the synthesis of linear and branched maleimide-based active polyethylene glycol, in which the main raw materials are mPEG-OH (5kD), mPEG-OH (10kD), mPEG-OH (20kD), mPEG -OH (30kD) was purchased from Sunbio Company in South Korea, and other reagents such as ethylenediamine, N-hydroxysuccinimide (HOSu), Lys, toluene, and methylene chloride were commonly used reagents.
[0042] 2. Synthesis of 1mPEG-MAL (20kD)
[0043]
[0044] Put 20g (1mmol) mPEG-OH (20kD) into a 200ml single-necked bottle, add 100ml toluene, reflux to separate water; then distill off the toluene, cool to room temperature, add 100ml DCM, and then add 1.18g (4mmol) of triphosgene (triphosgene), stirred at room temperature and reacted overnight; the next day, the reaction solution was washed into 200ml of anhydrous ether in a fume hood, filtered and vacuum-dried to obtain 15g of a white solid. Put 15g of...
Embodiment 3
[0081] Example 3: Synthesis of Pegylated GLP-1 Analog SEQIDNO.95
[0082] Active polyethylene glycol mPEG-MAL (20kD), mPEG4-Lys-MAL (20kD), mPEG2-Lys-MAL (20kD), mPEG2-Lys-MAL (40kD), mPEG2-Lys respectively prepared in Example 2 -MAL (60kD) and mPEG2-glycerol-MAL (40kD) purchased from U.S. NEKTAR company (article number: 2D3Y0T01), react with the GLP-1 analog SEQIDNO.95, and form a thioether bond after the Michael addition reaction to make the polypeptide and Polyethylene glycol is covalently bound to obtain the pegylated GLP-1 analog SEQ ID NO.95.
[0083]
[0084] Structure of mPEG2-glycerol-MAL(40kD)
[0085] 3.1 Preparation of PEX-165
[0086] Weigh 5.0gmPEG-MAL (20kD) and 1.26g GLP-1 analog SEQIDNO.95 (1.2 times excess), add 300ml 0.1M sodium phosphate buffer (pH7.7), and stir at room temperature for 2 hours.
[0087] The reaction solution was separated and purified using a reverse-phase HPLC preparative column (Luna, C18), and lyophilized to obtain 2.5 g of a white...
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