Introduction
ritaglutide is a triple agonist peptide of glucagon receptor (GCGR), glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon-like peptide -1 receptor (GLP-IR). It shows more excellent clinical effect than that of Smeaglutide for obesity, type 2 diabetes caused by obesity, cardiovascular disease, endocrine and metabolic diseases, etc, the original research institution, Eli Lilly (Eli Lilly and Company), has entered the third phase of clinical research on the therapeutic effect of the drug on cardiovascular disease, type 2 diabetes, and obesity. In the near future, it is expected to become another breakthrough weight-loss drug after simaglutide, and the market prospect is very considerable.
Common side effects include gastrointestinal symptoms such as nausea, vomiting, and diarrhea, similar to those of other GLP-1 classes of drugs. These symptoms are common in the early stages of treatment, but the patient usually gets used to them over time.
Preparation method
the method includes the following steps: synthesis of retaglutide side chain peptide resin by Fmoc/tBu solid phase method; Removal of Lys17 main chain protecting group Dde or Alloc, sequential coupling of main chain protected amino acids or protected amino acid fragments to obtain retaglutide peptide resin; Cleavage of peptide resin with acidic mixed solution to obtain crude peptide; HPLC purification, salt conversion, concentration, lyophilization of crude peptide, the nataglutide sperm peptide was obtained. According to the nataglutide synthesis method of the present invention, the purity of the nataglutide product is greater than 99 .0%, and the single impurity is less than 0 .10%. The synthetic method has the advantages of simple operation, high purity of crude peptide, easy purification and preparation, low cost of comprehensive synthesis, and is very suitable for industrial production.
Preparation of the side chain peptide resin of retamaglutide
take 750mmol AM Resin ( the degree of substitution is0.55mmol/g) after swelling and washing, the protection amino Acids Fmoc-Linker(1.5eq) condensation Reagent DIEA(3eq) , TBTU(1.5eq) add to the reaction kettle and use DMF dissolve, stir in coupling reaction at 25~35 ℃ for 120~300 minutes, filtration and washing to obtain Fmoc-Linker-AM Resin .
Use20%Pip/DMF solution removal Fmoc after protection, protective amino acids are added Fmoc-Ser(tBu)-OH(3eq) and HOBt(3eq) use appropriate amount DMF dissolve and cool0~15 DEG C. Take DIC(3eq), slowly add to the protective ammonia with stirring base acid DMF solution in the reaction was stirred at 0-15°C for 5-15 minutes to obtain an activated protected amino acid solution.
The activated protected amino acid solution is added to the resin to be reacted. Coupling reaction at 25~35 ℃ for 120~300 minutes, filtration and washing to obtain Fmoc-Ser(tBu)-Linker-AM Resin .
Using the same method as above, the protected amino acids or fully protected amino acid fragment peptides in Table 2 are sequentially inserted: Fmoc-
Pro-OH.H₂O , Fmoc-Pro-OH.H₂O , Fmoc-Pro-OH.H₂O , Fmoc-Ala-OH.H₂O , Fmoc-Gly-0H , Fmoc-Ser(tBu) -OH , Fmoc-Ser(tBu)-OH , Fmoc-Pro-OH.H₂O , Fmoc-Gly-Gly-0H , Fmoc-Glu(OtBu)- OH.H₂O , Fmoc-Leu-0H , Fmoc-Leu-0H , Fmoc-Tyr(tBu)-OH , Fmoc-Glu(OtBu)-OH. H₂O , Fmoc- Ile-0H , Fmoc-Phe-0H , Fmoc-Ala-OH.H₂O , Fmoc-Gln(Trt)-Aib-0H , Fmoc-Ala-0H.H₂O , Dde- Lys(Fmoc)-OH , Fmoc-AEEA-OH , Fmoc-Glu-0tBu , eicosandioic acid mono-tert-butyl ester, obtained Dde-Lys(AEEA- γ-Glu-(OtBu)-Eicosanedioicacid-0tBu)-Ala-Gln(Trt)-Aib-Ala-Ph e-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Linker-AM Resin .
Preparation of retamaglutide peptide resin
removal Lys¹⁷ site backbone protecting group Dde after that, each point of the main chain is coupled in turn and used.2 ~ 3% hydrazine hydrate/DMF dissolved liquid removal Dde protecting groups, in5~35 DEG C under the conditions of reaction 10min, total three times. After washing the peptide resin, it is connected to the main chain points: Fmoc-Lys(Boc)-OH , Fmoc-Asp(OtBu)-OH , Fmoc-Leu-OH , Fmoc-aMeLeu-OH , Fmoc-Ile-OH , Fmoc-Ser(tBu)-OH , Fmoc-Tyr(tBu)-OH , Fmoc-Asp(OtBu)-OH , Fmoc-Ser(tBu)-OH , Fmoc-Thr(tBu)-OH , Fmoc-Phe-OH , Fmoc-Thr(tBu)-OH , Fmoc-Gly-OH , Fmoc-Gln(Trt)-OH , Boc-Tyr(tBu)-Aib-OH deRutaglutide Peptide Resin6500.3g .
Preparation of crude nataglutide peptide
to the above-prepared Rutaglutide peptide resin, add an acidic mixed solution, the mixed solution ratio is. TFA:EDT/ Tis/phenol/H0 = 90:2.5:2.5:2.5:2.5, used quantity is8mL/ grams of resin; stirring reaction at 15~30 ℃ for 2~4 hours to obtain the reaction mixture was filtered using a sand-core funnel, the filtrate was collected, and then a small amount TFA washing resin3 times, combine filtrate and add MTBE precipitation, reuse MTBE wash sediment3 times, 3536.3g drained white-like powder, namely the crude product of Rutaglutide, the purity of the crude product is81.81 percent.
Preparation of retamaglutide sperm peptide
take the crude Rietaglutide, add water, stir, and adjust with ammonia. pH9.0 ~ 9.5 to complete dissolution, the resulting solution is used.0.45μm filtration of mixed microporous membrane; purification and preparation by reversed-phase preparative chromatography, collection of Reitaglutide fraction; combined fraction, sodium salt, decompression concentration shrink to obtain a solution of Rutaglutide; freeze-dried, Rutaglutide spermatopeptide1963.38.68g purity is99.51%, largest single an impurity0.10%, the total yield was 55.33%, and the molecular weight was 4730.76.
The above-mentioned embodiment shows that the purity of the crude product obtained by the synthesis method provided by the present invention has been greatly improved, and the purity of the natalu peptide peptide product is greater.99.0%, single impurity is less than 0.10%. At the same time, the present invention provides. method is also improved the final product quality, the process is simple and easy to control, the degree of industrialization is high, has a wide range of practical value and application prospects.
References: Synthesis of Rutaglutide and Rutaglutide CN 119823233
Post time: 2026-09-23