A kind of method for preparing linaclotide
A linaclotide, a pair of technology, applied in the field of preparation of linaclotide, can solve the problems of complex processing process, less source of raw materials, high cost, etc., achieve simple process operation, and avoid disulfide bond mismatch isomer impurities , the effect of reducing the difficulty of purification
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Embodiment 1
[0042] Example 1: Preparation of Fmoc-Cys(Mmt)-CTC resin
[0043] Accurately weigh 320 g of CTC resin (1.0 mmol / g) into a peptide resin synthesis reactor, and add 3 L of DCM to swell for 1 h. After the swelling was completed, the cells were washed three times with DMF, each 3L. Weigh 394 g of Fmoc-Cys(Mmt)-OH and 87 g of HOBt to dissolve in 2L DMF, add 110mL DIC to activate, add the solution to the reactor, and react for 4h. After the reaction was completed, the resin was washed three times with DMF, and then a pre-prepared capping reagent (2.5L DCM, 0.3L methanol, 0.2L DIEA) was added, and the end capping reaction was carried out for 1 h. After the end capping was completed, DMF was washed 4 times, DCM was washed twice, and methanol was washed 3 times, 3 L each time, and then the resin was vacuum-dried to obtain 507 g of Fmoc-Cys(Mmt)-CTC resin. Take a small amount of resin and measure Fmoc-Cys The substitution degree of (Mmt)-CTC resin was 0.61 mmol / g. The synthesis scale...
Embodiment 2
[0044] Example 2: Preparation of Fragment I Peptide Resin
[0045] 493 g (300 mmol) of Fmoc-Cys(Mmt)-CTC resin with a substitution degree of 0.61 mmol / g in Example 1 was weighed and placed in a peptide resin synthesis reactor, and 4 L of DCM was added to swell for 2 h. After the swelling was completed, washed with DMF for 3 times, each 4L, and then added 4L of 20% piperidine / DMF solution for deprotection twice for 10min and 10min respectively. After deprotection, the resin was washed 6 times with DMF, 4 L each time. Weigh Fmoc-Cys(Trt)-OH 352g and HOBt 81g to dissolve in 2L DMF, add 103mL DIC to activate, add the solution to the reactor, react for 2h, and monitor the reaction end point with Kaiser test. After the reaction was completed, the resin was washed 5 times with DMF, and then the next protected amino acid was deprotected and coupled. Repeat the above steps, and sequentially carry out the coupling of Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys(Mmt)...
Embodiment 3
[0046] Example 3: Preparation of Fragment I Linear Peptides
[0047] Weigh 700 g (200 mmol) of fragment I peptide resin obtained in Example 2 into a 10 L reaction flask, add 7 L of cleavage reagent TFA-TIS-DCM (5-5-90), and react at room temperature for 5 min. At the end of the reaction, the lysate was filtered into a suction filter bottle. The above-mentioned cleavage reaction was repeated 3 times, and the filtrate was combined with the same volume of 0.1mol / L NaHCO. 3 The filtrate was washed with an aqueous solution until the pH of the solution was 7-8, concentrated by rotary evaporation at 25 °C, the precipitated precipitate was collected by filtration, and vacuum-dried to obtain 261 g of fragment I linear peptide with a purity of 82.3% and a yield of 95%.
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