Preparation method and kit for synthesizing polypeptide containing two pairs of disulfide bonds, and preparation method of plecanatide
A disulfide bond, linear peptide technology, applied in the field of polypeptide preparation, can solve the problems of low yield, difficult separation and purification, low yield, etc., and achieve the effect of high yield
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Embodiment 1
[0070] Preparation of Fmoc-Leu-Wang resin with a degree of substitution of 0.50mmol
[0071] Weigh 100 g of Wang resin with a substitution degree of 1.0 mmol / g in a solid-phase reaction column, add DMF, and swell with nitrogen gas bubbles for 60 minutes; weigh 35.4 grams (100 mmol) of Fmoc-Leu-OH and 16.2 grams (120 mmol) of HOBt 1.2 g (10 mmol) of DMAP, dissolved in DMF, added 20.3 mL of DIC at 0°C, activated for 5 minutes, and added to the reaction column. After reacting for two hours, add 70mL acetic anhydride and 60mL pyridine, mix and seal for 24 hours, wash with DCM three times, dry the resin after shrinking with methanol to obtain 130 grams of Fmoc-Leu-Wang resin, and the detection degree of substitution is 0.50mmol / g.
[0072] Preparation of peptide resin
[0073] Weigh 50 g of Fmoc-Leu-Wang resin with a substitution degree of 0.50 mmol / g into a solid phase reaction column, add 50 mL of DMF, and swell with nitrogen gas for 60 minutes; then deprotect with 50 mL of DBLK...
Embodiment 2
[0081] The difference from Example 1 is: after the preparation of the linear pricanatide, different amounts of DHS50g were added during the oxidation reaction,
[0082] Preparation of Plicanatide
[0083] Add 90L of purified water, 200g of guanidine hydrochloride, and 300g of sodium bicarbonate in sequence in a 100L reactor to make the pH of the solution = 10, then add 50g of ethylene glycol, 50g of DHS (273.2mmol), and then lower the temperature of the solution to -10°C , and finally add 82.3 g of linear plicanatide, and after 48 hours of reaction, the mixture goes directly to the next step.
[0084] Purified Plicanatide
[0085] The mixture obtained above was directly loaded on a 15cm×25cm preparative column for high performance liquid phase purification. Reversed-phase octadecylsilane as the stationary phase, 0.1% acetic acid aqueous solution / acetonitrile as the mobile phase, the ratio is 70:30; prepared by isocratic elution; flow rate: 70-80ml / min; detection wavelength: ...
Embodiment 3
[0087] The difference from Example 1 is: after the preparation of linear pricanatide, different amounts of DHS68.6g were added during the oxidation reaction.
[0088] Preparation of Plicanatide
[0089] Add 90L of purified water, 200g of guanidine hydrochloride, and 300g of sodium bicarbonate in sequence in a 100L reactor to make the pH of the solution = 10, then add 50g of ethylene glycol, 68.6g (375mmol) of DHS, and then lower the temperature of the solution to -10 ℃, 82.3 g of linear plicanatide was finally added, and after 48 hours of reaction, the mixture was directly carried out to the next step.
[0090] Purified Plicanatide
[0091] The mixture obtained above was directly loaded on a 15cm×25cm preparative column for high performance liquid phase purification. Reversed-phase octadecylsilane as the stationary phase, 0.1% acetic acid aqueous solution / acetonitrile as the mobile phase, the ratio is 70:30; prepared by isocratic elution; flow rate: 70-80ml / min; detection wa...
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