Solid-phase preparation method of pracanatide

[0163] Example 1: Acm protected a fragment (fragment a [13,4]) Synthesis of

[0164] step one:

[0165] Were added sequentially in a solid phase reaction flask Wang resin (90.00 g, degree of substitution 0.60mmol / g), 1100mLDCM, swelling after suction filtration, the filter cake was obtained. Were added sequentially at 24 ± 4 ℃ Fmoc-Thr (tBu) -OH (15.51g), DIC (4.92g), HOBt (5.28g), 1100mLDCM, the reaction was stirred under nitrogen for 2h. Drained, 300mL * 5DCM washed five times, washed with 300mL * 3DMF 2 times. Was added acetic anhydride 90mL, 90 mL pyridine, stirred for 8h under DMF900mL, nitrogen. Drained again added 90 mL acetic anhydride, 90 mL pyridine, stirred at DMF900mL, nitrogen protection reaction 8h. Drained, 300mL * 5DCM washed five times, 350mL * 3MeOH washed three times. Dried at room temperature in vacuo to give 108.85g of Fmoc-Thr (tBu) Wang resin. After testing, the degree of substitution 0.30mmol / g;

[0166] In the solid phase reaction flask was added Fmoc-Thr (tBu) Wang resin (5.00g, degree of substitution 0.30mmol / g), 100mLDCM, swelling drained after 10 minutes. 30mLDMF filter cake was washed, drained. In the flask at 24 ± 4 ℃ successively added 35mLDCM / DMF (1: 1, volume ratio), Fmoc-Cys (Mmt) -OH (4.50mmol), DIC (0.60g) and HOBt (0.64g), under nitrogen protection stirred for 2h, to detect the coupling ninhydrin reaction was complete. Drained and the filter cake was washed with an appropriate amount of DMF, dry. Was added 70mL20% piperidine / DMF solution, mixed nitrogen stirred for 10 minutes, drained; added 70mL20% piperidine / DMF solution, mixing a nitrogen atmosphere for 10 minutes, drained. The filter cake was washed with DMF six times amount, drained. At 24 ± 4 ℃ repeating the above coupling reactions, deprotection steps, in accordance with the polypeptide sequence and each amino acid in turn is connected 4.50mmol of: Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-Cys (Acm) -OH, Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Mmt) -OH. Be the last Fmoc-Cys (Mmt) -OH amino acid after the coupling reaction, drained, washed three times cake was of DMF, and then washed three times with DCM and sucked dry.

[0167] Step 2:

[0168] 70mL vial were added a mixed solution (TFA: TIS: DCM = 1: 10: 89), deprotected for 10 minutes, drained. Repeating the above deprotection reaction 4 times. The filter cake was washed three times with an appropriate amount of DMF, DCM and then washed three times with drained, washed three times with DMF. Mixed solution was added 70mL (30% hydrogen peroxide: DMF, 5: 95, volume ratio) sequentially at 24 ± 4 ℃ bottle, the oxidation reaction times (i.e., repeated three times as follows: the oxidation reaction stirred for 60 minutes and then drained). The filter cake was washed three times with an appropriate amount of DMF, DCM and then washed three times with drained, washed three times with DMF.

[0169] At 24 ± 4 ℃, in the above-described reaction flask into 70mL cleavage agents (TFA: TIS: water, 90: 5: 5, volume ratio) at room temperature stirred for 3h. Filtered, the resin washed with TFA and the combined filtrate was concentrated under reduced pressure to give the residue was mixed with 400mL of ice-cold diisopropyl ether, sedimentation, centrifugation, the white precipitate was washed with isopropyl ether and centrifuged. A fragment obtained by vacuum drying, an off-white solid, HPLC96%, MS: 1698.5; 1700.4.

[0170] Synthetic linker Acm protected peptide resin of Example 2:

[0171] Step three:

[0172] Were added sequentially in a solid phase reaction flask Wang resin (30.00 g, degree of substitution 0.60mmol / g), 300mLDCM, swelling after suction filtration, the filter cake was obtained. At 24 ± 4 ℃ sequentially added Fmoc-Leu-OH (3.42g), DIC (1.25g), HOBt (1.27g), 180mLDCM, the reaction was stirred under nitrogen for 2h. Drained, washed with 100mL * 5DCM 5 times. Was added acetic anhydride 30mL, pyridine 30mL, the reaction was stirred 8h at DMF30mL, nitrogen. Dry. 30mL acetic anhydride was added again, 30mL of pyridine, and stirred at DMF30mL, nitrogen protection reaction 8h. Dry. 100mL * 5DCM washed five times, 60mL * 3MeOH washed three times. Cake was MeOH, DCM alternately washed, drained, and dried in vacuo to give Fmoc-Leu Wang resin at room temperature. After testing, the degree of substitution 0.23mmol / g;

[0173] At 24 ± 4 ℃. Are sequentially added to the Fmoc-Leu Wang resin in a solid phase reaction flask (5g, degree of substitution 0.23mmol / g) and 100mLDCM, drained after swelling. Was added 40mL20% piperidine / DMF solution, mixed and stirred for 10 minutes and drained; added 40mL20% piperidine / DMF solution, mixed and stirred for 10 minutes, drained. The filter cake was washed with DMF six times amount, drained. At 24 ± 4 ℃. The resin were added Fmoc-Cys (Acm) -OH (2.01mmol), DIC (0.27g), HOBt (0.29g), 40mLDCM, nitrogen and stirred until the coupling ninhydrin detection reaction was complete. Drained and the filter cake was washed with DMF six times. Repeat deprotection reaction step of connecting the coupling Fmoc-Gly-OH (2.01mmol), to give linker peptide resin was used directly in the next step.

[0174] Example 3: [16-4, disulfide S-S (4 → 12)] synthetic peptide resin

[0175] Step 4:

[0176] Linker peptide resin was added to the reaction flask 40mL20% piperidine / DMF solution, mixed and stirred for 10 minutes and drained; added 40mL20% piperidine / DMF solution, stirred and mixed for 10 minutes and drained. The filter cake was washed with an appropriate amount of DMF, drained inactive deprotected linker peptide resin. Activation in a clean bottle, were added successively a fragment [13-4, disulfide SS (4 → 12)] (1.00mmol), DIEA (2.1mmol) and 25mL DCM / NMP (1: 1, volume ratio) at 5 ± 4 ℃ successively added HATU (1.05mmol) and HOAt (1.05mmol), activated under nitrogen for 2 minutes. Under nitrogen activated linker peptide resin was transferred to a deprotection reaction flask. Under nitrogen for 24 ± 4 ℃ reaction 2h, drained; activation in a clean flask, a fragment [13-4, disulfide SS (4 → 12)] (1.00mmol), DIEA (2.1mmol) and 25mL DCM / NMP (1: 1, volume ratio) at 5 ± 4 ℃ successively added HATU (1.05mmol), HOAt (1.05mmol), activated under nitrogen for 2 minutes. Under nitrogen activated linker peptide resin was transferred to a deprotection reaction flask. Under nitrogen for 24 ± 4 ℃ ninhydrin reaction to detect the coupling reaction was complete, drained, the filter cake washed with DMF six times, and then MeOH, DCM was washed alternately three times, drained, and dried in vacuo at room temperature to give [16-4, disulfide SS (4 → 12)] peptide-resin was used directly in the next step.