| 222 mg |
|
[Example 32] (0345) (Synthesis method AF): Production of (3-carboxypropyl)carbamoyl-[D-Hyp24,Iva25,Pya(4)26,Cha27,36,Leu(Me)28, Lys30,Aib31]-PYY(23-36) (compound No. 349) Compound No. 349: (0346) Synthesis of (3-carboxypropyl)carbamoyl-[D-Hyp24,Iva25,Pya(4)26,Cha27,36,Leu(Me)28,Lys30,Aib31]-PYY(23-36) (0347) Using commercially available Sieber Amide resin (391 mg, 0.25 mmol) as a starting material, and ABI433A peptide synthesizer DCC/HOBt 0.25 mmol protocol, amino acids were condensed in the order of Cha, Arg(Pbf), Gln(Trt), Arg(Pbf), Thr(But), Aib, Lys(Boc), Asn(Trt), Leu(Me), Cha to give H-Cha-Leu(Me)-Asn(Trt)-Lys(Boc)-Aib-Thr(But)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Cha-Sieber Amide Resin (SEQ ID NO:183). In this case, the protocol was partly modified, and capping protocol with acetic anhydride was incorporated after every condensation procedure. In addition, the 30-position Lys(Boc) condensation was performed by double coupling. The obtained resin was swollen with DMF, and treated with Fmoc-Pya(4)-OH (388 mg, 1.0 mmol), HOAt in DMF (0.5 M, 2.0 mL, 1.0 mmol), DIPEA (174 muL, 1.0 mmol), DIPCDI (159 muL, 1.0 mmol) for 3 hr. Fmoc group was removed with 20% piperidine in DMF and the resin was treated with <strong>[857478-30-9]Fmoc-Iva-OH</strong> (339 mg, 1.0 mmol), HOAt in DMF (0.5 M, 2.0 mL, 1.0 mmol), DIPCDI (159 muL, 1.0 mmol) for 2 hr. The resin was washed with DMF, and further treated overnight with <strong>[857478-30-9]Fmoc-Iva-OH</strong> (339 mg, 1.0 mmol), HOAt in DMF (0.5 M, 2.0 mL, 1.0 mmol), DIPCDI (159 muL, 1.0 mmol). The resin was washed and subjected to a capping treatment with decanoic anhydride (368 muL, 1.0 mmol), DIEA (174 muL, 1.0 mmol) in DMF for 20 min. Fmoc group was removed with 20% piperidine in DMF and the resin was treated with Fmoc-D-Hyp-OH (409 mg, 1.0 mmol), HOAt in DMF (0.5 M, 2.0 mL, 1.0 mmol), DIPCDI (159 muL, 1.0 mmol) for 6 hr. The resin was washed, and the resin was subjected to a capping treatment with decanoic anhydride (368 muL, 1.0 mmol), DIEA (174 muL, 1.0 mmol) in DMF for 30 min. Fmoc group was removed with 20% piperidine in DMF and the resin was treated with Fmoc-Ser(But)-OH (383 mg, 1.0 mmol), HOAt in DMF (0.5 M, 2.0 mL, 1.0 mmol), DIPCDI (159 muL, 1.0 mmol) for 2 hr. The resin was washed, and the resin was subjected to a capping treatment with decanoic anhydride (368 muL, 1.0 mmol), DIEA (174 muL, 1.0 mmol) in DMF for 30 min. Fmoc group was removed with 20% piperidine in DMF and the resin was washed successively with DMF, MeOH and dried under reduced pressure. The total amount of the obtained H-Ser(But)-D-Hyp(But)-Iva-Pya(4)-Cha-Leu(Me)-Asn(Trt)-Lys(Boc)-Aib-Thr(But)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Cha-Sieber Amide resin was swollen again with DMF, and treated with CDI (203 mg, 1.25 mmol), DIEA (218 muL, 1.25 mmol) in DMF for 2 hr. The resin was washed with DMF, and treated with 4-aminobutyric acid (206 mg, 2.0 mmol), DIEA (523 muL, 3.0 mmol) in DMF for 24 hr. The resin was washed successively with DMF, MeOH, dried under reduced pressure and the obtained resin (1.13 g) was suspended in TFA: thioanisole: m-cresol: H2O: EDT: TIS (80:5:5:5:2.5:2.5) (10 mL) and the suspension was stirred at room temperature for 7 hr. The reaction solution was added to stirring diethyl ether under ice-cooling while removing the resin by a filter to obtain precipitation, and an operation to remove the supernatant after centrifugation was repeated 3 times. The residue was extracted with 50% aqueous acetic acid solution, passed through a disc filter with a pore diameter 0.45 mum to remove fine granules, and purified in 4 portions by HPLC. The HPLC conditions were YMC Pack R&D-ODS-5-B S-5 120A column (30x250 mm), Solution A: 0.1% TFA-water, Solution B: 0.1% TFA-containing acetonitrile, flow rate 15 mL/min, A/B: 74.5/25.5-64.5/35.5 or 74/26-64/36 linear concentration gradient elution (60 min). Each fraction was analyzed by HPLC to specify fractions containing only the object product. The fractions with low purity obtained by the first purification were concentrated, and subjected to HPLC purification again under the same conditions. All the fractions containing only the object product were combined and freeze-dried to give 345 mg of a white powder. (0348) The obtained purified sample (345 mg) was dissolved in CHCN/HO (10/20 mL), and AG 1x8 AcO resin (3.03 mL, 3.64 mmol equivalents) was added. The solution was stood for 1 hr while occasionally stirring with hand, passed through a disc filter with a pore diameter 0.45 mum to remove fine granules, concentrated in an evaporator to reduce the liquid amount to about 5 mL, and the solution was freeze-dried to give 222 mg of a white powder. MALDI-TOF-MS analysis, (M+H)1896.3 (Calculated 1896.1) HPLC elution time: 10.4 min elution condition (HPLC mode g): column: SHISEIDO CAPCELL PAK C18 MGII(4.6×100 mm) eluent: using Solution A: 0.1% TFA-water, Solution B: 0.1% TFA-containing acetonitrile, A/B: 80/.20 - 30/70 linear concentration gradient elution (25 min) flow rate: 1.0 mL/min |
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