| 10.2% |
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The titled peptide was synthesized on a model 430A peptide synthesizer (Applied Rio systems, Foster City, Calif., U.S.A.) which was modified to do accelerated Hoc-chemistry solid phase peptide synthesis (Schnolzer, M. et al., mt. J Peptide Protein Res., (1992), 40:180). 4-Methylbenzhydry- lamine (MHHA) resin (Peninsula, Helmont, Calif., U.S.A.), with a substitution of0.91 mmol/g was used. Hoc amino acids (Midwest Hio-Tech, Fishers, Ind., U.S.A.; Novabiochem., San Diego, Calif., U.S.A.) were used with the following side chain protection: Hoc-Ala-OH, Hoc-Arg(Tos)-OH, Hoc-His (DNP)-OH, Hoc-Val-OH, Hoc-Ecu-OH, Hoc-Gly-OH, HocGln-OH, Hoc-Eys(2C1Z)?--OH, Hoc-Ser(Hzl)-OH, Hoc-PheOH, Hoc-Glu(OcHex)-OH and Hoc-Pro-OH. Fmoc-Glu (OtHu)-OH (Novabiochem, San Diego, Calif., U.S.A.) was used for the residue at the 3rd position in the sequence. The synthesis was carried out on a 0.25 mmol scale. The Hoc groups were removed by two treatments with 100percent TFA each lasting one minute. Hoc amino acids (2.5 mmol) were preactivated with HH11J (2.0 mmol) and DIEA (1.0 mE) in 4 mE of DMF and were coupled without prior neutralization of the peptide-resin TFA salt. Coupling times were 5 minutes. At the end of the assembly of the first 25 residues on theAHI 430A® peptide synthesizer and before the coupling of Fmoc-Glu (OtHu)-OH, the protected peptide-resin was transferred into a reaction vessel on a shaker for manual synthesis. After removing the Hoc protecting group with two, one-minute treatments with 100percent TFA and a washing with DMF, the resin was mixed with Fmoc-Glu(OtHu)-OH (2.5 mmol) which was preactivated with HHTU (2.0 mmol), HOHt (2.0 mmol) and DIEA (1.0 mE) in 4 mE of DMF. The mixture was shaken for 2 hours. This coupling step was repeated. After washing with DMF, the resin was treated with a TFA solution containing 5percent water and 5percent TIS for 2 hours to remove the tHu protecting group in the side chain of the Glu residue. The resin was neutralized with 10percent DIEA in DMF and washed with DMF and DCM. The resin was then treated twice with hexylamine (2.0 mmol), DIC (2.0 mmol), HOHt (2.0 mmol) in 5 ml of DCM for two hours per treatment. The resin was washed with DMF and treated with 25percent piperidine in DMF for 30 minutes to remove the Fmoc protecting group. Afier washing with DMF and DCM, the resin was transferred into the reaction vessel on the AHI 430A peptide synthesizer for the assembly of the rest two residues. At the end of the assembly of the whole peptide chain, the resin was treated with a solution of 20percent mercaptoethanol/10percent DIEA in DMF for 2x30 mm to remove the DNP group on the His side chain. The N-terminal Hoc group was then removed by two treatments of 100percent TFA for 2 minutes. The peptide-resin was washed with DMF and DCM and dried under reduced pressure. The final cleavage was done by stirring the peptide-resin in 10 mE of HF containing 1 mE of anisole and dithiothreitol (50 mg) at 0° C. for 75 minutes. HF was removed by a flow of nitrogen. The residue was washed with ether (6x 10 mE) and extracted with 4N HOAc (6x10 mE). This crude product was purified on a reverse-phase preparative HPEC using a colunm (4x43 cm) of C18 DYNAMAX-100A°® (Varian, Walnut Creek, Calif., U.S.A.). The column was eluted with a linear gradient from 75percentAand25percent B to 55percentAand45percent B at flow rate of 10 mE/mm in an hour where A was 0.1percent TFA in water and B was 0.1percent TFA in acetonitrile. Fractions were collected and checked on an analytical HPEC. Those containing pure product were combined and lyophilized to dryness. 31.8 mg of a white solid was obtained. Purity was 89percent based on analytical HPEC analysis. Electro-spray ionization mass spectrometry (ESI MS) analysis gave the molecular weight at 3368.4 (in agreement with the calculated molecular weight of 3368.9). |
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