Reaktion #1283913
ord-81c8b3062ad94ddb9a4d5dc9eba7dacb
Reaktionsgleichung
Reagenzien
Reaktionsbedingungen
Aufarbeitung
- 1Sonstige(3.5 ml/min) to remove excess base
- 2Waschen(3.5 ml/min) to wash excess reagents out
Vorschrift
Fmoc-Leu-resin(0.093 mmol/g, 1.0 g) which was prepared from ULTROSYN B(Pharmacia LKB Biotechnology) and Fmoc-Leu by a symmetrical acid anhydride method, was previously swelled in DMF, and packed in a reaction column of a BIOLYNX 4175 peptide synthesizer(Pharmacia LKB Biotechnology) and a solid phase peptide synthesis was performed manually by the standard protocol; 20% piperidine/DMF was pumped through the reaction column for 10 min(flow rate: 3.5 ml/min) to deprotect Fmoc group. Then DMF was pumped for 10 min(3.5 ml/min) to remove excess base. Each 2.5 equivalents of Fmoc-DVal-OPfp and HOBT.H2O was dissolved in DMF(1 ml). The solution was drawn into the reaction system and recirculated for 1 h(3.5 ml/min) to achieve acylation of the amino groups present on the resin. DMF was pumped for 5 min (3.5 ml/min) to wash excess reagents out, to give Fmoc-DVal-Leu-resin. In a similar manner, Fmoc-Lys(Boc)-OPfp, Fmoc-DAsp(OtBu) and Fmoc-DTrp-OPfp were successively coupled with the corresponding resin bound peptide. In the case of the coupling reaction of DAsp, were used each 5 equivalents of Fmoc-DAsp(OtBu) and HOBT.H2O, and 4 equivalents of DIPC. Completion of each acylation reaction was judged by the Kaiser test [Anal. Biochem., 34, 595(1970)]. If the acylation reaction was imcomplete, additional recirculation was performed until the Kaiser test turned negative. Finally the Fmoc group of the resin bound pentapeptide was deprotected in a reaction column by pumping 20% piperidine/DMF for 10 min(3.5 ml/min). The resin was unpacked from a reaction column, washed on a glass filter successively with DMF, tert-amyl alcohol, acetic acid, tert-amyl alcohol, DMF and ether, and dried in vacuo to give DTrp-DAsp(OtBu)-Lys(Boc)-DVal-Leu-resin (1.06 g). 10% Hydradine monohydrate/1,4-dioxane/methanol(v/v, 9/1, 3.0 ml) was added to the resin and the mixture was allowed to stand at room temperature for 2 h with occasional shaking. The resin was filtered out and washed several times with each small amount of 1,4-dioxane/methanol(9/1). The combined filtrate and washings were neutralized with dry ice and concentrated in vacuo. Water(10 ml) was added to the residue to precipitate a solid, which was collected by filtration and dried in vacuo to give DTrp-DAsp(OtBu)-Lys(Boc) -DVal-Leu-NHNH,(65.3 mg) as a colorless powder.