4-Methylmorpholine → N,N'-Dicyclohexylurea

Participants

Reaction Steps

DCys(O3Bu4N)-Pro-DThg-Leu-resin prepared in the same manner described in Example 5, was acylated with Boc-DTrp(CHO) by the standard protocol described in Example 5. The resulting resin was washed successively with DMF, methanol, and dichloromethane and dried in vacuo to give Boc-DTrp(CHO)-DCys(O3Bu4N)-Pro-nThg-Leu-resin. The resin was added to TFA/water(95/5, 20 ml) and the mixture was stirred at room temperature for 1.5 h and filtered. The collected resin was washed with TFA and the combined filtrate and washings were concentrated under reduced pressure. The residue was precipitated with ether to give DTrp(CHO)-DCys(O3Bu4N)-Pro-DThg-Leu. A solution of the pentapeptide(28 mg) in DMF(1.4 ml) was added dropwise to a solution of DCC(9 mg), HOBT H2O(7 mg) and N-methylmorpholine(1.6 μl) in DMF(1.4 ml) under ice cooling over a period of 1 h. The reaction mixture was stirred under ice cooling for 1 h and at room temperature for 17 h. Acetic acid(10 μl) was added to the reaction mixture. The resulting mixture was stirred and concentrated under reduced pressure. The residue was triturated with water to give colorless dicyclohexyl urea which was collected by filtration and thoroughly washed with water. The combined filtrate and washings were lyophilized to give a colorless solid which was purified by thin layer chromatography on silica gel (Analtichem International, Empore sheet) with butanol/acetic acid/water(8/1/1) for development to give an ammonium salt of the title compound. A water solution of the ammonium salt was passed through a column of ion exchange resin(AMBERLITE IR-120B, H+ -form) and lyophilized. The resulting solid(12.93 mg) was dissolved in water(0.6 ml) and the equimolar amount of NaHCO3 was added. The solution was directly purified by reverse-phase short column chromatography(Waters, SEP-PAK C18 cartridge) with water and methanol for elution. The combined methanolic layers were concentrated under reduced pressure to give the title compound(10.74 mg) as a colorless powder.

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