N-Hydroxysuccinimide → 2,5-Dioxopyrrolidin-1-yl N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-leucinate

Participants

Reaction Conditions

Reaction Steps

Fmoc-L-Leu-OH (10 g,28.32 mmol) was dissolved in a mixture of DMF (10 ml)-CH2Cl2 (70 ml) and cooled in an ice-bath. N-Hydroxysuccinimide (3.58 g, 31.14 mmol, 1.1 equiv.) and dicyclohexylcarbodiimiide (6.06 g, 31.14 mmol, 1.1 equiv.) were added and the mixture was stirred at 0° C. for 1 hour and 25° C. for 14 hour. The reaction mixture was cooled in an ice-bath, filtered and the precipitate washed with CH2Cl2 (40 ml). The filtrate was evaporated to dryness and the residue dissolved in a mixture of dioxane (80 ml)-H2O (10 ml) to form a solution of Fmoc-Leu-OSu. A solution of anhydrous L-asparagine (5.606 g, 42.48 mmol, 1.5 equiv.) in 60 ml of Na2CO3 (2.50 g, 42.28 mmol, 1.5 equiv.) was added to the above solution of Fmoc-Leu-OSu. An additional 10 ml of dioxane was added and the reaction mixture was stirred at 25° C. for 2 hours. Ethyl acetate (200 ml) was added to the reaction mixture and the pH adjusted to about 2 by the addition of 5% aqueous HCl with stirring. The EtOAc layer was separated and the aqueous layer was extracted 4 more times with 100 ml each. The combined EtOAc extract was washed with saturated NaCl (100 ml), H2O (3×100 ml), dried over anhydrous Na2SO4, filtered and evporated to dryness. The residue was dissolved in DMF (60 ml) with warming (40-45° C.) and H2O added to the cloud point. The product crystallized after standing overnight. It was filtered, washed with ether and recrystallized from DMF-H2O. Lyophilization from dioxane gave 9.45 g (71.3%) of product. NMR and FAB mass spectroscopy confirmed the molecular formula C25H29N3O6 with an observed mass for (M+H)+, 468.3. The product Fmoc-Leu-Asn-OH was shown to be >95% pure by analytical HPLC. HPLC conditions were: column: Lichrosorb RP-8 (5μ); eluant: (A) 0.1M NaClO4 (pH 2.5)-(B) CH3CN; gradient: 40%-80% (B) in 20 minutes; flow 1 ml/l minute.; and detection: 210 nm.

Provenance & Evidence