3 Symmetrical Anhydrides

A solution of tert-butyloxycarbonyl-L-phenylalanine (5.30 g, 20 mmol) in dichloromethane (200 ml) is placed into a 500 ml round bottom flask provided with a drying tube filled with cotton. The solution is cooled in an ice water bath and stirred vigorously with a magnetic stirrer. The water soluble carbodiimide [3J N -ethyl-N' -3-dimethylaminopropy1carbodiimide hydrochloride (1.92 g, 10 mmol) is added and stirring is continued at ice bath temperature for 2 hours. The solvent is evaporated in vacuo at a bath temperature of 0 °C and the residue dissolved in a mixture of ethyl acetate (200 ml) and water (100 ml) to which a few pieces of ice have been added. The organic layer is washed twice with ice water (100 ml each time), then with 0.5 N KHC03 and finally with saturated sodium chloride solution, always with pieces of ice added. The solution is dried over MgS04 while cooled in an ice-water bath, filtered and the solvent removed in vacuo at a bath temperature of O°c. The residue is crystalline or crystallizes on storage in the cold (- 5 0q. The anhydride is washed with a 20: 1 mixture of hexane and ether and dried in vacuo. The product, 3.9 g (76%), melts at 95- 97 °C; [IXJ~3 - 32.8° (c 2, CHCI3) [4]. In the i.r. spectrum, in addition to the urethane carbonyl band (1710 cm -1) two carbonyl bands, characteristic for anhydrides, appear at 1750 cm -1 and 1830 cm - t .

1. Chen FMF, Kuroda K, Benoiton NL (1978) Synthesis, 928 2. The preparation of several additional tert-butyloxycarbonyl- and benzyloxycarbonyl-amino

acid anhydrides is described in ref. 1. Symmetrical anhydrides of benzyloxycarbonylamino

tert-Butyloxy­carbonyl-L­phenylalanine Anhydride [1,21

M. Bodanszky et al., The Practice of Peptide Synthesis© Springer-Verlag Berlin Heidelberg 1994

88 Symmetrical Anhydrides

acids were obtained also through the disproportionation of mixed anhydrides. (Wieland T, Kern W, Sehring R (1950) J Liebigs Ann Chern 569: 117; Wieland T, Flor F, Birr C, ibid. 1973: 1595

3. The expected formation of symmetrical anhydrides in the reaction of carbodiimides with protected amino acids (Khorana G (1955) Chern Ind 1087) was soon found to be useful in practical synthesis (Muramatsu I, Hagitani A (1959) Nippon Kagaku Zasshi 80: 1497; (1961) Chern Abstr 55: 6394)

4. Symmetrical anhydrides of benzyloxycarbonylamino acids could be obtained, with the help of dicycIohexyIcarbodiimide, in crystalline form (Schiissler H, Zahn H (1962) Chern Ber 95: 1076). Benzyloxycarbonyl-glycine (2.1 g, 10 mmol) in acetonitrile (60 ml) was treated with a solution of dicycIohexyIcarbodiimide (1.0 g, ca 5 mmol) dissolved in acetonitrile (10 ml). After 17 hours the precipitated urea derivative was removed by filtration and the solvent by evaporation in vacuo. The residue was crystallized from dry benzene to afford the anhydride of benzyloxycarbonylglycine in 75% yield, with m.p. 108-114 dc. The method cited in ref. 1 and described in detail above produced this anhydride with higher m.p. (118-119 0c) in 80% yield. Similarly higher yield and higher m.p. was noted when a water soluble carbodiimide rather than dicycIohexyIcarbodiimide was used for the preparation of benzyloxycarbonyl-L­valine anhydride (cf. ref. 1). Symmetrical anhydrides of N'-9-fluorenylmethyloxycarbonylamino acids (Fmoc-amino

acids) were prepared (Heimer EP, Chang CD, Lambros T, Meienhofer J (1981) Int J Peptide Protein Res 18: 237) through the reaction of the protected amino acids with a water soluble carbodiimide. A solution of the Fmoc-amino acid (20 mmol) in ethyl acetate (250 ml) is cooled in an ice-water bath while N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydro­chloride (2.11 g, 11 mmol) is added with stirring. Stirring is continued at O°C for one hour and at room temperature for an additional hour. The mixture is poured into ice water (250 ml), more ethyl acetate is added to dissolve the product and the organic phase is washed with 0.1 N HCI, water, 0.1 N KHC0 3 , water, dried over MgS04 and evaporated to dryness in vacuo. Crystallization from ethyl-acetate-hexane affords the pure symmetrical anhydrides. Yields range from 50 to 86%. The Fmoc-amino acid anhydrides have the characteristic bands (1830 and 1750 cm - 1) in their i.r. spectra.

For extended periods, symmetrical anhydrides of tert-butyloxycarbonyl-amino acids should be stored at or below - 20 ec, under anhydrous conditions (Yamashiro D (1987) Int J Peptide Protein Res 30: 9).