p-Toluenesulfonyl-L-isoleucine [2J (2.9 g, 10 mmol) is dissolved in dry ether (15 ml), phosphorus pentachloride [3,4J (2.2 g, 10.6 mmol) is added and the suspension is shaken until almost all the pels dissolves. The solution is decanted from the small amount of undissolved material and the ether evaporated in vacuo with a water aspirator. The phosphorus oxychloride remaining in the residue is removed by continuing the evaporation on an oil pump for about one hour. The acid chloride (about 3 g) is a colorless oil [5]. It is dissolved in dry ether and added, in several portions, to a suspension of glycine ethyl ester hydrochloride (2.5 g, 18 mmol) in dry ether (50 ml) containing triethylamine (3.5 ml = 2.52 g, 25 mmol). The mixture is allowed to stand at room temperature with occasional shaking for 12 hours. The precipitate is collected on a filter, washed with ether and then triturated and washed with water to extract triethylammonium chloride. The dry product (3.34 g, 90%) [6J melts at 159- 160 °C. Recrystallization of a sample from ethanol raises the m.p. by 1°C.
Tosyl-L-isoleucine (2.9 g, 10 mmol) is converted to the acid chloride as described above. A solution of the acid chloride in dry dioxane (15 ml) is added, in small portions, over a period of about one hour, to a vigorously stirred (or shaken) ice cold suspension of L-glutaminyl-L-asparagine [7J (2.6 g, 10 mmol) and magnesium oxide (0.84 g, 21 mmol) in water (25 ml). The pH of the reaction mixture should be kept above 8 during the addition of the acid chloride [8]. The magnesium salt of the tosyltripeptide separates and the reaction mixture gradually turns into a semisolid mass. More water (12 ml) is added and the Mg salt is collected on a filter and washed with water. It is resuspended, without drying, in distilled water (300 ml) and the suspension acidified with 5% HCI to pH 3. The protected tripeptide acid is thoroughly washed on a filter with water and dried. The first crop weighs about 2.2 g. A second crop is secured by the acidification of the filtrates and washings from the Mg salt. The total amount of the crude product (m.p. 219-220 °C, dec.) is about 2.9 g (55%). It is dissolved in a 0.5 N solution of KHC03 in water and the solution is acidified with N HC!. The purified peptide derivative forms long needles, which melt with decomposition at 223°C; [o:Jl/ - 31 0 (c 2, 0.5 N KHC0 3 ). On elemental analysis satisfactory values are obtained for C, H and N.
1. Katsoyannis PG, du Vigneaud V (1954) J Amer Chern Soc 76: 3114 2. Preparation of this tosylamino acid is described in this volume (p. 9). 3. The same acid chloride can be prepared by treating the tosylamino acid with thionyl chloride
rather than with phosphorus pentachloride. Thionyl chloride, however, is not suitable for the conversion of benzyloxycarbonylamino acids into their chlorides because already at room temperature and even more on heating benzyloxycarbonylamino acid chlorides cyclize to Ncarboxyanhydrides with the elimination of benzyl chloride. Treatment of Z-amino acids with PCl s should be carried out at 0 DC.
4. Phosphorus pentachloride is a dangerous material. Its contact with the skin and also with water should be carefully avoided.
5. The acid chloride prepared from tosyl-L-isoleucine with thionyl chloride was obtained in crystalline form by extraction of the distillation residue with boiling hexane. On cooling rectangular tables separated melting at 54-56°C. The crystalline material gave satisfactory Nand CI values on elemental analysis (Bodanszky M, du Vigneaud V (1959)) J Amer Chern Soc 81: 2504
6. The yield can be further increased by extraction of the combined filtrate and washings with water, 0.5 N HCI, 0.5 N KHC0 3 and water and evaporation (after drying over anhydrous Na2S04 ) to dryness. This second crop (about 0.25 g) after recrystallization from ethanol has the same m.p. as the first crop.
7. Swan JM, du Vigneaud V (1954) J Amer Chern Soc 76: 3110 8. On higher pH, however, most tosylamino acid chlorides decompose to p-toluenesulfon
amide, carbon monoxide, an aldehyde and an (alkali) chloride. Therefore, it is advantageous to use MgO as acid binding agent, because its poor solubility in water prevents highly alkaline conditions.
