TETRAHEDRON LETTERS Tetrahedron Letters 42 (2001) 3395–3397 Pergamon High isolated yields in thermolysin-catalysed synthesis of Z -L-aspartyl-L-phenylalanine methyl ester in toluene at controlled water activity Luigi De Martin, Cynthia Ebert, Lucia Gardossi* and Paolo Linda Dipartimento di Scienze Farmaceutiche, Universita ` degli Studi, Piazzale Europa 1, 34127 Trieste, Italy Received 16 January 2001; revised 8 March 2001; accepted 15 March 2001 Abstract—Z -L-Aspartyl-L-phenylalanine methyl and ethyl esters were synthesised enzymatically in toluene by means of the zinc protease thermolysin adsorbed onto Celite R-640 ® , which is a porous support able to control the hydration of the protein. The conversion of the two derivatised amino acids into the desired products was complete, leading to >90% isolated yields. Moreover, working with equimolar concentrations of the reactants no purification steps were required. Thermolysin adsorbed onto Celite R-640 ® is shown to be a practical tool to synthesise biologically active peptides in organic media. © 2001 Elsevier Science Ltd. All rights reserved. The benefits of using enzymes in organic synthesis and, in particular, for the preparation of optically pure drugs are well recognised. 1 In the last few years much attention has been paid to protease-catalysed peptide synthesis by using the reverse of the hydrolytic reaction, namely thermodynamically controlled synthesis. 1 The enzymatic approach offers some advantages over chem- ical methods since enzymatic reactions are often per- formed under mild conditions and without protection of the side chains of the amino acid derivatives. 2 Aspar- tame (-L-Asp-L-Phe-OMe) is one of the best known peptides obtained through enzymatic synthesis. This dipeptide is an artificial sweetener which is 200 times sweeter than sucrose and used throughout most of the world. 3 The precursor of aspartame (X-L-Asp-L-Phe-OMe, where X is an N -protecting group) has been synthesised by several research groups starting from N -protected L-aspartic acid (X-L-Asp) and L-phenylalanine methyl ester (L-PheOMe) using the zinc protease thermolysin. 4– 7 The first approach reported in the literature included the thermodynamically controlled synthesis of the pre- cursor of aspartame in water. 5,8 Since in water the equilibrium constant is shifted towards the hydrolysis products (K eq =1.5 M -1 ), unsatisfactory yields were obtained in the dilute aqueous medium. 3 However, over the last few years Halling and co-workers have obtained very high yields in the thermolysin-catalysed synthesis of Z -L-Gln-L-Leu-NH 2 and Z -L-Asp-L-Phe- OMe using aqueous suspensions of the substrates. 9–11 An alternative approach could be represented by the partial substitution of the aqueous medium with an organic solvent which can shift the thermodynamic equilibrium towards synthesis. 12 Therefore, reactions have been performed in either water/organic solvent mixtures 13,14 or in two phase systems. 6,15–17 In principle, the use of pure organic solvents in enzy- matic synthesis should be beneficial for the equilibrium shift, for the recovery of the product and the recycling of the catalyst. 18 Recently we reported that thermolysin is very active in hydrophobic organic solvents when the hydration of the protein is controlled by adsorbing the enzyme onto Celite R-640 ® rods. 19 In the present paper we present the thermolysin- catalysed synthesis of Z -L-aspartyl-L-phenylalanine methyl and ethyl esters in a hydrophobic solvent at controlled water activity (a w ), using equimolar concen- trations of the substrates. Keywords : aspartame; peptide synthesis; thermolysin; organic solvent; thermodynamically controlled synthesis; water activity. * Corresponding author. Fax: +39-040-52572; e-mail: linda@ univ.trieste.it 0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII:S0040-4039(01)00452-X