Protease-catalyzed synthesis of disaccharide amino acid esters in organic media Oh-Jin Park, Gyu-Jong Jeon, Ji-Won Yang* Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusung-dong, Yusung-gu, Taejon, 305-701 South Korea Received 20 July 1998; received in revised form 16 November 1998; accepted 20 November 1998 Abstract An enzymatic synthesis of sugar amino acid esters has been developed in pyridine by using a commercial protease, Optimase M-440, from Bacillus licheniformis. Optimase M-440 showed broad substrate specificity toward amino acid esters as acyl donors and disaccharides as nucleophiles. Analysis of acylation sites indicated sucrose was acylated only at three primary hydroxyls. Trehalose, consisting of two glucose units, was acylated at two primary (6-OH and 6'-OH) and one secondary (3-OH). With sucrose and trehalose, diesters along with monoesters were enzymatically synthesized. In both cases, no triester was formed. Molecular size of nucleophiles, glucose and sucrose, have effects on the extent of acylation with D-amino acids. N-blocked phenylalanine, leucine, and methionine exhibited higher activity toward sucrose than lysine, aspartic acid, and tyrosine. Various leaving groups of tBoc-L-phenylalanine were used and cyanomethyl ester gave the highest rate of reaction. Optimization of initial water activity of Optimase M-440 (A w = 0.2– 0.3) was necessary to maintain catalytic activity and to prevent undesirable hydrolysis of activated esters. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Optimase M-440; Amino acid esters; Acylation; Subtilisin; Organic solvents 1. Introduction Hydrolases (lipases, esterases, and proteases) are well known to catalyze reverse reactions (esterification, transes- terification, and lactonization) in organic media, which are impossible in the aqueous solution [1]. The properties have been utilized to synthesize pharmaceutical intermediates [2,3] and food ingredients [4]. Drawbacks of nonaqueous enzymology, such as low activity of enzymes in organic solvents, could be overcome by various strategies [5]. Many examples of using lipases and proteases in organic synthesis have been reported in the literature. Regioselective synthesis and modification of carbohy- drate conjugates is a problematical and arduous task due to the presence of multiple hydroxyl groups [6]. Despite the attractive properties of sugar esters, their industrial uses remain limited as they are difficult to synthesize chemically. Selective acylation of a given primary or secondary hy- droxyl, however, requires even more significant synthetic control via blocking and deblocking methodologies. Nowhere is regioselectivity of enzymes potentially more important than in the synthesis of sugar esters and their derivatives [7,8]. In addition to its use as a sweetener, sucrose is a renewable resource of great commercial impor- tance [9]. Dordick’s group reported regioselective acylation of sucrose with enzymes in the synthesis of biodegradable polymers containing sucrose [10 –12]. Although various studies concerning the enzymatic synthesis of vinyl sugar esters and sucrose fatty acid esters were published [13–17], there are only a few reports on the synthesis of sugar amino acid esters especially with disaccharides to the best of our knowledge [18,19]. Sugars and amino acids, along with fatty acids, are major basic components of life and are cheap renewable raw materials. Various functionalities of amino acids could be utilized to modify carbohydrates and to introduce new functional groups, which could be used as moieties for chemoenzymatic biodegradable polymer syn- thesis [20,21]. In a related work, Fernandez et al. were able to synthe- size peptides with unnatural amino acids by using various * Corresponding author. Tel.: +82-42-869-3924; fax: +82-42-869- 3910. E-mail address: jwyang@kaist.ac.kr (J.-W. Yang) Enzyme and Microbial Technology 25 (1999) 455– 462 0141-0229/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S0141-0229(99)00040-X