Process Biochemistry 46 (2011) 1698–1702 Contents lists available at ScienceDirect Process Biochemistry jo u rn al hom epa ge: www .elsevier.com/locate/procbio Short communication A highly efficient diastereoselective synthesis of -isosalicin by maltase from Saccharomyces cerevisiae Duˇ san Veliˇ ckovi ´ c a , Aleksandra Dimitrijevi ´ c a , Filip Bihelovi ´ c a , Dejan Bezbradica b , Ratko Jankov a , Nenad Milosavi ´ c a,∗ a Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000 Belgrade, Serbia b Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia a r t i c l e i n f o Article history: Received 15 February 2011 Received in revised form 10 May 2011 Accepted 11 May 2011 Keywords: -Isosalicin Glucosidase Salicyl alcohol Transglucosylation Bakers’ yeast a b s t r a c t In this report, -isosalicin, a potent anticoagulant and skin whitening agent, was synthesized by a highly efficient chemoselective and diastereoselective reaction, catalyzed by maltase from bakers’ yeast (Sac- charomyces cerevisiae). The highest yield of this one-step transglucosylation reaction was achieved with 50 mM of salicyl alcohol as a glucose acceptor. The key reaction factors were optimized using response surface methodology (RSM) with an enzyme concentration of 10 U/mL. The optimum temperature of the reaction was determined as 36.5 ◦ C, the optimal maltose concentration was 40% (w/v), the optimal pH was 6.5, and the optimal reaction time was 16 h. Under these conditions 75% of -isosalicin was obtained, with a yield of 10 g/L, and no by product formation was observed. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Development of stereoselective methods for the synthesis of glycosidic linkages presents a considerable challenge to synthetic chemists [1–3]. Chemical syntheses of glycosidic moieties are mainly based on time-consuming protection and deprotection strategies, activation or metal catalysis, but are often accompanied by the formation of unwanted diastereomers and low yields [4,5]. However, these difficulties can be overcome by the application of enzymatic syntheses [6]. Transglycosylation reactions are well known and widely used methods for glucoside syntheses. Glycosidases, responsible for catalytic hydrolysis of the glycosidic linkage, are increasingly being used in carbohydrate synthesis [7]. -Glucosidase (maltase) is one of the most abundant glucosyl hydrolases present in baker’s yeast and has been used for the synthesis of various glucosides [7–9]. Glucosides of o-hydroxybenzyl alcohol (salicyl alcohol) con- tinue attracting increasing attention due to a variety of biological activities such as anti-inflammatory and analgesic [10], anti- cancer [11], antipyretic [12] and allergy preventive activity [13]. -Isosalicin is particularly important due to its effect on blood coag- ulation, since -isosalicin is an even more efficient anticoagulant ∗ Corresponding author. Tel.: +381 11 333 6656; fax: +381 11 2184 330. E-mail address: nenadmil@chem.bg.ac.rs (N. Milosavi ´ c). than heparin [14]. Furthermore, -isosalicin is a potential skin whitening agent, due to its tyrosinase inhibitory activity [15]. Transglucosylation reactions require a narrow range of condi- tions (temperature, pH, concentration of reactants and duration of reaction) for maximum utilization of the biocatalytic activity of an enzyme. Response surface methodology (RSM) has been widely employed for the optimization of enzymatic processes as well as other catalytic studies and it is also useful in simul- taneous analysis of the effects of several independent variables [6]. In this study, the synthesis of -isosalicin [2-hydroxybenzyl- -d-glucopyranoside], based on chemo- and diastereoselective glucosylation of 2-hydroxybenzyl alcohol (salicyl alcohol) with - 1,4-glucosidase from Saccharomyces cerevisiae was investigated. The optimal condition for the synthesis of -isosalicin by maltase from baker’s yeast, with maltose as the glucose donor and salicyl alcohol as the glucose acceptor, was determined. During optimiza- tion of conditions, the reaction was monitored by both, TLC and HPLC. The product was isolated, and its structure was confirmed by spectroscopic methods ( 1 H and 13 C NMR, HRMS and optical rotation). 2. Materials and methods 2.1. Chemicals and enzyme All commercially available reagents and solvents were used as obtained without further purification. -1,4-Glucosidase (3.2.1.20) was isolated from baker’s yeast by a previously published procedure [16], and it showed a molecular weight of 63 kDa on SDS PAGE. Its specific activity against 4-nitrophenyl--d-glucopyranoside 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.05.007