Bio-solvents change regioselectivity in the synthesis of disaccharides using Biolacta b-galactosidase María P  erez-S  anchez, Manuel Sandoval, María J. Hern  aiz * Department of Pharmaceutical and Organic Chemistry, Faculty of Pharmacy, Complutense University of Madrid, Campus de Moncloa, 28040 Madrid, Spain article info Article history: Received 3 November 2011 Received in revised form 29 December 2011 Accepted 11 January 2012 Available online 16 January 2012 Keywords: Biocatalysis Solvent effects Bio-solvents Transglycosylation b-Galactosidase abstract Bio-solvents are good alternative solvents that avoid the use of classical organic solvents when per- forming enzymatic reactions. A noticeably change in regioselectivity was observed in the synthetic be- haviour of Biolacta b-galactosidase using bio-solvents derived from dimethylamide and glycerol as co- solvents. Under these conditions, the enzyme changes its well known tendency to produce b-(1/4) to b-(1/6) disaccharides. An evaluation of the bio-solvent concentration and the effects of the non proteic additives in commercially available Biolacta b-galactosidase was undertaken in order to optimize the reaction conditions to improve the yield of the b-(1/6) product. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Oligosaccharides are involved in a wide range of biological processes including bacterial and viral infection, cancer metastasis, the blood-clotting cascade and many other crucial intercellular recognition events. 1e4 As the understanding of these biological functions increases, the need for practical synthetic procedures of oligosaccharides in large quantities has become a major issue. Organic chemical methods for obtaining them have been devel- oped, 5e7 but they involve several elaborate protection and depro- tection procedures. Glycosyl hydrolases (glycosidases) can be used to synthesize oligosaccharides in a kinetically controlled reaction, where a glycosyl donor is used to transfer its glycosyl residue to a sugar acceptor present in the reaction medium. In spite of the increased amount of work carried out with glycosyl hydrolases, their main drawback is the lack of regioselectivity, which limits their use for synthetic purposes. In an important contribution to glycosidase-catalyzed oligosaccharide synthesis, Usui et al. 8 re- ported for the first time the use of Biolacta b-galactosidase to synthesize some b-D-(1/4) galactosyl disaccharides bearing a GlcNAc residue at the reducing end, although some b-D-(1/6) linkages were also formed. This was one of the first examples of a preparative scale b-D-galactosyltransfer where the linkage occurs preferentially at the O-4 position using a galactosyl hydrolase. It is also well documented that many enzymes can maintain their activity in a variety of organic solvents. This improves the solubility of the reagents, decreases unfavourable side reactions and shifts the equilibrium towards the product side, therefore minimising hydrolysis. 9 On the other hand, the presence of organic solvents in enzymatic reactions can alter the activity and specificity of the enzyme. There are numerous examples in the literature where enzyme specificity and enantioselectivity is closely related to the solvent’s physical properties, such as, density, dipole mo- ment and hydrophobicity. 10e13 Among the different families of the so- called bio-solvents, organic solvents derived from renewable sources are receiving an increasing attention. 14,15 For instance, glycosidases can catalyze transglycosylation reactions in aqueous- organic solvents mixtures. 16e18 However, the use of glycosidases in bio-solvents is still limited and a few studies about their use in transglycosylation reactions are available. 19e21 Recently, we have shown that in the presence of co-solvents derived from glycerol in transglycosylation reactions catalyzed by Biolacta b-galactosidase the regioselectivity is shifted towards the obtention of the b(1/6) regioisomer. 21 In this work, we report the influence of a series of bio-solvents derived from biomass on the activity and selectivity of the enzymatic synthesis of disaccharides catalyzed by Biolacta b- galactosidases. 2. Results and discussions 2.1. Transglycosylation reactions in bio-solvents The transglycosylation reaction of N-acetylglucosamine (GlcNAc) with p-nitrophenyl-b-D-galactopyranoside (pNP-b-Gal), * Corresponding author. E-mail address: mjhernai@farm.ucm.es (M.J. Hern aiz). Contents lists available at SciVerse ScienceDirect Tetrahedron journal homepage: www.elsevier.com/locate/tet 0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2012.01.020 Tetrahedron 68 (2012) 2141e2145