Regio-selective deprotection of peracetylated sugars via lipase hydrolysis Gloria Fernandez-Lorente, a Jose M. Palomo, b Jany Cocca, b Cesar Mateo, b Paola Moro, a Marco Terreni, a Roberto Fernandez-Lafuente b, * and Jose M. Guisan b, * a Pharmaceutical Biocatalysis Laboratories, Department of Pharmaceutical Chemistry, University of Pavia, Via Taramelli 12, Milan, Italy b Department of Biocatalysis, Institute of Catalysis, CSIC, Campus Universidad Auto ´noma, 28049 Madrid, Spain Received 31 March 2003; revised 19 May 2003; accepted 2 June 2003 Abstract—Purified lipases (via interfacial activation on hydrophobic supports) from different microbial extracts have been evaluated in the regio-selective hydrolysis of peracetylated sugars (peracetylated glucose, ribose and sucrose). Among the enzymes tested, lipases from Candida rugosa (CRL) and from Pseudomonas fluorescens (PFL) exhibited the best properties in these reactions.Then, we have prepared two different immobilized lipase preparations obtained by interfacial activation on hydrophobic supports or by covalent attachment on glutaraldehyde agarose. Interfacially activated lipases exhibited a higher activity than covalently attached enzymes (even by a 100-fold factor), giving the higher yields of mono deacetylated sugars (in some instances by more than a threefold factor) in short reaction times. In the hydrolysis of 1,2,3,5-tetra-O-acetyl-b-D-ribofuranose catalyzed by PFL adsorbed on octyl agarosa, hydrolyzed mainly the 3 position (30% of yield) while the CRL gave the hydrolysis only in position 5 (about 50% of yield). Depending on the enzyme immobilized preparation, we have been able also to obtain selective hydrolysis of 1,2,3,4,6-penta-O-acetyl-a/b-D-glucopyranose obtaining a free hydroxyl group in position 1, 4 or 6. Moreover, selective hydrolysis in the 4 0 position of peracetylated sucrose was achieved when the hydrolysis is performed with CRL immobilized on octyl-agarose (yield was 77%). q 2003 Elsevier Ltd. All rights reserved. 1. Introduction Pure regioisomers of O-acetyl-pyranosides presenting only one free hydroxyl group (AP) may be used as key intermediates for the synthesis of a large number of glycoderivatives (oligosacharides, sugar esters, glyco- peptides, etc.). 1,2 AP intermediates can be readily and selectively modified at this free hydroxyl group and they are soluble in most organic solvents. Moreover, the protected final products can be easily deacetylated by very mild chemical or enzymatic processes. However, the preparation of APs with only one free hydroxyl group by classical chemical synthesis requires multi-step reactions that may cause environmental problems for large-scale production, due to the use of organic solvents and toxic reagents. 3–9 These problems make the use of enzymatic catalysts such as lipases or esterases an attractive alternative route for AP preparation, 10,11 although in many instances, the reactions are very slow or proceed with poor selectivity and yield. In addition, these reactions often afford complex mixtures of tetra-, tri-, di-, and monoacetates, along with free mono- saccharide. 12 – 14 In the design of this kind of reactions, we have considered two different aspects: First, the enzyme must mainly recognize the peracetylated sugar as substrate, permitting the accumulation of mono deacetylated sugar. This is related to the specificity of the enzyme-immobilized preparation by the different available substrates. Second, it is convenient that the enzyme produces only one of the different possible regioisomers. This is related to the regioselectivity of the enzyme. When using lipases as biocatalyst of organic reactions, it is necessary to bear in mind that their mechanism of catalysis implies dramatic conformational changes of the enzyme molecule between a ‘closed’ and an ‘open’ form. 15 – 21 This equilibrium between two very disimilar molecular forms also exists in most immobilized lipases. Our hypothesis is that by using immobilization techniques that involve different areas of the enzyme, that confer different rigidity to the enzyme structure, it is possible to restringe the mobility of the enzyme and, in this way, altering their catalytic properties (Fig. 1). Thus, the same lipase molecule can present very different catalytic proper- ties after immobilization on different supports. This has shown in resolution of racemic mixtures with lipases, 22 – 25 to permit a strong modulation of the lipase properties. In this 0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0040-4020(03)00876-7 Tetrahedron 59 (2003) 5705–5711 * Corresponding authors. Tel.: þ34-915854809; fax: þ34-915854760; e-mail: rfl@icp.csic.es; jmguisan@icp.csic.es Keywords: modulation of lipase properties; selective deprotection of peracetylated sugars; lipase immobilization.