Journal of Molecular Catalysis B: Enzymatic 84 (2012) 102–107 Contents lists available at SciVerse ScienceDirect Journal of Molecular Catalysis B: Enzymatic j ourna l ho me pag e: www.elsevier.com/locate/molcatb In situ aldehyde generation for aldol addition reactions catalyzed by d-fructose-6-phosphate aldolase Maria Mifsud ∗ , Anna Szekrényi, Jesús Joglar, Pere Clapés Biotransformation and Bioactive Molecules Group, Instituto de Química Avanzada de Catalu˜ na, Consejo Superior de Investigaciones Científicas (IQAC-CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain a r t i c l e i n f o Article history: Available online 17 February 2012 Keywords: One-pot two-step oxidation/aldol reaction Biocatalytic oxidation Laccase/O2/TEMPO Alcohol oxidase d-Fructose-6-phosphate aldolase a b s t r a c t In situ coupling of aldehyde generation, by a mild alcohol oxidation, with an enzymatic aldol addi- tion reaction, mediated by d-fructose-6-phosphate aldolase (FSA) has been investigated as an approach to improve the performance of the process. Four sustainable oxidation methods compatible with the activity and stability of the enzymatic aldol addition have been assayed. Among them, the laccase/O 2 /2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and alcohol oxidase gave the best results for the N-Cbz-aminoethanol to N-Cbz-glycinal (53%) and furfuryl alcohol to furfural (89%), respectively, followed by the aldol addition with hydroxyacetone catalyzed by FSA A129S mutant. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The aldol addition reaction is one of the most powerful meth- ods of forming carbon–carbon bonds [1]. Biocatalysis by means of aldolases offers a unique stereoselective and green tool to perform this transformation [2,3]. The aldol addition reaction involves an acceptor aldehyde that must be stable throughout the reaction as well as during its prepa- ration, purification and manipulation conditions. Aldehydes are strong electrophiles that can undergo a number of side reactions with other nucleophiles present in the medium, including self- polymerization reactions and oxidation to acids. Moreover, they can deactivate or inhibit the aldolase either by non-selective bind- ing or by irreversible Schiff base formation with the amino group of the catalytic lysine residue in Class I aldolases. To overcome this problem an in situ aldehyde generation by the corresponding alcohol oxidation through a sequential reaction under conditions compatible with the enzymatic aldol addition would be ideal (Fig. 1). The combination of both concepts, biocatalysis and multi- step sequential, will have an economic and environmental benefit in the chemical production. Oxidation is a central reaction in organic chemistry [4]. Sev- eral oxidizing reagents including permanganate and dichromate have been traditionally employed to accomplish this transfor- mation. However, many methodologies use reaction conditions that are incompatible with the enzymatic activity and stability. Recently, much attention has been focused on the development of ∗ Corresponding author. Tel.: +34 93 4006112; fax: +34 93 2045904. E-mail address: mmgqbm@cid.csic.es (M. Mifsud). (bio)/catalytic methods [5] employing oxygen or hydrogen perox- ide as highly attractive oxidants in terms of minimum energy use and waste generation. In this sense, tremendous advances in tran- sition metal catalysis [6–9], and organocatalysis [10,11] applied to oxidation reactions have been achieved. Moreover, biocatalysis is emerging as an additional pillar for environmentally benign oxidation catalysis in water [12,13]. Thus, biocatalytic approaches such as laccase/O 2 /mediator system (LMS), alcohol oxidase (AO) and chloroperoxidase (CPO) are a priori good candidates. Examples for the oxidase/aldolase coupling reactions are described in the literature [14–16]. In 1994 Fessner et al. devel- oped an approach for the preparation of complex carbohydrates by enzymatic aldolization in which both the aldol donor and the acceptor components were generated in situ by air oxidation using microbial oxidases [14]. More recently, Siebum et al. briefly discussed the oxidation of 4-pentenol by alcohol oxidase (AO) followed by the condensation reaction catalyzed by 2-deoxyribose- 5-phosphate aldolase (DERA) [16]. Similarly Wong and co-workers integrated the galactose oxidase (GO) catalyzed oxidation of glyc- erol to l-glyceraldehyde coupled with an aldolase reaction to produce l-fructose [15]. Their main drawback is that the oxidases usually lack of a broad substrate selectivity, therefore limiting their synthetic applications. Moreover, in some instances the need of metal cofactors used by the oxidases and class II aldolases such as l-rhamnulose-1-phosphate (RhuA), may be incompatible. Thus, alternatives must be provided to make the oxidation and aldol addi- tion compatible to in situ transformations. In this work, we endeavored to study the coupling of an in situ aldehyde generation by a green oxidation methodology with an enzymatic aldol addition reaction. We proposed to investigate 1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.molcatb.2012.02.001