Chemoenzymatic Synthesis of Carbohydrates and Derivatives with Engineered D-Fructose-6-Phosphate Aldolase

Abstract

[spa] Los compuestos polihidroxilados, como carbohidratos y sus derivados son moléculas de gran importancia en procesos bioquímicos. La síntesis de estos compuestos es compleja debido principalmente a tres factores: i) su alta funcionalización, ii) su elevado número de centros estereogénicos iii) y su aislamiento. Por lo tanto, el desarrollo de metodologías simples y selectivas que permitan la preparación de colecciones de estas sustancias con diversitad estructural son de gran importancia. Las aldolasas, que catalizan una reacciones aldólica o retro-aldólica son unas de las liasas mas importantes para la formación estereoselectiva de enlaces carbono-carbono. Estos biocatalizadores son uno de los dos clases de enzimas que son utilizadas en la síntesis de monosacáridos, permitiendo la funcionalización y fromación de nuevos centros esterogenicos. La D-fructosa 6-fosfato aldolasa (FSA) de E. coli es la unica aldolasa que cataliza la formación reversible de D-fructosa 6-fosfato. La enzima puede utilizar varios dadores no fosforilados como dihidroxiacetona (DHA), hidroxiacetona (HA) y glicolaldehído (GO). El objecto de esta tesis es el rediseño racional de FSA para la síntesis de varios carbohidratos. Diferentes estrategias de ingeniería genética han sido aplicadas para ampliar la tolerancia frente a varios sustratos dadores y aceptores, que permitió la síntesis de aldosas. También se ha modificado la estereoquímica y mejorado el rendimiento de la reacción enzimática. Utilizando mutagénesis sitio específica se desarrolló una variante de FSA que acepta varios 1-hidroxi-2-alcanones y sus análogos como sustratos dadores con alta diastereoselectividad. Así se abren nuevas rutas para la síntesis de colecciones de compuestos inovadores, inaccesibles hasta el momento por biocatálisis. También se aplicó mutagénesis para el diseño de mutantes activos hacia las adiciones aldólicas de GO. Además combinando las diferentes mutaciones la reactividad de la FSA se ha visto mejorada frente a sustratos aceptores. Esta nueva actividad ha sido aplicada en la preparación de un gran número de aldosas mediante reacciones tándem biocatalíticas por dos adiciones secuenciales de GO. En la literatura se encuentran pocos ejemplos de reacciones catalíticas en cascada. Por ello, se realizó un estudio de la combinación de adiciones aldólicas organo- y biocatalíticas para la síntesis de desoxiazúcares.

[eng] Biocatalysis is the chemical transformation of organic substrates by enzymes. This is the driving force of some of the oldest transformations known to humans. Nowadays, methods involving biocatalysts are gaining importance in organic synthesis owing to their high efficiency and selectivity, and driven by the need to develop processes that are economical and environmentally sustainable. Recombinant DNA technology has paved the road for mass production of enzymes and recently their major limitation, i.e., narrow substrate spectrum is also being addressed by the development of enzyme engineering. Rational design and directed evolution has been used to tackle a number of limitations associated with the use of wild-type aldolases as catalysts in synthetic organic chemistry. Aldolases, which catalyze carbon-carbon bond formations (one of the most challenging transformations in synthetic organic chemistry) with high stereospecificity have substantial utility as biocatalysts in the synthesis of chiral complex bioactive compounds such as carbohydrates, amino acids and their derivatives. D-fructose 6-phosphate aldolase (FSA) from E. coli is a unique member of this class of enzymes for accepting non-phosphorylated analogues of dihydroxyacetone phosphate (DHAP), and constitutes an exception to the strict donor specificity of aldolases. This dihydroxyacetone-dependent aldolase accepts a wide range of donor analogues of dihydroxyacetone (DHA), becoming an extremely promising catalyst for direct stereoselective aldol reactions. In this thesis an unprecedented expansion in the nucleophile and electrophile substrate scope of FSA is presented with the assistance of enzyme engineering. Strategies for the structure guided redesign by site-directed and site-saturation mutagenesis was used to modify the enzyme to improve its tolerance for a wide range of donor and acceptor substrates. We report the structure-guided rational protein engineering of FSA to accept a unique variety of 1-hydroxy-2-alkanones and related ether components as novel donor substrates. The double-active-site variant FSA L107A/L163A was found to convert outstanding variety of donor structures with good reaction rates, retaining high diastereoselectivy for the D-threo configuration. This newly designed FSA variant opens new avenues towards the synthesis of novel product families that were before inaccessible by biological catalysis. In a similar fashion, structure-guided engineering of the enzyme’s active site was applied to design a set of FSA variants highly active for aldol additions of glycolaldehyde (GO). The A129 residue was chosen as target position to improve the activity and selectivity towards GO, resulting a variant with highly increased affinity for catalyzing reactions with GO. This mutation in combination with others in the active site led to FSA variants with improved acceptor tolerance. Thus, a toolbox of new FSA variants was built up, expanding the synthetic possibilities of this biocatalyst towards the preparation of aldose-like carbohydrate compounds. The ideal strategy for biocatalytic synthesis of aldose sugars would be the consecutive aldol additions of GO with high enantio- and diastereoselectivity. Thus, to exploit this newly found activity of the FSA mutants in catalyzing self-aldol reaction of GO, we have developed a procedure for the preparation of a number of aldose carbohydrates (D-idose and derivatives) and derivatives from formaldehyde, glycolaldehyde and other simple aldehydes by biocatalytic tandem reactions by two consecutive additions of GO. Furthermore, the stereochemical course of the reaction was also altered to allow the one-pot synthesis of L-glucose from the stereoselective trimerization of glycolaldehyde using a combination of FSA variants. Examples of catalytic cascade reactions are scarce in literature, hence as a proof of concept we intended to combine organo- and biocatalytic aldol additions for the synthesis of deoxysugars. Proline catalyzed self-aldol reaction of propionaldehyde was coupled with FSA catalyzed addition of hydroxyacetone and dihydroxyacetone, proving once again the versatility of this aldolase, in the catalysis of aldol-additions with high stereoselectivity.