Engineered d-fructose-6-phosphate aldolase (fsa) in organic synthesisaliphatic carbonil compounds as nucleophiles in biocatalytic aldol addition reaction

Resumen

Restricted substrate selectivity is among the major drawbacks of enzyme catalyzed aldol reactions. Particularly most of aldolases showed stringent nucleophile specificity that hampers many potential applications, especially those for the addition of simple non-functionalized aliphatic ketones including ethanal. D-Fructose-6-phospate-aldolase from E. coli (FSA, EC 4.1.2.–) is a remarkable aldolase with unprecedented tolerance for a large structural variety of nucleophilic and electrophilic structures by virtue of the malleability for the modification of its active site. In this thesis, we have obtained an unprecendented collection of FSA variants capable to catalyze aldol additions of ethanal, propanone, butanone, 3-pentanone, cyclobutanone and cyclopentanone to hydroxyaldehydes such as 3-hydropropanal, (S)-, (R)-3-hydroxybutanal and benzyloxyethanal obtaining pyranose analogues with enantio and diastereoselectivities >95%. To furnish functionalized N-heterocycles of the pyrrolidine and piperidine type including cycloalkane derivatives, aldol addition of these nucleophilic components to N-Cbz-aminoaldehydes such as N-Cbz protected 2-aminoethanal and 3-aminopropanal followed by an intramolecular reductive amination was conducted. The most efficents biocatalyst were FSA D6X variants (where X = N, S, T and H), including A165G. FSA variants also catalyzed the cross-aldol additions of propanal to formaldehyde, ethanal to chloroethanal and the homoaldol addition of propanal, furnishing low-molecular weight chiral molecules (e.g., Roche ester derivatives, β-hydroxyaldehydes, β-hydroxyketones and β-hydroxycarboxylic acids) used as building blocks for the synthesis of active compounds including pharmaceutics, and fragrances. In those reactions using ethanal or propanal as nucleophile components, the product obtained, an aldehyde, was utilized as electrophilic component for a sequential cascade aldol reactions. Thus, molecular complexity was rapidly buildup under mild conditions, without a need for tedious and time-consuming iterative steps for protection and deprotection of sensitive or reactive functional groups, yet with high chemical efficiency and often uncompromised stereochemical fidelity. The residue D6, located in the active site of FSA, is a critical residue for the tolerance of the novel nucleophile structures with the selected electrophiles. The reactions uncovered in this study open new entries to novel chiral synthons and product families difficult to access by pure chemical methods.