Abstract
The feasibility of a peroxidase–aldolase cascade reaction for the synthesis of therapeutically-valuable iminocyclitols is discussed herein. A two-enzyme system consisting of chloroperoxidase (CPO) and D-fructose-6-phosphate aldolase (FSA) was evaluated for the synthesis of a D-fagomine precursor (preFagomine) from a N-Cbz-3-aminopropanol. An in-depth, systematic, step-by-step kinetic modeling of seven reactions and two inactivation decays was proposed to elucidate the reaction mechanism, prepare suitable stabilized biocatalysts, and find the optimal conditions for its application. The model described accurately the data and predicted the outcome at different experimental conditions. The inactivation of FSA caused by CPO was identified as the main bottleneck in the reaction. A two-step reaction approach and the use of immobilized enzymes on magnetic nanoparticle clusters and functionalized agarose carriers increased the stability of FSA, with an 1839-fold higher preFagomine formation per mol of enzyme in comparison to a one-pot reaction using soluble enzymes.
Highlights
Iminocyclitols, referred to as iminosugars, are carbohydrate analogs in which a nitrogen atom has replaced the endocyclic oxygen
Chemical syntheses of D-fagomine with carbohydrate precursors as starting materials usually involve cumbersome protection-deprotection reactions, achieving moderate isolated yields (Fleet et al, 1987; Goujon et al, 2005; Kumari et al, 2009; Pandey and Kapur, 2000); the synthesis from amino aldehydes is described with final yields below 15% (Banba et al, 2001)
The aldol addition was catalyzed by Dfructose-6-phosphate aldolase (FSA, EC 4.1.2.-), a class I aldolase from E. coli that is not dependent on phosphorylated donor substrates
Summary
Iminocyclitols, referred to as iminosugars, are carbohydrate analogs in which a nitrogen atom has replaced the endocyclic oxygen These compounds are inhibitors of glycosidases with enormous therapeutic potential in many diseases by altering the glycosylation or catabolism of glycoproteins The aldol addition was catalyzed by Dfructose-6-phosphate aldolase (FSA, EC 4.1.2.-), a class I aldolase from E. coli that is not dependent on phosphorylated donor substrates. This substrate promiscuity has allowed many syntheses of iminocyclitols, which have been assayed against a panel of glycosidases (Herna et al, 2019; Sugiyama et al, 2007). Several FSA genetic variants with modified substrate specificity are described elsewhere (Gutierrez et al, 2011; Szekrenyi et al, 2014)
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