A novel protocol has been established to prepare the kanamycin ring II/III fragment, which has been validated as a minimum structural motif for the development of new aminoglycosides on the basis of its bactericidal activity even against resistant strains. Furthermore, its ability to act as a AAC-(6') and APH-(3') binder, and as a poor substrate for the ravenous ANT-(4'), makes it an excellent candidate for the design of inhibitors of these aminoglycoside modifying enzymes.

Glycoside hydrolases (GHs) are enzymes that hydrolyze glycosidic bonds, but some of them can also catalyze the synthesis of glycosides by transglycosylation. However, the yields of this reaction are generally low since the glycosides formed end up being hydrolyzed by these same enzymes. For this reason, mutagenic variants with null or drastically reduced hydrolytic activity have been developed, thus enhancing their synthetic ability. Two mutagenic variants, a glycosynthase engineered from a β-glucosidase (BGL-1-E521G) and a thioglycoligase from a β-xylosidase (BxTW1-E495A), both from the ascomycete Talaromyces amestolkiae, were used to synthesize three novel epigallocatechin gallate (EGCG) glycosides. EGCG is a phenolic compound from green tea known for its antioxidant effects and therapeutic benefits, whose glycosylation could increase its bioavailability and improve its bioactive properties. The glycosynthase BGL-1-E521G produced a β-glucoside and a β-sophoroside of EGCG, while the thioglycoligase BxTW1-E495A formed the β-xyloside of EGCG. Glycosylation occurred in the 5″ and 4″ positions of EGCG, respectively. In this work, the reaction conditions for glycosides' production were optimized, achieving around 90% conversion of EGCG with BGL-1-E521G and 60% with BxTW1-E495A. The glycosylation of EGCG caused a slight loss of its antioxidant capacity but notably increased its solubility (between 23 and 44 times) and, in the case of glucoside, also improved its thermal stability. All three glycosides showed better antiproliferative properties on breast adenocarcinoma cell line MDA-MB-231 than EGCG, and the glucosylated and sophorylated derivatives induced higher neuroprotection, increasing the viability of SH-S5Y5 neurons exposed to okadaic acid.

The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophilic acceptors. Based on the particularities of the GH3 glycosidase family active site, we hypothesized that converting a suitable member into a thioglycoligase could boost the acceptor range. Herein we show the engineering of an acidophilic fungal β-xylosidase into a thioglycoligase with broad acceptor promiscuity. The mutant enzyme displays the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters with conversion yields from moderate to high. Analyses also indicate that the pKa of the target compound was the main factor to determine its suitability as glycosylation acceptor. These results expand on the glycoconjugate portfolio attainable through biocatalysis.