Chemo-enzymatic Synthesis of Pseudo-trisaccharide Aminoglycoside Antibiotics with Enhanced Nonsense Read-through Inducer Activity.

Aminoglycosides (AGs) are broad-spectrum antibiotics used to treat bacterial infections. Over the last two decades, studies have reported the potential of AGs in the treatment of genetic disorders caused by nonsense mutations, owing to their ability to induce the ribosomes to read through these mutations and produce a full-length protein. However, the principal limitation in the clinical application of AGs arises from their high toxicity, including nephrotoxicity and ototoxicity. In this study, five novel pseudo-trisaccharide analogs were synthesized by chemo-enzymatic synthesis by acid hydrolysis of commercially available AGs, followed by an enzymatic reaction using recombinant substrate-flexible KanM2 glycosyltransferase. The relationships between their structures and biological activities, including the antibacterial, nephrotoxic, and nonsense readthrough inducer (NRI) activities, were investigated. The absence of 1-N-acylation, 3',4'-dideoxygenation, and post-glycosyl transfer modifications on the third sugar moiety of AGs diminishes their antibacterial activities. The 3',4'-dihydroxy and 6'-hydroxy moieties regulate the in vitro nephrotoxicity of AGs in mammalian cell lines. The 3',4'-dihydroxy and 6'-methyl scaffolds are indispensable for the ex vivo NRI activity of AGs. Based on the alleviated in vitro antibacterial properties and nephrotoxicity, and the highest ex vivo NRI activity among the five compounds, a kanamycin analog (6'-methyl-3''-deamino-3''-hydroxykanamycin C) was selected as a novel AG hit for further studies on human genetic disorders caused by premature transcriptional termination.

Enzymatic synthesis of novel unnatural phenoxodiol glycosides with a glycosyl donor flexible glycosyltransferase MeUGT1.

Isoflavonoids are of great interest due to their human health-promoting properties, which have resulted in studies on exploiting these phytochemicals as hotspots in diverse bio -industries. Biocatalytic glycosylation of isoflavonoid aglycones to glycosides has attracted marked interests because it enable the biosynthesis of isoflavonoid glycosides with high selectivity under mild conditions, and also provide an environmentally friendly option for the chemical synthesis. Thus, these inspired us to exploit new flexible and effective glycosyltransferases from microbes for making glycosides attractive compounds that are in high demand in several industries. Most recently, we have reported the functional characterization of a bacterial-origin recombinant glycosyltransferase (MeUGT1). Herein, more detailed kinetic characteristics of this biocatalyst, using a number of glycosyl donor substrates, were examined for further investigation of its biocatalytic applicability, enabling it feasible to biosynthesize new glycosides; phenoxodiol-4'-O-α-glucuronide, phenoxodiol-4'-O-α-(2''-N-acetyl)glucosaminide, phenoxodiol-4'-O-α-galactoside, phenoxodiol-4'-O-α-(2''-N-acetyl)galactosaminide and phenoxodiol-4'-O-α-(2''-deoxy)glucoside. The thorough kinetic analyses revealed that while the recombinant enzyme can utilize, albeit with different substrate preference and catalytic efficiency, a total five different nucleotide sugars as glycosyl donors, exhibiting its promiscuity towards glycosyl donors. This is the first report that a recombinant glycosyltransferase MeUGT1 that can regio-specifically glycosylate C4'-hydroxyl function of semi-synthetic phenoxodiol isoflavene to biosynthesize a series of unnatural phenoxodiol-4'-O-α-glycosides.

Enzymatic Biosynthesis of Simple Phenolic Glycosides as Potential Anti-Melanogenic Antioxidants.

Simple phenolics (SPs) and their glycosides have recently gained much attention as functional skin-care resources for their anti-melanogenic and antioxidant activities. Enzymatic glycosylation of SP aglycone make it feasible to create SP glycosides with updated bioactive potentials. Herein, a glycosyltransferase (GT)-encoding gene was cloned from the fosmid libraries of Streptomyces tenjimariensis ATCC 31603 using GT-specific degenerate PCR followed by in silico analyses. The recombinant StSPGT was able to flexibly catalyze the transfer of two glycosyl moieties towards two SP acceptors, (hydroxyphenyl-2-propanol [HPP2] and hydroxyphenyl-3-propanol [HPP3]), generating stereospecific α-anomeric glycosides as follows: HPP2-O-α-glucoside, HPP2-O-α-2″-deoxyglucoside, HPP3-O-α-glucoside and HPP3-O-α-2″-deoxyglucoside. This enzyme seems not only to prefer UDP-glucose and HPP2 as a favorable glycosyl donor and acceptor, respectively but also differentiates the positional difference of the hydroxyl function as acceptor catalytic sites. Paired in vitro and in vivo antioxidant assays represented SPs and their corresponding glycosides as convincing antioxidants in a time- and concentration-dependent manner by scavenging DPPH radicals and intracellular ROS. Even compared to the conventional agents, HPP2 and glycoside analogs displayed improved tyrosinase inhibitory activity in vitro and still suppressed in vivo melanogenesis. Both HPP2 glycosides are further likely to exert the best inhibitory activity against elastase, eventually highlighting these glycosides with enhanced anti-melanogenic and antioxidant activities as promising anti-wrinkle hits.

Glycosylation of Semi-Synthetic Isoflavene Phenoxodiol with a Recombinant Glycosyltransferase from Micromonospora echinospora ATCC 27932.

Glycosyltransferase (GT)-specific degenerate PCR screening followed by in silico sequence analyses of the target clone was used to isolate a member of family1 GT-encoding genes from the established fosmid libraries of soil actinomycetes Micromonospora echinospora ATCC 27932. A recombinant MeUGT1 was heterologously expressed as a His-tagged protein in E. coli, and its enzymatic reaction with semi-synthetic phenoxodiol isoflavene (as a glycosyl acceptor) and uridine diphosphate-glucose (as a glycosyl donor) created two different glycol-attached products, thus revealing that MeUGT1 functions as an isoflavonoid glycosyltransferase with regional flexibility. Chromatographic separation of product glycosides followed by the instrumental analyses, clearly confirmed these previously unprecedented glycosides as phenoxodiol-4'-α-O-glucoside and phenoxodiol-7-α-O-glucoside, respectively. The antioxidant activities of the above glycosides are almost the same as that of parental phenoxodiol, whereas their anti-proliferative activities are all superior to that of cisplatin (the most common platinum chemotherapy drug) against two human carcinoma cells, ovarian SKOV-3 and prostate DU-145. In addition, they are more water-soluble than their parental aglycone, as well as remaining intractable to the simulated in vitro digestion test, hence demonstrating the pharmacological potential for the enhanced bio-accessibility of phenoxodiol glycosides. This is the first report on the microbial enzymatic biosynthesis of phenoxodiol glucosides.