para-Nitrophenol (pNP)-tagged α2-8-linked sialosides containing different sialic acid forms were chemoenzymatically synthesized using an efficient one-pot three-enzyme α2-8-sialylation system. The resulting compounds allowed high-throughput substrate specificity studies of the α2-8-sialidase activity of a recombinant human cytosolic sialidase hNEU2 and various bacterial sialidases. The sialoside substrate profiles obtained can be used to guide the selection of suitable sialidases for sialylglycan analysis and for cell and tissue surface glycan modification. They can also be used to guide sialidase inhibitor design.

5,7-Di-N-acetyllegionaminic acid (Leg5,7Ac₂) is a bacterial nonulosonic acid (NulO) analogue of sialic acids, an important class of monosaccharides in mammals and in some bacteria. To develop efficient one-pot multienzyme (OPME) glycosylation systems for synthesizing Leg5,7Ac₂-glycosides, Legionella pneumophila cytidine 5'-monophosphate (CMP)-Leg5,7Ac₂ synthetase (LpCLS) was cloned and characterized. It was successfully used in producing Leg5,7Ac₂-glycosides from chemoenzymatically synthesized Leg5,7Ac₂ using a one-pot two-enzyme system or from its chemically synthesized six-carbon monosaccharide precursor 2,4-diacetamido-2,4,6-trideoxymannose (6deoxyMan2,4diNAc) in a one-pot three-enzyme system. In addition, LpCLS was shown to tolerate Neu5Ac7NAc, a C9-hydroxyl analogue of Leg5,7Ac₂ and also a stable analogue of 7-O-acetylneuraminic acid (Neu5,7Ac₂), to allow OPME synthesis of the corresponding α2-3-linked sialosides, from chemically synthesized six-carbon monosaccharide precursor 4-N-acetyl-4-deoxy-N-acetylmannosamine (ManNAc7NAc).

O-GalNAc glycans or mucin-type glycans are common protein post-translational modifications in eukaryotes. Core 2 O-GalNAc glycans are branched structures that are broadly distributed in glycoproteins and mucins of all types of cells. To better understand their biological roles, it is important to obtain structurally defined Core 2 O-GalNAc glycans. We present here regioselective one-pot multienzyme (OPME) chemoenzymatic strategies to systematically access a diverse array of sialyl Core 2 glycans. Regioselectivity can be achieved by using OPME systems containing a glycosyltransferase with restricted acceptor specificity or by differentiating the branches using altered glycosylation sequences. This work provides a general regioselective strategy to access diverse Core 2 O-GalNAc glycans which can be extended for the synthesis of other complex branched glycans.

A chemoenzymatic synthon was designed to expand the scope of the chemoenzymatic synthesis of carbohydrates. The synthon was enzymatically converted into carbohydrate analogues, which were readily derivatized chemically to produce the desired targets. The strategy is demonstrated for the synthesis of glycosides containing 7,9-di-N-acetyllegionaminic acid (Leg5,7Ac₂ ), a bacterial nonulosonic acid (NulO) analogue of sialic acid. A versatile library of α2-3/6-linked Leg5,7Ac₂ -glycosides was built by using chemically synthesized 2,4-diazido-2,4,6-trideoxymannose as a chemoenzymatic synthon for highly efficient one-pot multienzyme (OPME) sialylation followed by downstream chemical conversion of the azido groups into acetamido groups. The syntheses required 10 steps from commercially available d-fucose and had an overall yield of 34-52 %, thus representing a significant improvement over previous methods. Free Leg5,7Ac₂ monosaccharide was also synthesized by a sialic acid aldolase-catalyzed reaction.

A highly efficient chemoenzymatic method for synthesizing glycosphingolipids using α-Gal pentasaccharyl ceramide as an example is reported here. Enzymatic extension of the chemically synthesized lactosyl sphingosine using efficient sequential one-pot multienzyme (OPME) reactions allowed glycosylation to be carried out in aqueous solutions. Facile C18 cartridge-based quick (<30 minutes) purification protocols were established using minimal amounts of green solvents (CH₃CN and H₂O). Simple acylation in the last step led to the formation of the target glycosyl ceramide in 4 steps with an overall yield of 57%.

A sialyltransferase acceptor tagging and two-step enzymatic reaction strategy has been developed for multigram-scale chemoenzymatic synthesis of 2,7-anhydro-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac), a compound that can serve as a sole carbon source for the growth of Ruminococcus gnavus, a common human gut commensal. Different approaches of introducing hydrophobic UV-active tags to lactose as well-suited sialyltransferase acceptors have been explored and a simple two-step high-yield chemical synthetic procedure has been identified. The UV-active hydrophobic tag facilitates monitoring reaction progress and allows facile product purification by C18-cartridges. A two-step enzyme-catalyzed reaction procedure has been established to combine with C18 cartridge-based purification process for high-yield production of the desired product in multigram scales with the recycled use of chromophore-tagged lactoside starting material and sialoside intermediate. This study demonstrated an environmentally friendly highly-efficient synthetic and purification strategy for the production of 2,7-anhydro-Neu5Ac to explore its potential functions.

A novel chemoenzymatic synthon strategy has been developed to construct a comprehensive library of α2-3- and α2-6-linked sialosides containing 7-N- or 7,9-di-N-acetyl sialic acid, the stable analogue of naturally occurring 7-O-acetyl- or 7,9-di-O-acetyl-sialic acid. Diazido and triazido-mannose derivatives that were readily synthesized chemically from inexpensive galactose were shown to be effective chemoenzymatic synthons. Together with bacterial sialoside biosynthetic enzymes with remarkable substrate promiscuity, they were successfully used in one-pot multienzyme (OPME) sialylation systems for highly efficient synthesis of sialosides containing multiple azido groups. Conversion of the azido groups to N-acetyl groups generated the desired sialosides. The hydrophobic and UV-detectable benzyloxycarbonyl (Cbz) group introduced in the synthetic acceptors of sialyltransferases was used as a removable protecting group for the propylamine aglycon of the target sialosides. The resulting N-acetyl sialosides were novel stable probes for sialic acid-binding proteins such as plant lectin MAL II, which bond strongly to sialyl T antigens with or without an N-acetyl at C7 or at both C7 and C9 in the sialic acid.

Sialidase transition state analog inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en, DANA) has played a leading role in developing clinically used anti-influenza virus drugs. Taking advantage of the Neu5Ac2en-forming catalytic property of Streptococcus pneumoniae sialidase SpNanC, an effective one-pot multienzyme (OPME) strategy has been developed to directly access Neu5Ac2en and its C-5, C-9, and C-7-analogs from N-acetylmannosamine (ManNAc) and analogs. The obtained Neu5Ac2en analogs can be further derivatized at various positions to generate a larger inhibitor library. Inhibition studies demonstrated improved selectivity of several C-5- or C-9-modified Neu5Ac2en derivatives against several bacterial sialidases. The study provides an efficient enzymatic method to access sialidase inhibitors with improved selectivity.

A highly efficient streamlined chemoenzymatic strategy for total synthesis of four prioritized ganglioside cancer antigens GD2, GD3, fucosyl GM1, and GM3 from commercially available lactose and phytosphingosine is demonstrated. Lactosyl sphingosine (LacβSph) was chemically synthesized (on a 13 g scale), subjected to sequential one-pot multienzyme (OPME) glycosylation reactions with facile C18-cartridge purification, followed by improved acylation conditions to form target gangliosides, including fucosyl GM1 which has never been synthesized before.

DNA glycosylases of the base excision repair (BER) pathway are front-line defenders in removing compromising modifications of the DNA nucleobases. Aberrantly modified nucleobases mediate genomic mutations and inhibit DNA replication leading to adverse health consequences such as cancer, neurological diseases, and aging. In an effort to develop high-affinity transition state (TS) analogues as chemical biology probes for DNA glycosylases, oligonucleotides containing a propargyl-modified pyrrolidine TS mimic nucleotide were synthesized. A small library of TS mimic-containing oligonucleotides was generated using a structurally diverse set of five azides via copper(I)-catalyzed azide-alkyne cycloaddition "click" chemistry. The relative affinity ( K_d) was evaluated for BER glycosylases Escherichia coli MutY, bacterial formamidopyrimidine glycosylase (Fpg), and human OG glycosylase 1 (hOGG1) with the library of TS mimic DNA duplexes. All of the BER glycosylases were found to exhibit extremely high affinities (approximately picomolar K_d values) for the TS mimics. However, binding preferences, distinct for each glycosylase, for the TS mimic library members were observed, suggesting different modes of binding and transition state stabilization among the three glycosylases. Fpg bound all of the TS mimics with exceptionally high affinities, while the MutY binding affinity correlated inversely with the size of the appended moiety. Of note, we identified one member of the small TS mimic library that exhibited a particularly high affinity for hOGG1. These results strongly support the use of the propargyl-TS mimic oligonucleotides and elaboration via click chemistry in screening and identification of high-affinity ligands for BER glycosylases of interest.