Necrotizing enterocolitis (NEC) is one of the most common and devastating intestinal disorders in preterm infants. Therapies to meet the clinical needs for this special and highly vulnerable population are extremely limited. A specific human milk oligosaccharide (HMO), disialyllacto-N-tetraose (DSLNT), was shown to contribute to the beneficial effects of breastfeeding as it prevented NEC in a neonatal rat model and was associated with lower NEC risk in a human clinical cohort study. Herein, gram-scale synthesis of two DSLNT analogs previously shown to have NEC preventing effect is described. In addition, four novel disialyl glycans have been designed and synthesized by enzymatic or chemoenzymatic methods. Noticeably, two disialyl tetraoses have been produced by enzymatic sialylation of chemically synthesized thioethyl β-disaccharides followed by removal of the thioethyl aglycon. Dose-dependent and single-dose comparison studies showed varying NEC-preventing effects of the disialyl glycans in neonatal rats. This study helps to refine the structure requirement of the NEC-preventing effect of disialyl glycans and provides important dose-dependent information for using DSLNT analogs as potential therapeutics for NEC prevention in preterm infants.

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.

A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) for α2–6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective α2–6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activity compared to the wild-type PmST1. It retained the donor substrate promiscuity of the wild-type enzyme and was used in an efficient one-pot multienzyme (OPME) system to selectively catalyze the sialylation of the terminal galactose residue in a multigalactose-containing tetrasaccharide lacto-N-neotetraoside.

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%.

9-O-Acetylation is a common natural modification on sialic acids (Sias) that terminate many vertebrate glycan chains. This ester group has striking effects on many biological phenomena, including microbe-host interactions, complement action, regulation of immune responses, sialidase action, cellular apoptosis, and tumor immunology. Despite such findings, 9-O-acetyl sialoglycoconjugates have remained largely understudied, primarily because of marked lability of the 9-O-acetyl group to even small pH variations and/or the action of mammalian or microbial esterases. Our current studies involving 9-O-acetylated sialoglycans on glycan microarrays revealed that even the most careful precautions cannot ensure complete stability of the 9-O-acetyl group. We now demonstrate a simple chemical biology solution to many of these problems by substituting the oxygen atom in the ester with a nitrogen atom, resulting in sialic acids with a chemically and biologically stable 9-N-acetyl group. We present an efficient one-pot multienzyme method to synthesize a sialoglycan containing 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc) and compare it to the one with naturally occurring 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). Conformational resemblance of the two molecules was confirmed by computational molecular dynamics simulations. Microarray studies showed that the Neu5Ac9NAc-sialoglycan is a ligand for viruses naturally recognizing Neu5,9Ac2, with a similar affinity but with much improved stability in handling and study. Feeding of Neu5Ac9NAc or Neu5,9Ac2 to mammalian cells resulted in comparable incorporation and surface expression as well as binding to 9-O-acetyl-Sia-specific viruses. However, cells fed with Neu5Ac9NAc remained resistant to viral esterases and showed a slower turnover. This simple approach opens numerous research opportunities that have heretofore proved intractable.

•Sixteen new Neu5Ac9NAc-containing sialosides are successfully synthesized.•One-pot multienzyme (OPME) sialylation systems are highly efficient for the synthesis.•High-throughput microtiter plate assay results were confirmed by HPLC-based methods.•9-N-Acetyl Neu5Ac is a good mimic of 9-O-acetyl Neu5Ac to probe most sialidases.•Exceptions exist and mechanism needs further investigation.O-Acetylation of sialic acid (Sia) modulates its recognition by sialic acid-binding proteins and plays an important role in biological and pathological processes. 9-O-Acetylation is the most common modification of sialic acid in human. However, study of O-acetylated sialoglycans is hampered due to the instability of O-acetyl group towards pH changes and sensitivity to esterases. Our previous studies demonstrated a chemical biology method to this problem by replacing the oxygen atom in the C9 ester group of sialic acid by a nitrogen to form an amide. Here, we synthesized a library of sixteen new 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc)-containing α2–3- and α2–6-linked sialosides with various underlying glycans using efficient one-pot three-enzyme (OP3E) sialylation systems. Neu5Ac9NAc-containing compounds with a para-nitrophenol aglycon have been used together with their 9-O-acetyl analogs in microtiter plate-based high-throughput substrate specificity studies of nine different sialidases including those from humans and bacteria. In general, similar to 9-O-acetylation, 9-N-acetyl modification of sialic acid in the substrates lowers sialic acid-cleavage activity of most sialidases. In most cases, Neu5Ac9NAc is a good analog of 9-O-acetyl sialic acid. However, exceptions do exist. For example, 9-N- and 9-O-acetyl modifications have different effects on the sialosides cleave efficiencies of a commercially available C. perfringens sialidase as well as recombinant Streptococcus pneumoniae sialidase SpNanC and Bifidobacterium infantis sialidase BiNanH2. The mechanism for the difference awaits further investigation.Download high-res image (174KB)Download full-size image

Helicobacter pylori α1–3/4-fucosyltransferase (Hp3/4FT) was expressed in Escherichia coli at a level of 30 mg L−1 culture and used as a diverse catalyst in a one-pot multienzyme (OPME) system for high-yield production of L-fucose-containing carbohydrates including Lewis antigens such as Lewis a, b, and x, O-sulfated Lewis x, and sialyl Lewis x and human milk fucosides such as 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III, and lacto-N-difuco-hexaose (LNDFH) II and III. Noticeably, while difucosylation of tetrasaccharides was readily achieved using an excess amount of donor, the synthesis of LNFP III was achieved by Hp3/4FT-catalyzed selective fucosylation of the N-acetyllactosamine (LacNAc) component in lacto-N-neotetraose (LNnT).

Cu(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition (CuAAC), popularly known as the “click reaction”, serves as the most potent and highly dependable tool for facile construction of simple to complex architectures at the molecular level. Click-knitted threads of two exclusively different molecular entities have created some really interesting structures for more than 15 years with a broad spectrum of applicability, including in the fascinating fields of synthetic chemistry, medicinal science, biochemistry, pharmacology, material science, and catalysis. The unique properties of the carbohydrate moiety and the advantages of highly chemo- and regioselective click chemistry, such as mild reaction conditions, efficient performance with a wide range of solvents, and compatibility with different functionalities, together produce miraculous neoglycoconjugates and neoglycopolymers with various synthetic, biological, and pharmaceutical applications. In this review we highlight the successful advancement of Cu(I)-catalyzed click chemistry in glycoscience and its applications as well as future scope in different streams of applied sciences.

O-Sulfated sialyl Lewis x antigens play important roles in nature. However, due to their structural complexity, they are not readily accessible by either chemical or enzymatic synthetic processes. Taking advantage of a bacterial sialyltransferase mutant that can catalyze the transfer of different sialic acid forms from the corresponding sugar nucleotide donors to Lewis x antigens, which are fucosylated glycans, as well as an efficient one-pot multienzyme (OPME) sialylation system, O-sulfated sialyl Lewis x antigens containing different sialic acid forms and O-sulfation at different locations were systematically synthesized by chemoenzymatic methods.

A novel α1–2-fucosyltransferase from Thermosynechococcus elongatus BP-1 (Te2FT) with high fucosyltransferase activity and low donor hydrolysis activity was discovered and characterized. It was used in an efficient one-pot multienzyme (OPME) fucosylation system for the high-yield synthesis of human blood group H antigens containing β1–3-linked galactosides and an important human milk oligosaccharide (HMOS) lacto-N-fucopentaose I (LNFP I) on preparative and gram scales. LNFP I was shown to be selectively consumed by Bifidobacterium longum subsp. infantis but not Bifidobacterium animalis subsp. lactis and is a potential prebiotic.

Glycosyltransferase-catalyzed enzymatic and chemoenzymatic syntheses are powerful approaches for the production of oligosaccharides, polysaccharides, glycoconjugates, and their derivatives. Enzymes involved in the biosynthesis of sugar nucleotide donors can be combined with glycosyltransferases in one pot for efficient production of the target glycans from simple monosaccharides and acceptors. The identification of enzymes involved in the salvage pathway of sugar nucleotide generation has greatly facilitated the development of simplified and efficient one-pot multienzyme (OPME) systems for synthesizing major glycan epitopes in mammalian glycomes. The applications of OPME methods are steadily gaining popularity mainly due to the increasing availability of wild-type and engineered enzymes. Substrate promiscuity of these enzymes and their mutants allows OPME synthesis of carbohydrates with naturally occurring post-glycosylational modifications (PGMs) and their non-natural derivatives using modified monosaccharides as precursors. The OPME systems can be applied in sequence for synthesizing complex carbohydrates. The sequence of the sequential OPME processes, the glycosyltransferase used, and the substrate specificities of the glycosyltransferases define the structures of the products. The OPME and sequential OPME strategies can be extended to diverse glycans in other glycomes when suitable enzymes with substrate promiscuity become available. This Perspective summarizes the work of the authors and collaborators on the development of glycosyltransferase-based OPME systems for carbohydrate synthesis. Future directions are also discussed.

•N. meningitidis and H. pylori β3GlcNAcTs were actively expressed in E. coli.•Bovine β4GalT 1 was expressed in E. coli as a soluble active enzyme.•Thirteen UDP-sugars could be used as donor substrates for NmLgtA and Hpβ3GlcNAcT.•Ten trisaccharides could be used as acceptors for one of the three β4GalTs.•GalNAcβ1–3LacβProN3 and Galβ1–3Lac were produced by OPME reaction containing NmLgtA.β1–3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1–4-galactosyltransferases (β4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of β3GlcNAcTs and β4GalTs, donor substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1–3Galβ1–4GlcβProN3 and Galβ1–3Galβ1–4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents.

•Neisseria meningitidis serogroup A UDP-GlcNAc 2-epimerase (NmSacA) was cloned.•UDP-ManNAc derivatives were synthesized chemoenzymatically.•A capillary electrophoresis assay method was developed.•UDP-ManNAc and some derivatives were NmSacA substrates in the absence of UDP-GlcNAc.•UDP-GlcNAc improved while 2-acetamidoglucal and UDP inhibited NmSacA activity.Neisseria meningitidis serogroup A non-hydrolyzing uridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase (NmSacA) catalyzes the interconversion between UDP-GlcNAc and uridine 5′-diphosphate-N-acetylmannosamine (UDP-ManNAc). It is a key enzyme involved in the biosynthesis of the capsular polysaccharide [-6ManNAcα1-phosphate-]n of N. meningitidis serogroup A, one of the six serogroups (A, B, C, W-135, X, and Y) that account for most cases of N. meningitidis-caused bacterial septicemia and meningitis. N. meningitidis serogroup A is responsible for large epidemics in the developing world, especially in Africa. Here we report that UDP-ManNAc could be used as a substrate for C-terminal His6-tagged recombinant NmSacA (NmSacA-His6) in the absence of UDP-GlcNAc. NmSacA-His6 was activated by UDP-GlcNAc and inhibited by 2-acetamidoglucal and UDP. Substrate specificity study showed that NmSacA-His6 could tolerate several chemoenzymatically synthesized UDP-ManNAc derivatives as substrates although its activity was much lower than non-modified UDP-ManNAc. Homology modeling and molecular docking revealed likely structural determinants of NmSacA substrate specificity. This is the first detailed study of N. meningitidis serogroup A UDP-GlcNAc 2-epimerase.

Glycosphingolipids are a diverse family of biologically important glycolipids. In addition to variations on the lipid component, more than 300 glycosphingolipid glycans have been characterized. These glycans are directly involved in various molecular recognition events. Several naturally occurring sialic acid forms have been found in sialic acid-containing glycosphingolipids, namely gangliosides. However, ganglioside glycans containing less common sialic acid forms are currently not available. Herein, highly effective one-pot multienzyme (OPME) systems are used in sequential for high-yield and cost-effective production of glycosphingolipid glycans, including those containing different sialic acid forms such as N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (Kdn), and 8-O-methyl-N-acetylneuraminic acid (Neu5Ac8OMe). A library of 64 structurally distinct glycosphingolipid glycans belonging to ganglio-series, lacto-/neolacto-series, and globo-/isoglobo-series glycosphingolipid glycans is constructed. These glycans are essential standards and invaluable probes for bioassays and biomedical studies.

Lacto-N-neotetraose and its sialyl and fucosyl derivatives including Lewis x (Lex) pentasaccharide, sialyl Lewis x (sLex) hexasaccharide and internally sialylated derivatives were enzymatically synthesized from readily available lactoside, commercially available uridine 5′-diphosphate-glucose (UDP-Glc) and the corresponding monosaccharides using a highly efficient sequential one-pot multienzyme (OPME) strategy. The OPME strategy which combines bacterial glycosyltransferases and sugar nucleotide generation enzymes provides easy access to the biologically important complex oligosaccharides at preparative scale. Moreover, the same OPME strategy can be used for the regioselective introduction of sialic acid to the internal galactose unit of LNnT in a designed glycosylation route by simply changing the glycosylation sequence.

Arabidopsis thaliana glucuronokinase (AtGlcAK) was cloned and shown to be able to use various uronic acids as substrates to produce the corresponding uronic acid-1-phosphates. AtGlcAK or Bifidobacterium infantis galactokinase (BiGalK) was used with a UDP-sugar pyrophosphorylase, an inorganic pyrophosphatase, with or without a glycosyltransferase for highly efficient synthesis of UDP-uronic acids and glucuronides. These improved cost-effective one-pot multienzyme (OPME) systems avoid the use of nicotinamide adenine dinucleotide (NAD+)-cofactor in dehydrogenase-dependent UDP-glucuronic acid production processes and can be broadly applied for synthesizing various glucuronic acid-containing molecules.

•Several mutants of Photobacterium sp. α2–6-sialyltransferase (Psp2,6ST) were obtained.•A366G mutant has an increased expression level.•A366G mutant also has an improved catalytic activity in sialylating Tn antigens.•A one-pot two-enzyme sialylation system improved STn antigen synthetic efficiency.•Psp2,6ST A366G mutant is a powerful catalyst for synthesizing α2–6-sialosides.In order to improve the catalytic efficiency of recombinant Photobacterium sp. JT-ISH-224 α2–6-sialyltransferase Psp2,6ST(15–501)-His6 in sialylating α-GalNAc-containing acceptors for the synthesis of tumor-associated carbohydrate antigens sialyl Tn (STn), protein crystal structure-based mutagenesis studies were carried out. Among several mutants obtained by altering the residues close to the acceptor substrate binding pocket, mutant A366G was shown to improve the sialyltransferase activity of Psp2,6ST(15–501)-His6 toward α-GalNAc-containing acceptors by 21–115% without significantly affecting its sialylation activity to β-galactosides. Furthermore, the expression level was improved from 18–40 mg L−1 for the wild-type enzyme to 72–110 mg L−1 for the A366G mutant. In situ generation of CMP-sialic acid in a one-pot two-enzyme system was shown effective in overcoming the high donor hydrolysis of the enzyme. Mutant A366G performed better than the wild-type Psp2,6ST(15–501)-His6 for synthesizing Neu5Acα2–6GalNAcαOSer/Thr STn antigens.

Oligo(ethylene glycol)-linked light fluorous tags have been found to be optimal for conjugating to glycans for both high-yield enzymatic glycosylation reactions using one-pot multienzyme (OPME) systems and quick product purification using fluorous solid-phase extraction (FSPE) cartridges. The combination of OPME glycosylation systems and the FSPE cartridge purification scheme provides a highly effective strategy for facile synthesis and purification of glycans.

•One-pot three-enzyme chemoenzymatic sialylation method is efficient and convenient.•New sialosides containing C7-modified sialic acids are produced.•Novel probes for determining sialidase substrate specificity are obtained.•Substrates selective for bacterial sialidases but not human NEU2 are identified.•Information obtained can guide the design of selective bacterial sialidase inhibitors.Modifications at the glycerol side chain of sialic acid in sialosides modulate their recognition by sialic acid-binding proteins and sialidases. However, limited work has been focused on the synthesis and functional studies of sialosides with C7-modified sialic acids. Here we report chemical synthesis of C4-modified ManNAc and mannose and their application as sialic acid precursors in a highly efficient one-pot three-enzyme system for chemoenzymatic synthesis of α2–3- and α2–6-linked sialyl para-nitrophenyl galactosides in which the C7-hydroxyl group in sialic acid (N-acetylneuraminic acid, Neu5Ac, or 2-keto-3-deoxynonulosonic acid, Kdn) was systematically substituted by –F, –OMe, –H, and –N3 groups. Substrate specificity study of bacterial and human sialidases using the obtained sialoside library containing C7-modified sialic acids showed that sialosides containing C7-deoxy Neu5Ac were selective substrates for all bacterial sialidases tested but not for human NEU2. The information obtained from sialidase substrate specificity can be used to guide the design of new inhibitors that are selective against bacterial sialidases.

•Sialyltransferases couple sialic acid to glycans on glycoproteins and glycolipids.•Crystal structures of sialyltransferase from Photobacterium damselae have been determined.•Structure contains an N-terminal Ig-like domain with the GT-B sialyltransferase fold.•Binary structure with CMP-3F(a)Neu5Ac reveals a non-productive pre-Michaelis complex.Sialyltransferase structures fall into either GT-A or GT-B glycosyltransferase fold. Some sialyltransferases from the Photobacterium genus have been shown to contain an additional N-terminal immunoglobulin (Ig)-like domain. Photobacterium damselae α2–6-sialyltransferase has been used efficiently in enzymatic and chemoenzymatic synthesis of α2–6-linked sialosides. Here we report three crystal structures of this enzyme. Two structures with and without a donor substrate analog CMP-3F(a)Neu5Ac contain an immunoglobulin (Ig)-like domain and adopt the GT-B sialyltransferase fold. The binary structure reveals a non-productive pre-Michaelis complex, which are caused by crystal lattice contacts that prevent the large conformational changes. The third structure lacks the Ig-domain. Comparison of the three structures reveals small inherent flexibility between the two Rossmann-like domains of the GT-B fold.

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 facile one-pot two-enzyme chemoenzymatic approach has been established for the gram (Neu4,5Ac2α3Lac, 1.33 g) and preparative scale (Neu4,5Ac2α3LNnT) synthesis of monotreme milk oligosaccharides. Other O-acetyl-5-N-acetylneuraminic acid (Neu4,5Ac2)- or 4-O-acetyl-5-N-glycolylneuraminic acid (Neu4Ac5Gc) -containing α2-3-sialosides have also been synthesized in the preparative scale. Used as an effective probe, Neu4,5Ac2α3GalβpNP was found to be a suitable substrate by human influenza A viruses but not bacterial sialidases.

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.

A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) for α2–6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective α2–6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activity compared to the wild-type PmST1. It retained the donor substrate promiscuity of the wild-type enzyme and was used in an efficient one-pot multienzyme (OPME) system to selectively catalyze the sialylation of the terminal galactose residue in a multigalactose-containing tetrasaccharide lacto-N-neotetraoside.

Glycans play diverse roles in a wide range of biological processes. Research on glycan-binding events is essential for learning their biological and pathological functions. However, the functions of terminal and internal glycan epitopes exhibited during binding with glycan-binding proteins (GBPs) and/or viruses need to be further identified. Therefore, a focused library of 36 biantennary asparagine (Asn)-linked glycans with some presenting tandem glycan epitopes was synthesized via a combined Core Isolation/Enzymatic Extension (CIEE) and one-pot multienzyme (OPME) synthetic strategy. These N-glycans include those containing a terminal sialyl N-acetyllactosamine (LacNAc), sialyl Lewis x (sLex) and Siaα2–8-Siaα2–3/6-R structures with N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc) sialic acid form, LacNAc, Lewis x (Lex), α-Gal, and Galα1–3-Lex; and tandem epitopes including α-Gal, Lex, Galα1–3-Lex, LacNAc, and sialyl LacNAc, presented with an internal sialyl LacNAc or 1–2 repeats of an internal LacNAc or Lex component. They were synthesized in milligram-scale, purified to over 98% purity, and used to prepare a glycan microarray. Binding studies using selected plant lectins, antibodies, and viruses demonstrated, for the first time, that when interpreting the binding between glycans and GBPs/viruses, not only the structure of the terminal glycan epitopes, but also the internal epitopes and/or modifications of terminal epitopes needs to be taken into account.

A novel α1–2-fucosyltransferase from Thermosynechococcus elongatus BP-1 (Te2FT) with high fucosyltransferase activity and low donor hydrolysis activity was discovered and characterized. It was used in an efficient one-pot multienzyme (OPME) fucosylation system for the high-yield synthesis of human blood group H antigens containing β1–3-linked galactosides and an important human milk oligosaccharide (HMOS) lacto-N-fucopentaose I (LNFP I) on preparative and gram scales. LNFP I was shown to be selectively consumed by Bifidobacterium longum subsp. infantis but not Bifidobacterium animalis subsp. lactis and is a potential prebiotic.

O-Sulfated sialyl Lewis x antigens play important roles in nature. However, due to their structural complexity, they are not readily accessible by either chemical or enzymatic synthetic processes. Taking advantage of a bacterial sialyltransferase mutant that can catalyze the transfer of different sialic acid forms from the corresponding sugar nucleotide donors to Lewis x antigens, which are fucosylated glycans, as well as an efficient one-pot multienzyme (OPME) sialylation system, O-sulfated sialyl Lewis x antigens containing different sialic acid forms and O-sulfation at different locations were systematically synthesized by chemoenzymatic methods.

Glycosyltransferase-catalyzed enzymatic and chemoenzymatic syntheses are powerful approaches for the production of oligosaccharides, polysaccharides, glycoconjugates, and their derivatives. Enzymes involved in the biosynthesis of sugar nucleotide donors can be combined with glycosyltransferases in one pot for efficient production of the target glycans from simple monosaccharides and acceptors. The identification of enzymes involved in the salvage pathway of sugar nucleotide generation has greatly facilitated the development of simplified and efficient one-pot multienzyme (OPME) systems for synthesizing major glycan epitopes in mammalian glycomes. The applications of OPME methods are steadily gaining popularity mainly due to the increasing availability of wild-type and engineered enzymes. Substrate promiscuity of these enzymes and their mutants allows OPME synthesis of carbohydrates with naturally occurring post-glycosylational modifications (PGMs) and their non-natural derivatives using modified monosaccharides as precursors. The OPME systems can be applied in sequence for synthesizing complex carbohydrates. The sequence of the sequential OPME processes, the glycosyltransferase used, and the substrate specificities of the glycosyltransferases define the structures of the products. The OPME and sequential OPME strategies can be extended to diverse glycans in other glycomes when suitable enzymes with substrate promiscuity become available. This Perspective summarizes the work of the authors and collaborators on the development of glycosyltransferase-based OPME systems for carbohydrate synthesis. Future directions are also discussed.

Oligo(ethylene glycol)-linked light fluorous tags have been found to be optimal for conjugating to glycans for both high-yield enzymatic glycosylation reactions using one-pot multienzyme (OPME) systems and quick product purification using fluorous solid-phase extraction (FSPE) cartridges. The combination of OPME glycosylation systems and the FSPE cartridge purification scheme provides a highly effective strategy for facile synthesis and purification of glycans.

Lacto-N-neotetraose and its sialyl and fucosyl derivatives including Lewis x (Lex) pentasaccharide, sialyl Lewis x (sLex) hexasaccharide and internally sialylated derivatives were enzymatically synthesized from readily available lactoside, commercially available uridine 5′-diphosphate-glucose (UDP-Glc) and the corresponding monosaccharides using a highly efficient sequential one-pot multienzyme (OPME) strategy. The OPME strategy which combines bacterial glycosyltransferases and sugar nucleotide generation enzymes provides easy access to the biologically important complex oligosaccharides at preparative scale. Moreover, the same OPME strategy can be used for the regioselective introduction of sialic acid to the internal galactose unit of LNnT in a designed glycosylation route by simply changing the glycosylation sequence.

Arabidopsis thaliana glucuronokinase (AtGlcAK) was cloned and shown to be able to use various uronic acids as substrates to produce the corresponding uronic acid-1-phosphates. AtGlcAK or Bifidobacterium infantis galactokinase (BiGalK) was used with a UDP-sugar pyrophosphorylase, an inorganic pyrophosphatase, with or without a glycosyltransferase for highly efficient synthesis of UDP-uronic acids and glucuronides. These improved cost-effective one-pot multienzyme (OPME) systems avoid the use of nicotinamide adenine dinucleotide (NAD+)-cofactor in dehydrogenase-dependent UDP-glucuronic acid production processes and can be broadly applied for synthesizing various glucuronic acid-containing molecules.