Liuqing Wen

Facile Enzymatic Synthesis of Ketoses

Studies of rare ketoses have been hampered by a lack of efficient preparation methods. A convenient, efficient, and cost-effective platform for the facile synthesis of ketoses is described. This method enables the preparation of difficult-to-access ketopentoses and ketohexoses from common and inexpensive starting materials with high yield and purity and without the need for a tedious isomer separation step.

Chemoenzymatic Synthesis of a Library of Human Milk Oligosaccharides

Human milk oligosaccharides (HMOs) are a family of diverse unconjugated glycans that exist in human milk as one of the major components. Characterization, quantification, and biofunctional studies of HMOs remain a great challenge due to their diversity and complexity. The accessibility of a homogeneous HMO library is essential to solve these issues which have beset academia for several decades. In this study, an efficient chemoenzymatic strategy, namely core synthesis/enzymatic extension (CSEE), for rapid production of diverse HMOs was reported. On the basis of 3 versatile building blocks, 3 core structures were chemically synthesized via consistent use of oligosaccharyl thioether and oligosaccharyl bromide as glycosylation donors in a convergent fragment coupling strategy. Each of these core structures was then extended to up to 11 HMOs by 4 robust glycosyltransferases. A library of 31 HMOs were chemoenzymatically synthesized and characterized by MS and NMR. CSEE indeed provides a practical approach to harvest structurally defined HMOs for various applications.

Identification of the binding roles of terminal and internal glycan epitopes using enzymatically synthesized N-glycans containing tandem epitopes

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.

Two-Step Chemoenzymatic Detection of N-Acetylneuraminic Acid−α(2-3)-Galactose Glycans

Sialic acids are typically linked α(2-3) or α(2-6) to the galactose that located at the non-reducing terminal end of glycans, playing important but distinct roles in a variety of biological and pathological processes. However, details about their respective roles are still largely unknown due to the lack of an effective analytical technique. Herein, a two-step chemoenzymatic approach for the rapid and sensitive detection of N-acetylneuraminic acid-α(2-3)-galactose glycans is described.

Efficient enzymatic synthesis of l-rhamnulose and l-fuculose

L-Rhamnulose (6-deoxy-L-arabino-2-hexulose) and L-fuculose (6-deoxy-L-lyxo-2-hexulose) were prepared from L-rhamnose and L-fucose by a two-step strategy. In the first reaction step, isomerization of L-rhamnose to L-rhamnulose, or L-fucose to L-fuculose was combined with a targeted phosphorylation reaction catalyzed by L-rhamnulose kinase (RhaB). The by-products (ATP and ADP) were selectively removed by silver nitrate precipitation method. In the second step, the phosphate group was hydrolyzed to produce L-rhamnulose or L-fuculose with purity exceeding 99% in more than 80% yield (gram scale).

Chemoenzymatic synthesis of ADP-d-glycero-β-d-manno-heptose and study of the substrate specificity of HldE

An efficient one-pot three enzymes strategy for chemoenzymatic synthesis of ADP-d-glycero-β-d-manno-heptose (ADP-d, d-heptose) was reported using chemically synthesized d, d-heptose-7-phosphate and the ADP-d, d-heptose biosynthetic enzymes HldE and GmhB. Moreover, the result of investigating substrate specificity of the kinase action of HldE revealed that HldE had highly restricted substrate specificity towards structurally modified heptose-7-phosphate analogs.

Comparative analysis of Cu (I)-catalyzed alkyne-azide cycloaddition (CuAAC) and strain-promoted alkyne-azide cycloaddition (SPAAC) in O-GlcNAc proteomics

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is emerging as an essential protein post‐translational modification in a range of organisms. It is involved in various cellular processes such as nutrient sensing, protein degradation, gene expression, and is associated with many human diseases. Despite its importance, identifying O‐GlcNAcylated proteins is a major challenge in proteomics. Here, using peracetylated N‐azidoacetylglucosamine (Ac4GlcNAz) as a bioorthogonal chemical handle, we described a gel‐based mass spectrometry method for the identification of proteins with O‐GlcNAc modification in A549 cells. In addition, we made a labeling efficiency comparison between two modes of azide‐alkyne bioorthogonal reactions in click chemistry: copper‐catalyzed azide‐alkyne cycloaddition (CuAAC) with Biotin‐Diazo‐Alkyne and stain‐promoted azide‐alkyne cycloaddition (SPAAC) with Biotin‐DIBO‐Alkyne. After conjugation with click chemistry in vitro and enrichment via streptavidin resin, proteins with O‐GlcNAc modification were separated by SDS‐PAGE and identified with mass spectrometry. Proteomics data analysis revealed that 229 putative O‐GlcNAc modified proteins were identified with Biotin‐Diazo‐Alkyne conjugated sample and 188 proteins with Biotin‐DIBO‐Alkyne conjugated sample, among which 114 proteins were overlapping. Interestingly, 74 proteins identified from Biotin‐Diazo‐Alkyne conjugates and 46 verified proteins from Biotin‐DIBO‐Alkyne conjugates could be found in the O‐GlcNAc modified proteins database dbOGAP (http://cbsb.lombardi.georgetown.edu/hulab/OGAP.html). These results suggested that CuAAC with Biotin‐Diazo‐Alkyne represented a more powerful method in proteomics with higher protein identification and better accuracy compared to SPAAC. The proteomics credibility was also confirmed by the molecular function and cell component gene ontology (GO). Together, the method we reported here combining metabolic labeling, click chemistry, affinity‐based enrichment, SDS‐PAGE separation, and mass spectrometry, would be adaptable for other post‐translationally modified proteins in proteomics.

Enzymatic synthesis of 3-deoxy-d-manno-octulosonic acid (KDO) and its application for LPS assembly

The studies of 3-deoxy-d-manno-octulosonic acid (KDO) have been hindered due to its limited availability. Herein, an efficient enzymatic system for the facile synthesis of KDO from easy-to-get starting materials is described. In this one-pot three-enzyme (OPME) system, d-ribulose 5-phosphate, which was prepared from d-xylose, was employed as starting materials. The reaction process involves the isomerization of d-ribulose 5-phosphate to d-arabinose 5-phosphate catalyzed by d-arabinose 5-phosphate isomerase (KdsD), the aldol condensation of d-arabinose 5-phosphate and phosphoenolpyruvate (PEP) catalyzed by KDO 8-phosphate synthetase (KdsA), and the hydrolysis of KDO-8-phosphate catalyzed by KDO 8-phosphate phosphatase (KdsC). By using this OPME system, 72% isolated yield was obtained. The obtained KDO was further transferred to lipid A by KDO transferase from Escherichia coli (WaaA).

A General Chemoenzymatic Strategy for the Synthesis of Glycosphingolipids

A concise, prototypical, and stereoselective strategy for the synthesis of therapeutically and immunologically significant glycosphingolipids has been developed. This strategy provides a universal platform for glycosphingolipid synthesis by block coupling of enzymatically prepared free oligosaccharideglycans to lipids using glycosyl N-phenyltrifluoroacetimidates as efficient activated intermediates. As demonstrated here, two different types of glycosphingolipids were obtained in excellent yields using the method.

Toward Automated Enzymatic Synthesis of Oligosaccharides

Oligosaccharides together with oligonucleotides and oligopeptides comprise the three major classes of natural biopolymers. Automated systems for oligonucleotide and oligopeptide synthesis have significantly advanced developments in biological science by allowing nonspecialists to rapidly and easily access these biopolymers. Researchers have endeavored for decades to develop a comparable general automated system to synthesize oligosaccharides. Such a system would have a revolutionary impact on the understanding of the roles of glycans in biological systems. The main challenge to achieving automated synthesis is the lack of general synthetic methods for routine synthesis of glycans. Currently, the two main methods to access homogeneous glycans and glycoconjugates are chemical synthesis and enzymatic synthesis. Enzymatic glycosylation can proceed stereo- and regiospecifically without protecting group manipulations. Moreover, the reaction conditions of enzyme-catalyzed glycosylations are extremely mild when compared to chemical glycosylations. Over the past few years methodology toward the automated chemical synthesis of oligosaccharides has been developed. Conversely, while automated enzymatic synthesis is conceptually possible, it is not as well developed. The goal of this survey is to provide a foundation on which continued technological advancements can be made to promote the automated enzymatic synthesis of oligosaccharides.