A gram-scale synthesis of β-L-carbafucose for engineering antibody glycans V. Narasimharao Thota, Anthony Fers-Lidou, Matthew Nodwell, Anthony W. McDonagh, Pierre-André Gilormini, Yang Wang, Carolyn Leung, David J. Vocadlo, Robert Britton Communications Chemistry, 2025 Afucosylated antibodies often exhibit superior properties compared to their fucosylated counterparts including, among others, enhanced antibody-dependent cell cytotoxicity (ADCC). While several recombinant and biochemical strategies have been identified for generating afucosylated antibodies, small molecule metabolic inhibitors provide a potentially more straightforward option. We recently reported that β-L-carbafucose is an inhibitor of antibody fucosylation and is not incorporated into the antibody glycans. To support the further study of β-L-carbafucose, a gram-scale synthesis was needed. Here, we report our investigation of three distinct synthetic routes, including a highly efficient chromatography-free synthesis. Further, we demonstrate multi-gram production of afucosylated Herceptin (Trastuzumab®) in 10 L bioreactors using β-L-carbafucose. We expect this new synthetic process will support the widespread adoption of β-L-carbafucose for producing afucosylated antibodies for discovery and development purposes. Afucosylated antibodies, known for enhanced antibody-dependent cell cytotoxicity, require efficient production methods for broader application. Here, the authors explore three synthetic routes for β-L-carbafucose as a metabolic inhibitor of antibody fucosylation, achieving a chromatography-free synthesis, and demonstrating a multi-gram production of afucosylated Herceptin using β-L-carbafucose.
Efficient Synthesis of p-Tolyl 6-Deoxy-d-Heptopyranose Thioglycosides V. Narasimharao Thota, Todd L. Lowary Israel Journal of Chemistry, 2025 Capsular polysaccharides from a range of bacteria, notably campylobacters, possess 6‐deoxy‐heptose residues. Synthetic fragments of these polysaccharides have myriad applications in both fundamental and applied research, but few efficient routes for accessing these monosaccharides in a form suitable for glycosylation reactions are available. The synthesis of thioglycosides of 6‐deoxy‐ d ‐ manno ‐, 6‐deoxy‐ d ‐ talo ‐, and 6‐deoxy‐ d ‐ altro ‐heptoses from a single commercially available and inexpensive monosaccharide, methyl α‐ d ‐mannopyranoside, is reported here. The route involves C‐6 homologation and, in some cases, configurational inversion at C‐3 or C‐4. These routes provide access to glycosyl donors of three of the four naturally occurring 6‐deoxy‐ d ‐heptopyranoses, complementing previous work that has provided glycosyl donors of fourth such monosaccharide, 6‐deoxy‐ d ‐ ido ‐heptopyranose.
Klebsiella pneumoniae O-polysaccharide biosynthesis highlights the diverse organization of catalytic modules in ABC transporter-dependent glycan assembly Steven D. Kelly, Danielle M. Williams, Shawna Zhu, Taeok Kim, Manas Jana, Jeremy Nothof, V. Narasimharao Thota, Todd L. Lowary, Chris Whitfield Journal of Biological Chemistry, 2024 Klebsiella pneumoniae provides influential prototypes for lipopolysaccharide O antigen (OPS) biosynthesis in Gram-negative bacteria. Sequences of OPS-biosynthesis gene clusters in serotypes O4 and O7 suggest fundamental differences in the organization of required enzyme modules compared to other serotypes. Furthermore, some required activities were not assigned by homology shared with characterized enzymes. The goal of this study was therefore to resolve the serotype O4 and O7 pathways to expand our broader understanding of glycan polymerization and chain termination processes. The O4 and O7 antigens were produced from cloned genetic loci in recombinant Escherichia coli. Systematic in vivo and in vitro approaches were then applied to assign each enzyme in each of the pathways, defining the necessary components for polymerization and chain termination. OPS assembly is accomplished by multiprotein complexes formed by interactions between polymerase components variably distributed in single and multimodule proteins. In each complex, a terminator function is present in a protein containing a characteristic coiled-coil molecular ruler, which determines glycan chain length. In serotype O4, we discovered a CMP-α-3-deoxy-ᴅ-manno-octulosonic acid-dependent chain-terminating glycosyltransferase that is the founding member of a new glycosyltransferase family (GT137) and potentially identifies a new glycosyltransferase fold. The O7 OPS is terminated by a methylphosphate moiety, like the K. pneumoniae O3 antigen, but the methyltransferase-kinase enzyme pairs responsible for termination in these serotypes differ in sequence and predicted structures. Together, the characterization of O4 and O7 has established unique enzyme activities and provided new insight into glycan-assembly strategies that are widely distributed in bacteria.
A metabolic inhibitor blocks cellular fucosylation and enables production of afucosylated antibodies Pierre-André Gilormini, V. Narasimharao Thota, Anthony Fers-Lidou, Roger A. Ashmus, Matthew Nodwell, Jacob Brockerman, Chu-Wei Kuo, Yang Wang, Taylor E. Gray, Nitin, Anthony W. McDonagh, Shih-Yun Guu, Nursah Ertunc, Dominick Yeo, Wesley F. Zandberg, Kay-Hooi Khoo, Robert Britton, David J. Vocadlo Proceedings of the National Academy of Sciences of the United States of America, 2024 The fucosylation of glycoproteins regulates diverse physiological processes. Inhibitors that can control cellular levels of protein fucosylation have consequently emerged as being of high interest. One area where inhibitors of fucosylation have gained significant attention is in the production of afucosylated antibodies, which exhibit superior antibody-dependent cell cytotoxicity as compared to their fucosylated counterparts. Here, we describe β-carbafucose, a fucose derivative in which the endocyclic ring oxygen is replaced by a methylene group, and show that it acts as a potent metabolic inhibitor within cells to antagonize protein fucosylation. β-carbafucose is assimilated by the fucose salvage pathway to form GDP-carbafucose which, due to its being unable to form the oxocarbenium ion-like transition states used by fucosyltransferases, is an incompetent substrate for these enzymes. β-carbafucose treatment of a CHO cell line used for high-level production of the therapeutic antibody Herceptin leads to dose-dependent reductions in core fucosylation without affecting cell growth or antibody production. Mass spectrometry analyses of the intact antibody and N -glycans show that β-carbafucose is not incorporated into the antibody N -glycans at detectable levels. We expect that β-carbafucose will serve as a useful research tool for the community and may find immediate application for the rapid production of afucosylated antibodies for therapeutic purposes.
Synthesis of 6-deoxy-D-ido-heptopyranose-containing fragments of the Campylobacter jejuni strain CG8486 capsular polysaccharide V. Narasimharao Thota, Todd L. Lowary Carbohydrate Research, 2024 Campylobacters are important causes of gastrointestinal illness and the capsular polysaccharides (CPS) they produce are key virulence factors and targets for vaccine development. We report here the synthesis of two fragments of the Campylobacter jejuni CG8486 strain CPS that contain a rare 6-deoxy-d-ido-heptopyranose residue and, in one target, two O-methyl phosphoramidate (MeOPN) motifs. The synthetic approach features the stereoselective construction of the β-d-ido-heptopyranoside linkage via glycosylation with a β-d-galacto-heptopyranoside donor followed by a one-pot sequential C-2 and C-3 inversion. During the syntheses, we uncovered a number of interesting conformational effects with regard to the 6-deoxy-ido-heptopyranose ring, the glycosidic linkage connecting the two monosaccharides, and the MeOPN groups.
One-Pot Regioselective Diacylation of Pyranoside 1,2- cis Diols Taeok Kim, Michael R. Bell, V. Narasimharao Thota, Todd L. Lowary Journal of Organic Chemistry, 2022 A one-pot strategy for functionalizing pyranoside 1,2-cis-diols with two different ester protecting groups is reported. The approach employs regioselective acylation via orthoester hydrolysis promoted by a carboxylic acid, e.g., levulinic acid, acetic acid, benzoic acid, or chloroacetic acid. Upon removal of water and introduction of a coupling agent, the carboxylic acid is esterified to the hydroxyl group liberated during hydrolysis. Although applied to 1,2-cis-diols on pyranoside scaffolds, the method should be applicable to such motifs on any six-membered ring.
Synthesis of the Campylobacter jejuni 81-176 Strain Capsular Polysaccharide Repeating Unit Reveals the Absolute Configuration of its O-Methyl Phosphoramidate Motif V. Narasimharao Thota, Michael J. Ferguson, Ryan P. Sweeney, Todd L. Lowary Angewandte Chemie International Edition, 2018 The O‐methyl phosphoramidate (MeOPN) motif is a non‐stoichiometric modification of capsular polysaccharides (CPS) in ≈70 % of all Campylobacter jejuni strains. Infections by C. jejuni lead to food‐borne illnesses and the CPS they produce are key virulence factors. The MeOPN phosphorus atom in these CPS is stereogenic and is found as a single stereoisomer. However, to date, the absolute stereochemistry at this atom has been undefined. We report the synthesis of the three repeating units found in C. jejuni 81‐176 CPS; one of these possesses a MeOPN group. In the course of these studies we established that the stereochemistry of the phosphorus atom in this MeOPN group is R. These studies represent the first unequivocal proof of stereochemistry of this group in any C. jejuni CPS. The compounds produced are anticipated to be useful tools in investigations targeting the function and biosynthesis of this structurally‐interesting modification, which so far has only been identified in campylobacter.
Synthesis of β-C-galactosyl d- and l-alanines V. Narasimharao Thota, Jacquelyn Gervay-Hague, Suvarn S. Kulkarni Organic and Biomolecular Chemistry, 2012 Synthesis of β-C-D-galactosyl D- and L-alanines is carried out via a highly stereoselective Grignard reaction of glycosyl iodides, Sharpless dihydroxylation and S(N)2 displacement of the corresponding mesylate or tosylate. Alternatively, attempted triflation of the intermediate alcohols triggers a stereoselective debenzylative cyclization leading to interesting bicyclic trans-fused compounds.
