Streptococcus suis is a largely neglected but emerging bacterial zoonotic pathogen of global concern for animal welfare, antibiotic resistance development, and human health. No effective vaccines are now available. Here, we identified and characterized the function and structure of two cell wall polysaccharide variants in pathogenic S. suis strains using genetic deletion and (heterologous) complementation, lectin staining, glycan composition analysis, and specialized NMR spectroscopy. Both glycan variants were anionic polymers that differed in the presence of glucose in the side chain as a result of allelic variation in a glycosyltransferase gene. Deletion of this variable glycosyltransferase revealed an identical glycan "core" and affected S. suis morphology and lysozyme resistance. Immunization of pigs with this core domain elicited antibodies that recognized antigenically diverse pathogenic S. suis strains and induced complement deposition on encapsulated pathogenic S. suis strains. This study provides valuable insights for developing next-generation glycoconjugate vaccines, whereby a single-glycan target could protect against the emerging zoonotic pathogen S. suis.

Solution-state NMR spectroscopy is a powerful experimental technique that provides insight into the molecular structure and dynamics of saccharides in aqueous solution. Computational tools commonly used for modeling carbohydrate conformations, such as molecular dynamics (MD) simulations and quantum mechanical (QM) calculations, provide information on the inherent dynamics of conformational changes and structure-dependent NMR parameters, respectively; however, understanding how NMR parameters depend on dynamic conformational behavior remains challenging. Herein, we present an integrated MD and QM approach to accurately determine NMR parameters, in particular 1H and 13C NMR chemical shifts of saccharides. Classical MD simulations are used to sample conformational space, and representative structures are subsequently subjected to QM calculations to obtain NMR parameters, which are averaged according to populations in different conformational states. This approach reproduces experimental NMR chemical shifts with high accuracy (MAE = 0.96 ppm for 13C and 0.066 ppm for 1H relative to experimental data) across 11 monosaccharide entities. In parallel, we establish a quantitative relationship between conformational properties of monosaccharides and the chemical shift values. In this context, empirical relationships between chemical shifts and torsional angles enable mapping of structural descriptors onto NMR observables. Furthermore, we demonstrate that the use of conformation-dependent chemical shifts allows quantitative description of conformational equilibria within monosaccharide molecules. Average chemical shift values assigned to discrete conformers (e.g., ring conformers or hydroxymethyl rotamers) and to atoms in the vicinity of torsion angle transitions are analyzed; populations in distinct conformational states are optimized by minimizing deviations from experimental NMR chemical shifts. This approach enables determination of gt:gg:tg populations of hydroxymethyl group rotamers in β-d-Glcp-OMe, α-d-Manp-OMe and α-d-Galp-OMe, as well as the chair:inverted chair ratio for the β-d-Arap-OMe six-atom membered ring. Overall, this study establishes a framework in which NMR chemical shifts serve as quantitative probes of carbohydrate conformation, complementing traditional J coupling-based analyses.

Lipopolysaccharides are important components of the gram-negative bacterial cell envelope that are involved in immune evasion and act as a protective barrier. Employing cryo-electron microscopy, we resolved the structure and dynamics of FepE, the copolymerase component of the Wzy-dependent pathway, responsible for the length modulation of very long O-antigen molecules. Comparison of the interior volumes of related copolymerases’ periplasmic domains with the volume of hydrated sugars suggests that the size of the periplasmic domain controls the length of the O-antigen, implying that polysaccharide chain polymerization occurs inside the copolymerase periplasmic domain. Moreover, we show the opening of the FepE complex as well as other large mechanistically relevant movements. The opening of the complex presents an attractive corridor for the release of completed polysaccharide chains.

A method for direct N-functionalization of unprotected amino sugars using alcohols as alkylating agents is presented. The method relies on an iridium-catalyzed hydrogen borrowing strategy, offering a direct and highly effective approach for modifying unprotected carbohydrates. This approach avoids the need for additional protection/deprotection steps, minimizing waste generation. Different amino sugars and a broad variety of alcohols can be employed, including long-chain aliphatic alcohols for the synthesis of sugar-based surfactants. A synthetic pathway for obtaining unprotected C6 amino sugars is also presented.

Background: Shigella flexneri 2a (SF2a) and 6 (SF6) are two of the most common S. flexneri serotypes. They have distant O-specific polysaccharide (O-SP) structures. Previous studies showed no cross-reactivity or cross-protection between the two serotypes in a guinea pig model of infection. However, partial cross-reactivity and cross-protection were reported in humans immunized with a SF2a lattice-type conjugate vaccine candidate comprising the chemically detoxified lipopolysaccharide (LPS) attached to recombinant Pseudomonas aeruginosa Exoprotein A (rEPA). Objectives: This study aimed at deciphering the possible cross-reactivity with heterologous SF6 strains of antibodies induced in humans by SF2a-TT15, a sun-type SF2a conjugate vaccine candidate featuring a non- O -acetylated synthetic oligosaccharide (OS) as surrogate of the detoxified LPS. Special focus was on the impact of the O-SP non-stoichiometric O -acetylation on cross-reactivity. Methods: Serum IgG antibody titers to LPSs from SF6 strains harboring different degrees of O-SP O -acetylation, and from Escherichia coli O147 (EC147) which shares an identical but non- O -acetylated O-SP with SF6, were measured by ELISA in 63 serum samples of volunteers receiving 2 µg and 10 µg OS doses of SF2a-TT15 or placebo in the frame of a phase I clinical study. Antibody in-lymphocyte-supernatants (ALS), avidity, and serum bactericidal activity (SBA) were measured in a subset of volunteers. Results: SF2a-TT15 induced cross-reacting IgG antibodies to all SF6 LPSs and EC147 LPS. A ≥4-fold rise in anti-SF6 IgG titers was more frequent with the 10 µg dose than with 2 µg (50% vs 22%, p=0.045). Cross-reactivity rate was higher with the low O -acetylated SF6 O-SP than with the high O -acetylated one (50% versus 21%, p<0.05). Anti-SF6 responses correlated with homologous anti-SF2a LPS responses. Similar cross-reactivity was detected in ALS samples at day 7 after vaccination. Cross-reacting antibodies were partially functional against the heterologous SF6 parental strains, as shown by bactericidal activity and increased avidity. Conclusions: SF2a-TT15 induces stronger SF6 cross-reactive IgG responses than the previously tested detoxified O -acetylated SF2a LPS-rEPA conjugate. While both serotypes are included in most multivalent Shigella vaccine candidates, cross-reactivity and cross-protection between SF2a and SF6 could enhance the immunogenicity and efficacy of a Shigella multivalent vaccine candidate, particularly in infants in low-and-middle-income countries, the primary target population for a Shigella vaccine. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial NCT02797236 ### Funding Statement This work was supported by grant number OPP1195433 from the Bill & Melinda Gates Foundation conferred to DC and by grants from the Swedish Research Council (no. 2022-03014) and The Knut and Alice Wallenberg Foundation. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study protocol (#2015-060) including exploratory immunological endpoints was reviewed and approved by the Tel Aviv Sourasky Medical Center Institutional Ethics Committee, Institut Pasteur Institutional Review Board, and the Israeli Ministry of Health. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Comprehensive data are currently included in the article Tables & Figures and Supplementary Material. Any additional data supporting the findings of this study without participant personal identifiers will be made available on request from the corresponding author. This manuscript will be further submitted to a journal that has an open access format and offers the CC-BY license.

The bacterium Yersinia enterocolitica serotype O:3 is targeted by two distinct agents, the bacteriophage φR1-37 and the bacteriocin-like enterocoliticin (a tailocin), which both utilize the lipopolysaccharide (LPS) outer core (OC) hexasaccharide as their primary host receptor. In order to understand this convergent recognition mechanism, we first characterized the enterocoliticin system, reporting the complete sequence of its large, biosynthetic gene cluster. Most of the 42 predicted gene products were functionally annotated by homology to known gene products. We then focused on identifying the receptor-binding proteins (RBPs) responsible for host attachment of both agents in order to elucidate a possible shared mechanism of binding. For phage φR1-37, the receptor binding complex was identified as the inseparable Gp298 tail fiber protein and its Gp297 trimerization chaperone, confirming its function as the RBP. Based on sequence identity with Gp298, the Orf39 gene product of the enterocoliticin cluster was predicted to be its corresponding RBP. An analytical comparison of the predicted RBPs revealed a highly conserved homologous region spanning 80–85 amino acid residues, which presents the only structural explanation for their identical receptor specificity. To resolve the binding mechanism, we generated high-confidence trimeric structural models for the Gp298 and Orf39 proteins using AlphaFold3-multimer. These models validated the high structural similarity of the RBP domains, despite global dissimilarity of the complete trimeric structures. Further docking simulations with a pentasaccharide ligand (generated by CarbBuilder) provided suggestive molecular models for the protein-carbohydrate interactions within the OC region. Intriguingly, a database search using the identified binding site motif revealed their wide and diverse presence in various phage tail proteins, suggesting that this motif is a specialized, common structure for carbohydrate recognition. This work identifies a conserved, novel sugar-binding motif as the molecular basis of host recognition for these key anti-Yersinia biologics.

Solution-state NMR spectroscopy is a powerful experimental technique that provides insight into the molecular structure and dynamics of saccharides in aqueous solution. Computational tools commonly used for modeling carbohydrate conformations, such as molecular dynamics (MD) simulations and quantum mechanical (QM) calculations, provide information on the inherent dynamics of conformational changes and structure-dependent NMR parameters, respectively; however, understanding how NMR parameters depend on dynamic conformational behavior remains challenging. Herein, we present an integrated MD and QM approach to accurately determine NMR parameters, in particular 1H and 13C NMR chemical shifts of saccharides. Classical MD simulations are used to sample conformational space, and representative structures are subsequently subjected to QM calculations to obtain NMR parameters, which are averaged according to populations in different conformational states. This approach reproduces experimental NMR chemical shifts with high accuracy (MAE = 0.96 ppm for 13C and 0.066 ppm for 1H relative to experimental data) across 11 monosaccharide entities. In parallel, we establish a quantitative relationship between conformational properties of monosaccharides and the chemical shift values. In this context, empirical relationships between chemical shifts and torsional angles enable mapping of structural descriptors onto NMR observables. Furthermore, we demonstrate that the use of conformation-dependent chemical shifts allows quantitative description of conformational equilibria within monosaccharide molecules. Average chemical shift values assigned to discrete conformers (e.g., ring conformers or hydroxymethyl rotamers) and to atoms in the vicinity of torsion angle transitions are analyzed; populations in distinct conformational states are optimized by minimizing deviations from experimental NMR chemical shifts. This approach enables determination of gt:gg:tg populations of hydroxymethyl group rotamers in beta-d-Glcp-OMe, alpha-d-Manp-OMe and alpha-d-Galp-OMe, as well as the chair:inverted chair ratio for the beta-d-Arap-OMe six-atom membered ring. Overall, this study establishes a framework in which NMR chemical shifts serve as quantitative probes of carbohydrate conformation, complementing traditional J coupling-based analyses.

In mammals, glucose transporters (GLUTs) mediate organism-wide sugar distribution, yet the molecular basis of substrate specificity remains unclear. The bacterial xylose transporter XylE serves as a model for GLUTs. However, although xylose and glucose bind with a similar affinity, xylose is transported, but glucose acts as an inhibitor. Here, using saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, we distinguished transported sugars from sugar inhibitors. Our findings revealed that only transported sugars generate STD NMR signals, which are abolished for xylose when XylE is trapped in either outward- or inward-facing conformations. Engineering the sugar-binding pocket and gating helix TM7b enabled glucose transport by XylE and corresponding STD signals. Using complementary molecular dynamics simulations, together with structural, biochemical and STD NMR analysis of related parasitic and mammalian GLUTs, we identified TM7b as a key determinant of occluded state formation. We conclude that, rather than the initial substrate-binding event observed in experimental structures, formation of a substrate-induced transition-state intermediate is the primary determinant of specificity in transporters.

Listeria monocytogenes is a ubiquitous, psychrotrophic human pathogen that can cause listeriosis, a serious illness for vulnerable populations. Some foods, such as Hispanic-style fresh cheeses like queso fresco, pose a specific risk because there are no widely accepted or available methods for L. monocytogenes mitigation that are both effective and able to maintain the properties of the products. Listeria-specific bacteriophages encode endolysins that can cleave the peptidoglycan layer of L. monocytogenes cells externally, showing promise as a potential solution to this problem. PlyP100, from the GRAS Listeria phage P100, is one such endolysin that can prevent the growth of L. monocytogenes in both lab culture conditions and a miniaturized queso fresco model. In this work, we aimed to understand the structural and functional properties of PlyP100. An AlphaFold prediction suggested the presence of three separate domains (D1, D2, and D3). By solving the crystal structure of D1 and assessing various domain truncations, we present evidence that D1 is responsible for catalytic activity, D3 is sufficient for cell wall binding, and D2 is necessary for full function of the enzyme against live cells. Additionally, we performed point mutations in D1 and compared PlyP100 to proteins with similar structures, including Streptococcus pneumoniae LytA and Listeria endolysin Ply511, to understand its specific enzymatic mechanism and target strain specificity. These insights into the structure and function of PlyP100 will aid future work aiming to engineer better endolysins as safe food antimicrobials.

The serological properties of the O-antigen polysaccharide region of the lipopolysaccharides are used to differentiate E. coli strains into serogroups. In this study, we report the structure elucidation of the O-specific chain of E. coli O179 using NMR data, the program CASPER and analysis of biosynthetic information available in the E. coli O-antigen Database (ECODAB). The presence of genes that encode enzymes involved in the biosynthesis of the GDP-Man and UDP-GlcA within the O-antigen gene cluster of the bacteria indicates that the corresponding residues could be present in the polysaccharide. Furthermore, the occurrence of four genes that encode for glycosyltransferases indicates that the polysaccharide is composed of pentasaccharide repeating units; a bioinformatics approach based on predictive glycosyltransferase functions present in ECODAB revealed that the β-d-Manp-(1→4)-β-d-Manp-(1→3)-d-GlcpNAc structural element could be present in the O-specific chain. NMR spectroscopy data obtained from homonuclear and heteronuclear 2D NMR spectra (1H,1H-TOCSY, 1H,13C-HSQC, 1H,13C-H2BC and 1H,13C-HMBC) were analyzed using the CASPER program, revealing the following arrangement of monosaccharide residues as the most probable structure: →4)-α-d-GlcpA-(1→3)-[β-d-Glcp-(1→2)]β-d-Manp-(1→4)-β-d-Manp-(1→3)-β-d-GlcpNAc-(1→, which was further confirmed using 2D homonuclear 1H,1H-COSY and 1H,1H-NOESY spectra. The functions of the α-gluconosyltransferase and the β-glucosyltransferase were predicted using structural alignment of AlphaFold-predicted 3D structures. This O-antigen polysaccharide shares structural similarities with those of E. coli O6 and O188, S. boydii type 16, and the capsular polysaccharide of E. coli K43, explaining the serological cross-reactivities observed with strains belonging these O- and K-antigen groups.

Streptococcus suis is a largely neglected but emerging bacterial zoonotic pathogen of global concern for animal welfare, antibiotic resistance development and human health. No effective vaccines are currently available. Here, we identified and characterized the function and structure of two cell wall polysaccharide variants in pathogenic S. suis strains using genetic deletion and (heterologous) complementation, lectin staining, glycan composition analysis and specialized NMR spectroscopy. Both glycan variants were anionic polymers that differed in the presence of glucose in the side-chain as a result of allelic variation in a glycosyltransferase gene. Deletion of this variable glycosyltransferase revealed an identical glycan ‘core’ and affected S. suis morphology and lysozyme resistance. Immunization of pigs with this core domain induced antibodies recognizing a wide range of antigenically-diverse pathogenic S. suis strains. This study provides new insights for developing next-generation glycoconjugate vaccines, whereby a single-glycan target could protect against the emerging zoonotic pathogen S. suis . ### Competing Interest Statement A.A.C.J. and R.G. are employed by MSD Animal Health where a vaccine based on another antigen is currently under development. The other authors declare no competing interests. Netherlands Center for One Health, LSHM19137 Health~Holland, LSHM19137 Swedish Research Council, https://ror.org/03zttf063, 2022-03014 Knut and Alice Wallenberg Foundation, https://ror.org/004hzzk67 China Scholarship Council, https://ror.org/04atp4p48, CSC201909110078 Marie Sk łodowska-Curie grant, 861194 United States Department of Energy, DE-SC0015662

This study introduces a novel enzymatic cascade featuring five recombinant enzymes for the efficient synthesis of fucosylated glycosides, using sucrose exclusively as the sugar donor substrate. In our approach, we employed a sucrose synthase sourced from tomato to generate GDP-glucose from sucrose and GDP. By repurposing CDP-tyvelose 2-epimerase from Salmonella enterica, chosen for its catalytic efficiency from a panel of 27 CDP-tyvelose 2-epimerase candidates, it was possible to epimerize GDP-glucose into GDP-mannose. The subsequent transformation of GDP-d-mannose to GDP-l-fucose was achieved by GDP-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-d-mannose epimerase/reductase, also derived from S. enterica. In the final stage, Helicobacter pylori α1,3-fucosyltransferase was employed to fucosylate para-nitrophenyl β-lactoside, resulting in the production of para-nitrophenyl 3-fucosyllactoside with a conversion of more than 40%. Analysis of the synthesized compound by LC-MS and NMR analyses substantiated its structure. This investigation not only highlights the utility of this five-enzyme fucosylation cascade but also establishes a novel methodological paradigm for the biocatalytic production of α-l-fucosides for biochemical research and for biotechnological applications.

Methyl 2,3-di-O-benzyl-α-d-(4-2H)-glucopyranoside, C21H25DO6, is an intermediate used in synthesis of oligosaccharides. The hexopyranose ring has the 4C1 chair conformation in the crystal structure. The exocyclic groups of the hexose sugar show for the glycosidic torsion angle ϕ =−52.8° and for the hydroxymethyl group the gauche-gauche conformation with ω = −64.7°, one of the two main orientations of the latter group in hexopyranose sugars that have the gluco-configuration, i.e., with an equatorial hydroxyl group at C4. The benzene rings of the benzyl groups are arranged with an angle of 56.9° to each other within the molecule and show intramolecular as well as intermolecular C-H···π interactions. A chain of intermolecular hydrogen bonds exists along the b-axis involving O4 and O6 atoms. The experimentally observed peak in the infrared spectrum at 2159 cm− 1 was ascribed to the stretching of the C4–D4 bond based on DFT calculations.

In posttranslational modifications of proteins and peptides by glycosylation, the two major classes are N-linked and O-linked glycans. The sugar residue proximal to the peptide chain is in N-glycans linked to L-asparagine, and in O-linked glycans, it is linked to either L-serine, L-threonine, or L-tyrosine, although other amino acids may be glycosylated. Identifying and assigning the 1H and 13C nuclear magnetic resonance (NMR) chemical shifts of these glycoconjugates are a prerequisite for structural characterization as well as for subsequent conformational and interaction studies thereof. The web-based computer program CASPER ( http://www.casper.organ.su.se/casper ) is a tool that provides prediction of 1H and 13C NMR chemical shift for glycans, as well as those linked to L-Asn, L-Ser, L-Thr, or L-Tyr, for which the predicted NMR chemical shifts of the glycan show good agreement to those from NMR experiments of glycopeptides and glycoproteins. This highlights that an approximation in which a single amino acid is present at the reducing end of the glycan structure is sufficient to predict NMR data well, as shown for different N-linked and O-linked glycans of various complexity.

Streptococcus uberis is a causative pathogen of bovine mastitis with high genetic diversity. Rhamnose-rich polysaccharides (RPS) are abundant surface structures covalently anchored to peptidoglycan and represent promising vaccine candidates for several streptococcal pathogens. It was previously reported that the RPS of S. uberis strain 233 is composed of a repeating → 2)-α-l-Rhap-(1 → 3)-α-l-Rhap-(1 → disaccharide backbone decorated with α-d-Glcp side-chains. In this study, we identified a hitherto unknown glycerol phosphate (GroP) modification at the 6-OH of the Glc residue in S. uberis 233 RPS using nuclear magnetic resonance analysis. Comparative genomic analysis of 592 S. uberis genomes revealed significant diversity in the RPS biosynthesis gene cluster with six major RPS genotypes. RPS genotypes 1–4, representing 97.5

Outer membrane (OM) proteins play a vital role in the physiology of Gram-negative bacteria, and outer membrane protein F (OmpF) is one of the most studied porins in Escherichia coli. In this study, we have developed a comprehensive E. coli OM model with lipopolysaccharides (LPS), enterobacterial common antigen (ECA), and capsular polysaccharides (CPS) in the outer leaflet and with phospholipids in the inner leaflet. Using extensive all-atom molecular dynamics simulations of OmpF in this realistic asymmetric OM environment, we have investigated the structure and dynamics of OmpF within the OM and its interactions with the OM. The results demonstrate that the presence of ECA and CPS enhances the rigidity and stability of the OM while reducing the pore size of OmpF and increasing its cation selectivity. The complex and diverse interactions between OmpF and LPS/ECA/CPS contribute to these effects, resulting in a rigid and compact OmpF structure. These findings provide new insights into the complex interplay between bacterial OM components and OmpF porin, with potential implications for understanding bacterial resistance and developing novel antimicrobial strategies.

Streptococcus mutans, the causative agent of human dental caries, expresses a cell wall attached Serotype c- specific Carbohydrate (SCC) that is critical for cell viability. SCC consists of a repeating →3)α-Rha(1→2)α-Rha(1→ polyrhamnose backbone, with glucose (Glc) side-chains and glycerol phosphate (GroP) decorations. This study reveals that SCC has one major and two minor Glc modifications. The major Glc modification, α-Glc, attached to position 2 of 3-rhamnose, is installed by SccN and SccM glycosyltransferases and is the site of the GroP addition. The minor Glc modifications are β-Glc linked to position 4 of 3-rhamnose installed by SccP and SccQ glycosyltransferases, and α-Glc attached to position 4 of 2-rhamnose installed by SccN working in tandem with an unknown enzyme. Both the major and the minor β-Glc modifications control bacterial morphology, but only the GroP and major Glc modifications are critical for biofilm formation.

Group A Streptococcus (Strep A) is a human-exclusive bacterial pathogen killing annually more than 500,000 patients, and no current licensed vaccine exists. Strep A bacteria are highly diverse, but all produce an essential, abundant, and conserved surface carbohydrate, the Group A Carbohydrate, which contains a rhamnose polysaccharide (RhaPS) backbone. RhaPS is a validated universal vaccine candidate in a glycoconjugate prepared by chemical conjugation of the native carbohydrate to a carrier protein. We engineered the Group A Carbohydrate biosynthesis pathway to enable recombinant production using the industry standard route to couple RhaPS to selected carrier proteins within Escherichia coli cells. The structural integrity of the produced recombinant glycoconjugate vaccines was confirmed by Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry. Purified RhaPS glycoconjugates elicited carbohydrate-specific antibodies in mice and rabbits and bound to the surface of multiple Strep A strains of diverse M-types, confirming the recombinantly produced RhaPS glycoconjugates as valuable vaccine candidates.

β-1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme in the biosynthesis of glycosaminoglycans (GAG) that transfers the first galactose unit to xylose in the linker region. Searching for new inhibitors of the GAG biosynthesis, we used saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy to evaluate the binding interactions between β4GalT7 and several pentosides in the presence of UDP donors. These investigations verified the glycosylation specificity of β4GalT7 and revealed that the naphthalene and the uridine moieties were significant contributors to the binding of the acceptor and the donor, respectively, while the galactose part was less important. Based on these findings, we set out to investigate conjugates of UDP and naphthoxylosides to function as transition state analogues. These compounds were synthesized using a one-pot procedure and tested as inhibitors in a β4GalT7 assay. Interestingly, one truncated analogue, a bisphosphonate-xyloside construct, showed a significant inhibition (IC50: 188 μM). These findings open for the design of a new class of inhibitors of the GAG biosynthesis.

In galactosylation by β4GalT7 of 2-naphthyl xylosyl sulfoxides, higher affinity for the enzyme was observed for the acceptor substrate having the ( R ) S -configuration than for the diastereomer with the ( S ) S -configuration.