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Structure–Activity Relationship of Fluorinated Sialic Acid Inhibitors for Bacterial Sialylation
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  • Sam J. Moons
    Sam J. Moons
    Cluster of Molecular Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen 6525 AJ, The Netherlands
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  • Emiel Rossing
    Emiel Rossing
    Cluster of Molecular Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen 6525 AJ, The Netherlands
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  • Jurriaan J. A. Heming
    Jurriaan J. A. Heming
    Cluster of Molecular Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen 6525 AJ, The Netherlands
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  • Mathilde A. C. H. Janssen
    Mathilde A. C. H. Janssen
    Cluster of Molecular Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen 6525 AJ, The Netherlands
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  • Monique van Scherpenzeel
    Monique van Scherpenzeel
    Translational Metabolic Laboratory, Department of Neurology, Donders Center for Brain Cognition and Behavior, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands
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  • Dirk J. Lefeber
    Dirk J. Lefeber
    Translational Metabolic Laboratory, Department of Neurology, Donders Center for Brain Cognition and Behavior, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands
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  • Marien I. de Jonge
    Marien I. de Jonge
    Laboratory of Medical Immunology, Radboud Center for Infectious Diseases, Radboud Institute for Molecular Sciences, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands
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  • Jeroen D. Langereis*
    Jeroen D. Langereis
    Laboratory of Medical Immunology, Radboud Center for Infectious Diseases, Radboud Institute for Molecular Sciences, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands
    *Email: [email protected]
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  • Thomas J. Boltje*
    Thomas J. Boltje
    Cluster of Molecular Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen 6525 AJ, The Netherlands
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0001-9141-8784
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Bioconjugate Chemistry

Cite this: Bioconjugate Chem. 2021, 32, 6, 1047–1051
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https://doi.org/10.1021/acs.bioconjchem.1c00194
Published May 27, 2021

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Abstract

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Bacterial pathogens such as Nontypeable Haemophilus influenzae (NTHi) can evade the immune system by taking up and presenting host-derived sialic acids. Herein, we report a detailed structure–activity relationship of sialic acid-based inhibitors that prevent the transfer of host sialic acids to NTHi. We report the synthesis and biological evaluation of C-5, C-8, and C-9 derivatives of the parent compound 3-fluorosialic acid (SiaNFAc). Small modifications are tolerated at the C-5 and C-9 positions, while the C-8 position does not allow for modification. These structure–activity relationships define the chemical space available to develop selective bacterial sialylation inhibitors.

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Subjects

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Gram negative bacterium Nontypeable Haemophilus influenzae (NTHi) is a commensal organism in the upper respiratory tract microbiome, which can become an opportunistic pathogen in children and the elderly. (1,2) NTHi has evolved the ability to evade the immune system by expressing host-like molecular structures at its cell surface such as host-derived sialic acids. (3) The switch of NTHi from a symbiotic colonizing bacterium to an opportunistic pathogen is associated with uptake and expression of host sialic acids on its lipooligosaccharide (LOS). (4)
Sialic acids are complex nine-carbon sugars abundantly expressed on human glycoconjugates and can be released by the action of sialidases. These released sialic acids can be taken up by NTHi using a tripartite ATP-independent periplasmic (TRAP) transporter system and utilized for LOS sialylation or as a carbon source. (5,6) NTHi expresses SiaP to take up sialic acid, SiaB to generate cytidine monophosphate (CMP) sialic acid, which is the donor substrate for sialyltransferases LsgB, Lic3A, and Lic3A2 that enable LOS sialylation (4) (Figure 1). Alternatively, sialic acid is converted by aldolase NanA to enable the utilization of sialic acid as a carbon source. A clear link between virulence and sialic acid utilization by NTHi has been established, and LOS sialylation has been shown to confer NTHi resistance to serum killing and increases biofilm formation. (7,8) Conversely, NTHi strains defective in sialic acid utilization are no longer virulent in animal models for otitis media highlighting the therapeutic potential of the NTHi sialic acid utilization pathway. (9)

Figure 1

Figure 1. Sialic acid utilization in NTHi. Host-derived sialic acids are taken up by the SiaPQM transporter system. Intracellular sialic acid is either used as a carbon source (via NanA) or used to sialylate the lipooligosaccharide (LOS). To this end, sialic acid is CMP activated by SiaB and incorporated by sialyltransferases Lic3A, Lic3A2, and/or LsgB. (10)

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We have recently reported selective sialic acid-based inhibitors of sialylation in NTHi or host-derived cells. (11) The peracetylated sialic acid inhibitor enabled passive diffusion of the membrane of host cells but not NTHi. Conversely, unprotected sialic acid inhibitors showed active uptake via the TRAP transporter of NTHi (SiaP) but were not taken up by host cells. Selective inhibition of NTHi LOS sialylation using fluorinated sialic acid (SiaFNAc) in NTHi led to a reduced serum resistance confirming that targeting this evasive mechanism in NTHi is a promising therapeutic strategy. In mammalian cells, we have optimized the sialic acid inhibitor potency by modifying the C-5 position of the sialic acid scaffold. We found that C-5 carbamate modified sialic acids showed increased metabolic conversion by N-acylneuraminate cytidylyltransferase (CMAS) leading to higher intracellular concentrations of the active CMP-sialic acid derivative. (12) This prompted us to also carry out an extensive structure–activity relationship of fluorinated sialic acids for testing in NTHi as well. Herein, we report the synthesis and biological evaluation of C-5, C-8, and C-9 derivatives of fluorinated sialic acid.

Results and Discussion

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Design, Synthesis, and Evaluation of C-5 Modified 3-Fluorosialic Acid Derivatives

Since the C-5 carbamate derivatives of 3-fluorosialic acid showed increased metabolism to the corresponding CMP derivative in mammalian cells, we set out to prepare a set of C-5 modified 3-fluorosialic acids to evaluate the effect on the use in NTHi. (12) This requires derivatives devoid of protecting groups as sialic acid is actively taken up by the NTHi TRAP system. Hence, we opted to use a chemoenzymatic strategy starting from d-mannosamine·HCl (Scheme 1). Acylation reactions proceeded with moderate to excellent yields (52–96%) to afford a set of carbamate (1a–1d), (13) amide (1e–1j), (13−16) and formyl (1k) modified mannosamines. A subsequent reaction with fluoropyruvate catalyzed by neuraminic acid lyase (NAL) afforded 3-fluorosialic acids 2a–2k in poor to good yield (22–99%) and with the desired axial substitution in line with earlier reports. (17) To evaluate an entirely different chemotype, we also prepared methanesulfonyl derivative 2l. In this case, NAL did not accept the methanesulfonyl mannosamine as a substrate, so a chemical procedure starting from sialic acid glycal 3 was used instead. Introduction of the mesyl group and subsequent electrophilic fluorination with SelectFluor, followed by ester hydrolysis, afforded derivative 2l.

Scheme 1

Scheme 1. Synthesis of C-5 Modified SiaFNAc Inhibitorsa
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a(i) The synthesis of 1a–1j has been described previously; (13−16)1k: MeOH, NaOMe, TEA, methyl formate; (ii) Sodium fluoropyruvate, H2O, Neu5Ac aldolase, 37 °C; (iii) 1. MsCl, DIPEA, DCM, 0 °C; 2. NaOMe, MeOH; (iv) 1. Selectfluor, DMF, H2O; 2. NaOH, H2O.

To evaluate the efficacy of sialic acid-based inhibitors on LOS sialylation, we used an assay to measure LOS sialylation in NTHi. Neuraminic acid incorporation into NTHi LOS was measured using a metabolic sialic acid glycoengineering approach with azidoacetyl modified sialic acid (SiaNAz). NTHi bacteria were grown in sialic acid-deficient medium supplemented with SiaNAz, and incorporation into LOS was detected by reacting them with biotin-alkyne using copper-catalyzed alkyne–azide cycloaddition (CuAAC) followed by staining with fluorescent streptavidin. Evaluation of the ability of inhibitors 2a–2l to block the metabolic incorporation of SiaNAz was done by growing NTHi in defined media containing 100 μM SiaNAz and increasing concentrations of 2a–2l (0.1–1000 μM). (18) It is important to note that the inhibitors were used to outcompete an unnatural sialic acid (SiaNAz) and therefore likely differ from their ability to outcompete the natural substrate, SiaNAc. Nevertheless, it is expected that the relative ability of 2a–2l to outcompete SiaNAz is consistent when translated to their ability to inhibit the incorporation of SiaNAc.
The derivative most closely related to the natural substrate SiaNAc is SiaFNAc, and it also proved to be the best inhibitor with an EC50 of 0.085 μM (Figure 2). Previously, we reported an IC50 of 0.7 μM. (11) The difference in inhibitory capacity is likely caused by the change in growth conditions (shaking versus static growth), resulting in a more efficient inhibition of SiaNAz incorporation. Modification of the C-5 amide to various carbamates reduced their inhibitory potency compared to SiaFNAc. The methyl and allyl carbamates 2a and 2c did show inhibition in contrast to ethyl and propargyl carbamates 2b and 2d. The activity of the carbamate series is very different from the mammalian setting where the C-5 ethyl and propargyl carbamates proved to be much better inhibitors than the corresponding natural C-5 acetamido counterpart. Interestingly, in NTHi, it is clear that the amide series performs better with the acetamido proving to be the most potent inhibitor. Hence, a set of amides derivatives varying the chain length of the amide was evaluated next (2e–2g). Increasing the chain length led to a decrease of inhibitory potency with the inhibitory potency order of acetyl > propionyl > azidoacetyl. The electronics of the amide were also varied by introducing a mono-, di-, or trifluoroacetamido group at C-5 (2h–2j). A clear trend was found indicating that increasing the degree of fluorination leads to a corresponding decrease in inhibitory potency. Hence, increasingly bulky and electron poor amides lead to a loss of inhibitory potency. Finally, the formyl and mesyl derivatives 2k and 2l were tested. Even though these groups differ substantially from the parent acetamido compound, they did still show inhibition albeit with much decreased potency. These results demonstrate that the C-5 position of SiaFNAc is amenable to modification, yet all modifications lead to loss of inhibitory potency compared to the SiaFNAc parent structure and are in line with earlier research, which shows preferential utilization of SiaNAc compared to the C-5 modified SiaNGc by NTHi. (19)

Figure 2

Figure 2. Inhibitory potency and EC50 curves of C-5 modified SiaFNAc inhibitors on the incorporation of 100 μM SiaNAz by NTHi. EC50 values were calculated based on the fitted S-curves, see the Supporting Information for more details.

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To investigate the mechanism of sialylation inhibition by SiaFNAc, we performed a nucleotide sugar analysis on NTHi grown in minimal media with DMSO or 100 μM SiaFNAc. In the samples treated with SiaFNAc, CMP-SiaFNAc was clearly detected and represented the major portion of the nucleotide sugar pool (Figure S1). This is consistent with a mechanism where SiaFNAc is transported by SiaPQM and converted by SiaB into CMP-SiaFNAc which subsequently blocks the activity of the NTHi sialyltransferases. This mechanism is analogous to the mechanism observed for the mammalian sialylation pathway. (12,20,21) However, in the mammalian setting, we have observed that C-5 carbamate derivatives of SiaFNAc lead to higher intracellular concentrations of their corresponding CMP derivatives and a more potent inhibition of sialylation. (12) This is attributed to their more efficient conversion by CMAS. Possibly, SiaB in NTHi is less tolerant of C-5 modified sialic acids leading to a lower metabolic conversion to CMP analogues.

Design, Synthesis, and Evaluation of 3-Fluorosialic Acid Derivatives Modified at the Glycerol Side Chain

The glycerol side chain of sialic acid is one of the features that makes sialic acid stand out from other mammalian monosaccharides and known to undergo modification such as acylation, sulfation, and methylation. (22,23) We were interested in probing to what extent the glycerol side chain contributes to the recognition and activity of SiaFNAc. Hence, we investigated the impact of modifications at the glycerol side chain of SiaFNAc by preparing truncated derivatives 8 and 9 and 9-azido derivative 11 (Scheme 2). Sialic acid glycal 5 was converted using Malaprade oxidation (24) followed by a sodium borohydride reduction to afford truncated derivatives 6 and 7. Subsequent electrophilic fluorination using SelectFluor (25) followed by ester hydrolysis afforded octulosonic acid 8 and heptulosonic acid 9. 9-Azido derivative 11 was also obtained from sialic acid glycal 5 by C-9 tosylation, azidation, and subsequent fluorination and hydrolysis.

Scheme 2

Scheme 2. Synthesis of C-8 and C-9 Modified SiaFNAc Inhibitorsa
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a(i) 1. NaIO4, MeOH; 2. NaBH4, MeOH; (ii) 1. Selectfluor, DMF, H2O; 2. NaOH, MeOH; (iii) 1. TsCl, pyridine; 2. NaN3, acetone, H2O.

Testing of derivatives 8, 9, and 11 was carried out using the aforementioned protocol. The inhibitory potency of 8 and 9 demonstrates that truncation of the glycerol sides chain was tolerated to some extent (Figure 3). Introduction of a C-9 azide onto the SiaFNAc scaffold led to a very notable reduction of the inhibitory potency (11). In contrast, truncation of the carbon skeleton by one carbon (octulosonic acid 8) led to a less dramatic loss of activity. However, heptulosonic acid 9 did not show any inhibitory activity.

Figure 3

Figure 3. Inhibitory potency of SiaFNAc inhibitors modified at the glycerol side chain.

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Conclusion

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We have prepared and tested a number of C-5, C-8, and C-9 derivatives of 3-fluorosialic acid. In contrast to sialylation in mammalian cells, C-5 carbamate derivatives are much less potent inhibitors than the corresponding C-5 amides in NTHi. This indicates that the enzymes involved in the NTHi LOS sialylation pathway differ substantially from those in the mammalian biosynthesis and are less tolerant for modification of the sialic acid scaffold. Alteration to the glycerol side chain was tolerated to some extent. These results provide clear guidelines for future inhibitor development and may also be the basis for the incorporation of unnatural sialic acids lacking the 3-fluoride.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.1c00194.

  • General procedures, experimental procedure for synthesis and biological evaluation, and NMR data (PDF)

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Author Information

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  • Corresponding Authors
    • Jeroen D. Langereis - Laboratory of Medical Immunology, Radboud Center for Infectious Diseases, Radboud Institute for Molecular Sciences, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands;  Email: [email protected]
    • Thomas J. Boltje - Cluster of Molecular Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen 6525 AJ, The Netherlands;  Orcidhttps://orcid.org/0000-0001-9141-8784;  Email: [email protected]
  • Authors
    • Sam J. Moons - Cluster of Molecular Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen 6525 AJ, The Netherlands
    • Emiel Rossing - Cluster of Molecular Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen 6525 AJ, The Netherlands
    • Jurriaan J. A. Heming - Cluster of Molecular Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen 6525 AJ, The Netherlands
    • Mathilde A. C. H. Janssen - Cluster of Molecular Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen 6525 AJ, The Netherlands
    • Monique van Scherpenzeel - Translational Metabolic Laboratory, Department of Neurology, Donders Center for Brain Cognition and Behavior, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands
    • Dirk J. Lefeber - Translational Metabolic Laboratory, Department of Neurology, Donders Center for Brain Cognition and Behavior, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands
    • Marien I. de Jonge - Laboratory of Medical Immunology, Radboud Center for Infectious Diseases, Radboud Institute for Molecular Sciences, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands
  • Author Contributions

    S.J.M. and E.R. contributed equally.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by ERC-Stg (GlycoEdit, 758913) awarded to T.J.B.

References

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This article references 25 other publications.

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    A survey of Haemophilus influenzae strains indicated that around one-third of capsular strains and over two-thirds of non-typeable strains included sialic acid in their lipopolysaccharides (LPS). Mutation of the CMP-Neu5Ac synthetase gene (siaB) resulted in a sialylation-deficient phenotype. Isogenic pairs, wild type and siaB mutant of two non-typeable strains were used to demonstrate that sialic acid influences resistance to the killing effect of normal human serum but has little effect on attachment to, or invasion of, cultured human epithelial cells or neutrophils. We det. for the first time the site of attachment of sialic acid in the LPS of a non-typeable strain and report that a small proportion of glycoforms include two sialic acid residues in a disaccharide unit.
  9. 9
    Figueira, M. A., Ram, S., Goldstein, R., Hood, D. W., Moxon, E. R., and Pelton, S. I. (2007) Role of complement in defense of the middle ear revealed by restoring the virulence of nontypeable Haemophilus influenzae siaB mutants. Infect. Immun. 75, 325– 333,  DOI: 10.1128/IAI.01054-06
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    Role of complement in defense of the middle ear revealed by restoring the virulence of nontypeable Haemophilus influenzae siaB mutants
    Figueira, Marisol A.; Ram, Sanjay; Goldstein, Richard; Hood, Derek W.; Moxon, E. Richard; Pelton, Stephen I.
    Infection and Immunity (2007), 75 (1), 325-333CODEN: INFIBR; ISSN:0019-9567. (American Society for Microbiology)
    Nontypeable (NT) H. influenzae is an important cause of otitis media in children. The authors have shown previously that NT H. influenzae mutants defective in their ability to sialylate lipopolysaccharide (LPS), called siaB mutants, show attenuated virulence in a chinchilla model of exptl. otitis media (EOM). The authors show here that complement is a key arm of host innate immunity against NT H. influenzae-induced EOM. Depleting complement in chinchillas by use of cobra venom factor (CoVF) rendered two otherwise avirulent siaB mutants fully virulent and able to cause EOM with severity similar to that of wild-type strains. Clearance of infection caused by siaB mutants in CoVF-treated animals coincided with reappearance of complement C3. Wild-type strains were more resistant to direct complement-mediated killing than their siaB mutants. The serum-resistant strain bound less C3 and C4 than the serum-sensitive strain. Neither NT H. influenzae strain tested bound factor H (alternative complement pathway regulator). Selective activation of the alternative pathway resulted in more C3 binding to siaB mutants. LPS sialylation had a more profound impact on the amt. of alternative pathway-mediated C3 binding (∼5-fold decrease in fluorescence) when LPS was the main C3 target, as occurred on the more serum-resistant strain. In contrast, only an ∼1.5-fold decrease in fluorescence intensity of C3 binding was seen with the serum-sensitive strain, where surface proteins predominantly bound C3. Differences in binding sites for C3 and C4 may account for variations in serum resistance between NT H. influenzae strains, which in turn may impact their virulence. These data demonstrate a central role for complement in innate immune defenses against NT H. influenzae infections and specifically EOM.
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    Caught in a TRAP: substrate-binding proteins in secondary transport
    Fischer, Marcus; Zhang, Qian Yi; Hubbard, Roderick E.; Thomas, Gavin H.
    Trends in Microbiology (2010), 18 (10), 471-478CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
    A review. Substrate-binding protein (SBP)-dependent secondary transporters are ubiquitous in prokaryotes yet poorly characterized. Recently, the structures of over 10 prokaryotic SBPs have been solved, which we compare here to consider their impact on our understanding of transporter function and evolution. Seven structures are from tripartite ATP-independent periplasmic (TRAP) transporters of the DctP-type, which have similar overall structures distinct from SBPs used by ATP-binding cassette (ABC) transporters, despite recognizing a range of substrates. A defining feature of substrate recognition in the DctP-TRAP SBPs is the formation of a salt bridge between a highly conserved arginine and a carboxylate group in the substrate, suggesting that these transporters might have evolved specifically for uptake of diverse org. acids. Remarkably, two of the DctP-TRAP SBPs are clearly dimers and the potential impact of this on transporter function will be discussed. Other SBPs used in secondary transporters are structurally similar to ABC SBPs, demonstrating that multiple families of SBPs have evolved to function with secondary transporters.
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    11
    Selective Inhibition of Sialic Acid-Based Molecular Mimicry in Haemophilus influenzae Abrogates Serum Resistance
    Heise, Torben; Langereis, Jeroen D.; Rossing, Emiel; de Jonge, Marien I.; Adema, Gosse J.; Bull, Christian; Boltje, Thomas J.
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    Pathogens such as non-typeable Haemophilus influenzae (NTHi) evade the immune system by presenting host-derived sialic acids. NTHi cannot synthesize sialic acids and therefore needs to utilize sialic acids originating from host tissue. Here we report sialic acid-based probes to visualize and inhibit the transfer of host sialic acids to NTHi. Inhibition of sialic acid utilization by NTHi enhanced serum-mediated killing. Furthermore, in an in vitro model of the human respiratory tract, we demonstrate efficient inhibition of sialic acid transfer from primary human bronchial epithelial cells to NTHi using bioorthogonal chem.
  12. 12
    Heise, T., Pijnenborg, J. F., Büll, C., van Hilten, N., Kers-Rebel, E. D., Balneger, N., Elferink, H., Adema, G. J., and Boltje, T. J. (2019) Potent metabolic sialylation inhibitors based on C-5-modified fluorinated sialic acids. J. Med. Chem. 62, 1014– 1021,  DOI: 10.1021/acs.jmedchem.8b01757
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    12
    Potent Metabolic Sialylation Inhibitors Based on C-5-Modified Fluorinated Sialic Acids
    Heise, Torben; Pijnenborg, Johan F. A.; Buell, Christian; van Hilten, Niek; Kers-Rebel, Esther D.; Balneger, Natasja; Elferink, Hidde; Adema, Gosse J.; Boltje, Thomas J.
    Journal of Medicinal Chemistry (2019), 62 (2), 1014-1021CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Sialic acid sugars on mammalian cells regulate numerous biol. processes, while aberrant expression of sialic acid is assocd. with diseases such as cancer and pathogenic infection. Inhibition of the sialic acid biosynthesis may therefore hold considerable therapeutic potential. To effectively decrease the sialic acid expression, we synthesized C-5-modified 3-fluoro sialic acid sialyltransferase inhibitors. We found that C-5 carbamates significantly enhanced and prolonged the inhibitory activity in multiple mouse and human cell lines. As an underlying mechanism, we have identified that carbamate-modified 3-fluoro sialic acid inhibitors are more efficiently metabolized to their active cytidine monophosphate analogs, reaching higher effective inhibitor concns. inside cells.
  13. 13
    Bloemendal, V. R., Moons, S. J., Heming, J. J., Chayoua, M., Niesink, O., van Hest, J. C., Boltje, T. J., and Rutjes, F. P. (2019) Chemoenzymatic Synthesis of Sialic Acid Derivatives Using Immobilized N-Acetylneuraminate Lyase in a Continuous Flow Reactor. Adv. Synth. Catal. 361, 2443– 2447,  DOI: 10.1002/adsc.201900146
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    13
    Chemoenzymatic Synthesis of Sialic Acid Derivatives Using Immobilized N-Acetylneuraminate Lyase in a Continuous Flow Reactor
    Bloemendal, Victor R. L. J.; Moons, Sam J.; Heming, Jurriaan J. A.; Chayoua, Mohamed; Niesink, Olaf; van Hest, Jan C. M.; Boltje, Thomas J.; Rutjes, Floris P. J. T.
    Advanced Synthesis & Catalysis (2019), 361 (11), 2443-2447CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The synthesis of N-acetylneuraminic acid (Neu5Ac) derivs. is drawing more and more attention in glycobiol. research because of the important role of sialic acids in e. g. cancer, bacterial, and healthy cells. Chem. prepn. of these carbohydrates typically relies on multistep synthetic procedures leading to low overall yields. Herein we report a continuous flow process involving N-acetylneuraminate lyase (NAL) immobilized on Immobead 150P (Immobead-NAL) to prep. Neu5Ac derivs. Batch expts. with Immobead-NAL showed equal activity as the native enzyme. Moreover, by using a fivefold excess of either N-acetyl-D-mannosamine (ManNAc) or pyruvate the conversion and isolated yield of Neu5Ac were significantly improved. To further increase the efficiency of the process, a flow setup was designed providing a chemoenzymic entry into a series of N-functionalized Neu5Ac derivs. in conversions of 48-82%, and showing excellent stability over 1 wk of continuous use.
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    Saxon, E., Luchansky, S. J., Hang, H. C., Yu, C., Lee, S. C., and Bertozzi, C. R. (2002) Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation. J. Am. Chem. Soc. 124, 14893– 14902,  DOI: 10.1021/ja027748x
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    Investigating Cellular Metabolism of Synthetic Azidosugars with the Staudinger Ligation
    Saxon, Eliana; Luchansky, Sarah J.; Hang, Howard C.; Yu, Chong; Lee, Sandy C.; Bertozzi, Carolyn R.
    Journal of the American Chemical Society (2002), 124 (50), 14893-14902CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The structure of sialic acid on living cells can be modulated by metab. of unnatural biosynthetic precursors. Here we investigate the conversion of a panel of azide-functionalized mannosamine and glucosamine derivs. into cell-surface sialosides. A key tool in this study is the Staudinger ligation, a highly selective reaction between modified triarylphosphines and azides that produces an amide-linked product. A preliminary study of the mechanism of this reaction, and refined conditions for its in vivo execution, are reported. The reaction provided a means to label the glycoconjugate-bound azidosugars with biochem. probes. Finally, we demonstrate that the cell-surface Staudinger ligation is compatible with hydrazone formation from metabolically introduced ketones. These two strategies provide a means to selectively modify cell-surface glycans with exogenous probes.
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    Haloacetylation of D-mannosamine gave amides I (R = F, Cl, Br), which were acetylated to give the corresponding tetra-O-acetates (β-anomers). Data are given for I and their tetraacetates on the in vitro inhibition of thymidine incorporation into Lizio leukemia cells and on the in vivo antitumor activity in mice against Ehrlich tumor.
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    Biological Properties of N-Acyl and N-Haloacetyl Neuraminic Acids: Processing by Enzymes of Sialic Acid Metabolism, and Interaction with Influenza Virus
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    The synthesis of some mechanistic probes for sialic acid processing enzymes and the labeling of a sialidase from Trypanosoma rangeli
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    Sialyl hydrolases, trans-sialidases, and sialyl transferases are biol. important enzymes that are responsible for the incorporation and removal of sialic acid residues, which decorate many cell surface glycoconjugates. Two fluorinated sialic acid derivs. have been synthesized as mechanism-based inactivators, to probe the catalytic mechanisms through which sialidases and trans-sialidases operate. Both compds. are known to be covalent inactivators of a trans-sialidase from Trypanosoma cruzi. Here, 3-fluoro-sialosyl fluoride has been found to covalently label the catalytic nucleophile of a sialidase from T. rangeli, and the residue involved is shown to be Tyr346 within the sequence DENSGYSSVL. This is the first demonstration that sialidases operate through a covalent glycosyl-enzyme intermediate, strongly suggesting a common catalytic mechanism amongst all members of the sialidase super-family. CMP-3-fluoro-sialic acid is a competitive inhibitor of sialyl transferases and was synthesized via a two-step enzymic process from com. available N-acetyl mannosamine, 3-fluoropyruvic acid, and cytidine triphosphate in around 84% yield.
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    Büll, C., Heise, T., Beurskens, D. L. M., Riemersma, M., Ashikov, A., Rutjes, F. P., van Kuppevelt, T. H., Lefeber, D. J., den Brok, M. H., and Adema, G. J. (2015) Sialic acid glycoengineering using an unnatural sialic acid for the detection of sialoglycan biosynthesis defects and on-cell synthesis of siglec ligands. ACS Chem. Biol. 10, 2353– 2363,  DOI: 10.1021/acschembio.5b00501
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    Sialic Acid Glycoengineering Using an Unnatural Sialic Acid for the Detection of Sialoglycan Biosynthesis Defects and On-Cell Synthesis of Siglec Ligands
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  1. Sam J. Moons, Emiel Rossing, Mathilde A. C. H. Janssen, Torben Heise, Christian Büll, Gosse J. Adema, Thomas J. Boltje. Structure–Activity Relationship of Metabolic Sialic Acid Inhibitors and Labeling Reagents. ACS Chemical Biology 2022, 17 (3) , 590-597. https://doi.org/10.1021/acschembio.1c00868
  2. Nicholas Banahene, Herbert W. Kavunja, Benjamin M. Swarts. Chemical Reporters for Bacterial Glycans: Development and Applications. Chemical Reviews 2022, 122 (3) , 3336-3413. https://doi.org/10.1021/acs.chemrev.1c00729
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  4. Emiel Rossing, Johan F. A. Pijnenborg, Thomas J. Boltje. Chemical tools to track and perturb the expression of sialic acid and fucose monosaccharides. Chemical Communications 2022, 58 (87) , 12139-12150. https://doi.org/10.1039/D2CC04275D
  5. Bartłomiej Dudek, Jacek Rybka, Gabriela Bugla-Płoskońska, Agnieszka Korzeniowska-Kowal, Bożena Futoma-Kołoch, Aleksandra Pawlak, Andrzej Gamian. Biological functions of sialic acid as a component of bacterial endotoxin. Frontiers in Microbiology 2022, 13 https://doi.org/10.3389/fmicb.2022.1028796
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  • Abstract

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    Figure 1

    Figure 1. Sialic acid utilization in NTHi. Host-derived sialic acids are taken up by the SiaPQM transporter system. Intracellular sialic acid is either used as a carbon source (via NanA) or used to sialylate the lipooligosaccharide (LOS). To this end, sialic acid is CMP activated by SiaB and incorporated by sialyltransferases Lic3A, Lic3A2, and/or LsgB. (10)

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    Scheme 1

    Scheme 1. Synthesis of C-5 Modified SiaFNAc Inhibitorsa
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    a(i) The synthesis of 1a–1j has been described previously; (13−16)1k: MeOH, NaOMe, TEA, methyl formate; (ii) Sodium fluoropyruvate, H2O, Neu5Ac aldolase, 37 °C; (iii) 1. MsCl, DIPEA, DCM, 0 °C; 2. NaOMe, MeOH; (iv) 1. Selectfluor, DMF, H2O; 2. NaOH, H2O.

    Figure 2

    Figure 2. Inhibitory potency and EC50 curves of C-5 modified SiaFNAc inhibitors on the incorporation of 100 μM SiaNAz by NTHi. EC50 values were calculated based on the fitted S-curves, see the Supporting Information for more details.

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    Scheme 2

    Scheme 2. Synthesis of C-8 and C-9 Modified SiaFNAc Inhibitorsa
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    a(i) 1. NaIO4, MeOH; 2. NaBH4, MeOH; (ii) 1. Selectfluor, DMF, H2O; 2. NaOH, MeOH; (iii) 1. TsCl, pyridine; 2. NaN3, acetone, H2O.

    Figure 3

    Figure 3. Inhibitory potency of SiaFNAc inhibitors modified at the glycerol side chain.

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  • References

    This article references 25 other publications.

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      Non-typeable Haemophilus influenzae, an under-recognized pathogen
      Van Eldere, Johan; Slack, Mary P. E.; Ladhani, Shamez; Cripps, Allan W.
      Lancet Infectious Diseases (2014), 14 (12), 1281-1292CODEN: LIDABP; ISSN:1473-3099. (Elsevier Ltd.)
      A review. Non-typeable Haemophilus influenzae (NTHi) is a major cause of mucosal infections such as otitis media, sinusitis, conjunctivitis, and exacerbations of chronic obstructive pulmonary disease. In some regions, a strong causal relation links this pathogen with infections of the lower respiratory tract. In the past 20 years, a steady but const. increase has occurred in invasive NTHi worldwide, with perinatal infants, young children, and elderly people most at risk. Individuals with underlying comorbidities are most susceptible and infection is assocd. with high mortality. β-lactamase prodn. is the predominant mechanism of resistance. However, the emergence and spread of β-lactamase-neg. ampicillin-resistant strains in many regions of the world is of substantial concern, potentially necessitating changes to antibiotic treatment guidelines for community-acquired infections of the upper and lower respiratory tract and potentially increasing morbidity assocd. with invasive NTHi infections. Standardised surveillance protocols and typing methodologies to monitor this emerging pathogen should be implemented. International scientific organisations need to raise the profile of NTHi and to document the pathobiol. of this microbe.
    2. 2
      Langereis, J. D. and de Jonge, M. I. (2015) Invasive Disease Caused by Nontypeable Haemophilus influenzae. Emerging Infect. Dis. 21, 1711-8,  DOI: 10.3201/eid2110.150004
      There is no corresponding record for this reference.
    3. 3
      Langereis, J. D. and de Jonge, M. I. (2020) Unraveling Haemophilus influenzae virulence mechanisms enable discovery of new targets for antimicrobials and vaccines. Curr. Opin. Infect. Dis. 33, 231– 237,  DOI: 10.1097/QCO.0000000000000645
      3
      Unraveling Haemophilus influenzae virulence mechanisms enable discovery of new targets for antimicrobials and vaccines
      Langereis, Jeroen D.; de Jonge, Marien I.
      Current Opinion in Infectious Diseases (2020), 33 (3), 231-237CODEN: COIDE5; ISSN:0951-7375. (Lippincott Williams & Wilkins)
      A review. The human upper respiratory tract is colonized with a variety of bacterial microorganisms including Haemophilus influenzae. The species H. influenzae consists of typeable and nontypeable H. influenzae (NTHi) variants. Typeable H. influenzae are subdivided into types a through f, based on the polysaccharide capsule, whereas the NTHi strains do not express a polysaccharide capsule. In this review, we highlight the current advances in the field of H. influenzae, with the focus on bacterial virulence mechanisms that facilitate bacterial colonization and disease, particularly for NTHi. In the past decade, it has become apparent that NTHi has the ability to cause invasive infections. Recently, a no. of adhesins have been shown to be crucial for bacterial colonization and invasion and these proteins were investigated as vaccine antigens. Although NTHi lacks a polysaccharide capsule, it expresses lipooligosaccharide that contribute to adhesion and evasion of complement-mediated killing, both contributing to bacterial virulence, which could potentially be targeted by novel antimicrobial drugs or vaccines. The unraveling of H. influenzae virulence mechanisms resulted in the identification of promising targets for novel antimicrobials and vaccine antigens aiming to prevent or treat both typeable and nontypeable H. influenzae infections.
    4. 4
      Apicella, M. A. (2012) Nontypeable Haemophilus influenzae: the role of N-acetyl-5-neuraminic acid in biology. Front. Cell. Infect. Microbiol. 2, 19,  DOI: 10.3389/fcimb.2012.00019
      4
      Nontypeable Haemophilus influenzae: the role of N-acetyl-5-neuraminic acid in biology
      Apicella Michael A
      Frontiers in cellular and infection microbiology (2012), 2 (), 19 ISSN:.
      Nontypeable Haemophilus influenzae (NTHi) is an exclusive human pathogen, which has evolved a number of unique mechanisms to survive within the human environment. An important part of this is the ability of the organism to take up and incorporate sialic acid into its surface structures. This protects the organism against host adaptive and innate immune factor as well as serving as a mechanism for sustaining itself within biofilms. Recent evidence suggests that this also may be the source of the evolution of human antibodies to non-human sialic acid structures, which can lead to inflammation in the host. In very rare instances, evolution of antibodies to sialylated lipooligosaccharide (LOS) mimics of human antigens can result in autoimmune disease.
    5. 5
      Severi, E., Randle, G., Kivlin, P., Whitfield, K., Young, R., Moxon, R., Kelly, D., Hood, D., and Thomas, G. H. (2005) Sialic acid transport in Haemophilus influenzae is essential for lipopolysaccharide sialylation and serum resistance and is dependent on a novel tripartite ATP-independent periplasmic transporter. Mol. Microbiol. 58, 1173– 1185,  DOI: 10.1111/j.1365-2958.2005.04901.x
      5
      Sialic acid transport in Haemophilus influenzae is essential for lipopolysaccharide sialylation and serum resistance and is dependent on a novel tripartite ATP-independent periplasmic transporter
      Severi, Emmanuele; Randle, Gaynor; Kivlin, Polly; Whitfield, Kate; Young, Rosie; Moxon, Richard; Kelly, David; Hood, Derek; Thomas, Gavin H.
      Molecular Microbiology (2005), 58 (4), 1173-1185CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
      Sialylation of the lipopolysaccharide (LPS) is an important mechanism used by the human pathogen Haemophilus influenzae to evade the innate immune response of the host. We have demonstrated that N-acetylneuraminic acid (Neu5Ac or sialic acid) uptake in H. influenzae is essential for the subsequent modification of the LPS and that this uptake is mediated through a single transport system which is a member of the tripartite ATP-independent periplasmic (TRAP) transporter family. Disruption of either the siaP (HI0146) or siaQM (HI0147) genes, that encode the two subunits of this transporter, results in a complete loss of uptake of [14C]-Neu5Ac. Mutant strains lack sialylated glycoforms in their LPS and are more sensitive to killing by human serum than the parent strain. The SiaP protein has been purified and demonstrated to bind a stoichiometric amt. of Neu5Ac by electrospray mass spectrometry. This binding was of high affinity with a Kd of ∼0.1 μM as detd. by protein fluorescence. The inactivation of the SiaPQM TRAP transporter also results in decreased growth of H. influenzae in a chem. defined medium contg. Neu5Ac, supporting an addnl. nutritional role of sialic acid in H. influenzae physiol.
    6. 6
      Vimr, E., Lichtensteiger, C., and Steenbergen, S. (2000) Sialic acid metabolism’s dual function in Haemophilus influenzae. Mol. Microbiol. 36, 1113– 1123,  DOI: 10.1046/j.1365-2958.2000.01925.x
      6
      Sialic acid metabolism's dual function in Haemophilus influenzae
      Vimr, Eric; Lichtensteiger, Carol; Steenbergen, Susan
      Molecular Microbiology (2000), 36 (5), 1113-1123CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Science Ltd.)
      Many bacterial commensals and pathogens use the sialic acids as carbon and nitrogen sources. In Escherichia coli, the breakdown of these sugars is catalyzed by gene products of the nan (N-acylneuraminate) operon; other microorganisms may use a similar catabolic strategy. Despite the known ligand and antirecognition functions of the sialic acids, the contribution of their catabolism to infection or host colonization has never been directly investigated. The authors addressed these questions with Haemophilus influenzae type b, which metabolizes relatively few carbohydrates, using the infant-rat infection model. The predicted H. influenzae homolog (HI0142) of the E. coli sialic acid aldolase structural gene, nanA, was subcloned and mutagenized by insertion of a kanamycin resistance cassette. Phenotypic investigation of the resulting H. influenzae aldolase mutants showed that: (i) HI0142 is essential for sialic acid degrdn.; (ii) the products of the open reading frames (ORFs) flanking HI0142 (HI0140, 41, 44 and 45) are likely to have the same functions as those of their counterparts in E. coli; (iii) sialylation of the lipooligosaccharide (LOS) epitope recognized by monoclonal antibody 3F11 is dependent on an environmental source of sialic acid; (iv) a nanA mutant hypersialylates its LOS sialyl acceptor, corresponding to an apparent increased fitness of the mutant in the infant-rat model; and (v) expression of the LOS sialyl acceptor is altered in cells grown without exogenous sialic acid, indicating the direct or indirect effect of sialic acid metab. on LOS antigenicity. Taken together the data show the dual role of sialic acid catabolism in nutrition and cell surface modulation.
    7. 7
      Swords, W. E., Moore, M. L., Godzicki, L., Bukofzer, G., Mitten, M. J., and VonCannon, J. (2004) Sialylation of lipooligosaccharides promotes biofilm formation by nontypeable Haemophilus influenzae. Infect. Immun. 72, 106– 113,  DOI: 10.1128/IAI.72.1.106-113.2004
      7
      Sialylation of lipooligosaccharides promotes biofilm formation by nontypeable Haemophilus influenzae
      Swords, W. Edward; Moore, Miranda L.; Godzicki, Luciana; Bukofzer, Gail; Mitten, Michael J.; VonCannon, Jessica
      Infection and Immunity (2004), 72 (1), 106-113CODEN: INFIBR; ISSN:0019-9567. (American Society for Microbiology)
      Nontypeable H. influenzae (NTHi) is a major cause of opportunistic respiratory tract infections, including otitis media and bronchitis. The persistence of NTHi in vivo is thought to involve bacterial persistence in a biofilm community. Therefore, there is a need for further definition of bacterial factors contributing to biofilm formation by NTHi. Like other bacteria inhabiting host mucosal surfaces, NTHi has on its surface a diverse array of lipooligosaccharides (LOS) that influence host-bacterial interactions. In this study, we show that LOS contg. sialic (N-acetyl-neuraminic) acid promotes biofilm formation by NTHi in vitro and bacterial persistence within the middle ear or lung in vivo. LOS from NTHi in biofilms was sialylated, as detd. by comparison of electrophoretic mobilities and immunochem. reactivities before and after neuraminidase treatment. Biofilm formation was significantly reduced in media lacking sialic acid, and a siaB (CMP-sialic acid synthetase) mutant was deficient in biofilm formation in 3 different in vitro model systems. The persistence of an asialylated siaB mutant was attenuated in a gerbil middle ear infection model system, as well as in a rat pulmonary challenge model system. These data show that sialylated LOS glycoforms promote biofilm formation by NTHi and persistence in vivo.
    8. 8
      Hood, D. W., Makepeace, K., Deadman, M. E., Rest, R. F., Thibault, P., Martin, A., Richards, J. C., and Moxon, E. R. (1999) Sialic acid in the lipopolysaccharide of Haemophilus influenzae: strain distribution, influence on serum resistance and structural characterization. Mol. Microbiol. 33, 679– 692,  DOI: 10.1046/j.1365-2958.1999.01509.x
      8
      Sialic acid in the lipopolysaccharide of Haemophilus influenzae: strain distribution, influence on serum resistance and structural characterization
      Hood, Derek W.; Makepeace, Katherine; Deadman, Mary E.; Rest, Richard F.; Thibault, Pierre; Martin, Adele; Richards, James C.; Moxon, E. Richard
      Molecular Microbiology (1999), 33 (4), 679-692CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Science Ltd.)
      A survey of Haemophilus influenzae strains indicated that around one-third of capsular strains and over two-thirds of non-typeable strains included sialic acid in their lipopolysaccharides (LPS). Mutation of the CMP-Neu5Ac synthetase gene (siaB) resulted in a sialylation-deficient phenotype. Isogenic pairs, wild type and siaB mutant of two non-typeable strains were used to demonstrate that sialic acid influences resistance to the killing effect of normal human serum but has little effect on attachment to, or invasion of, cultured human epithelial cells or neutrophils. We det. for the first time the site of attachment of sialic acid in the LPS of a non-typeable strain and report that a small proportion of glycoforms include two sialic acid residues in a disaccharide unit.
    9. 9
      Figueira, M. A., Ram, S., Goldstein, R., Hood, D. W., Moxon, E. R., and Pelton, S. I. (2007) Role of complement in defense of the middle ear revealed by restoring the virulence of nontypeable Haemophilus influenzae siaB mutants. Infect. Immun. 75, 325– 333,  DOI: 10.1128/IAI.01054-06
      9
      Role of complement in defense of the middle ear revealed by restoring the virulence of nontypeable Haemophilus influenzae siaB mutants
      Figueira, Marisol A.; Ram, Sanjay; Goldstein, Richard; Hood, Derek W.; Moxon, E. Richard; Pelton, Stephen I.
      Infection and Immunity (2007), 75 (1), 325-333CODEN: INFIBR; ISSN:0019-9567. (American Society for Microbiology)
      Nontypeable (NT) H. influenzae is an important cause of otitis media in children. The authors have shown previously that NT H. influenzae mutants defective in their ability to sialylate lipopolysaccharide (LPS), called siaB mutants, show attenuated virulence in a chinchilla model of exptl. otitis media (EOM). The authors show here that complement is a key arm of host innate immunity against NT H. influenzae-induced EOM. Depleting complement in chinchillas by use of cobra venom factor (CoVF) rendered two otherwise avirulent siaB mutants fully virulent and able to cause EOM with severity similar to that of wild-type strains. Clearance of infection caused by siaB mutants in CoVF-treated animals coincided with reappearance of complement C3. Wild-type strains were more resistant to direct complement-mediated killing than their siaB mutants. The serum-resistant strain bound less C3 and C4 than the serum-sensitive strain. Neither NT H. influenzae strain tested bound factor H (alternative complement pathway regulator). Selective activation of the alternative pathway resulted in more C3 binding to siaB mutants. LPS sialylation had a more profound impact on the amt. of alternative pathway-mediated C3 binding (∼5-fold decrease in fluorescence) when LPS was the main C3 target, as occurred on the more serum-resistant strain. In contrast, only an ∼1.5-fold decrease in fluorescence intensity of C3 binding was seen with the serum-sensitive strain, where surface proteins predominantly bound C3. Differences in binding sites for C3 and C4 may account for variations in serum resistance between NT H. influenzae strains, which in turn may impact their virulence. These data demonstrate a central role for complement in innate immune defenses against NT H. influenzae infections and specifically EOM.
    10. 10
      Fischer, M., Zhang, Q. Y., Hubbard, R. E., and Thomas, G. H. (2010) Caught in a TRAP: substrate-binding proteins in secondary transport. Trends Microbiol. 18, 471– 478,  DOI: 10.1016/j.tim.2010.06.009
      10
      Caught in a TRAP: substrate-binding proteins in secondary transport
      Fischer, Marcus; Zhang, Qian Yi; Hubbard, Roderick E.; Thomas, Gavin H.
      Trends in Microbiology (2010), 18 (10), 471-478CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
      A review. Substrate-binding protein (SBP)-dependent secondary transporters are ubiquitous in prokaryotes yet poorly characterized. Recently, the structures of over 10 prokaryotic SBPs have been solved, which we compare here to consider their impact on our understanding of transporter function and evolution. Seven structures are from tripartite ATP-independent periplasmic (TRAP) transporters of the DctP-type, which have similar overall structures distinct from SBPs used by ATP-binding cassette (ABC) transporters, despite recognizing a range of substrates. A defining feature of substrate recognition in the DctP-TRAP SBPs is the formation of a salt bridge between a highly conserved arginine and a carboxylate group in the substrate, suggesting that these transporters might have evolved specifically for uptake of diverse org. acids. Remarkably, two of the DctP-TRAP SBPs are clearly dimers and the potential impact of this on transporter function will be discussed. Other SBPs used in secondary transporters are structurally similar to ABC SBPs, demonstrating that multiple families of SBPs have evolved to function with secondary transporters.
    11. 11
      Heise, T., Langereis, J. D., Rossing, E., de Jonge, M. I., Adema, G. J., Büll, C., and Boltje, T. J. (2018) Selective inhibition of sialic acid-based molecular mimicry in Haemophilus influenzae abrogates serum resistance. Cell Chem. Biol. 25, 1279– 1285.e8,  DOI: 10.1016/j.chembiol.2018.05.018
      11
      Selective Inhibition of Sialic Acid-Based Molecular Mimicry in Haemophilus influenzae Abrogates Serum Resistance
      Heise, Torben; Langereis, Jeroen D.; Rossing, Emiel; de Jonge, Marien I.; Adema, Gosse J.; Bull, Christian; Boltje, Thomas J.
      Cell Chemical Biology (2018), 25 (10), 1279-1285.e8CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
      Pathogens such as non-typeable Haemophilus influenzae (NTHi) evade the immune system by presenting host-derived sialic acids. NTHi cannot synthesize sialic acids and therefore needs to utilize sialic acids originating from host tissue. Here we report sialic acid-based probes to visualize and inhibit the transfer of host sialic acids to NTHi. Inhibition of sialic acid utilization by NTHi enhanced serum-mediated killing. Furthermore, in an in vitro model of the human respiratory tract, we demonstrate efficient inhibition of sialic acid transfer from primary human bronchial epithelial cells to NTHi using bioorthogonal chem.
    12. 12
      Heise, T., Pijnenborg, J. F., Büll, C., van Hilten, N., Kers-Rebel, E. D., Balneger, N., Elferink, H., Adema, G. J., and Boltje, T. J. (2019) Potent metabolic sialylation inhibitors based on C-5-modified fluorinated sialic acids. J. Med. Chem. 62, 1014– 1021,  DOI: 10.1021/acs.jmedchem.8b01757
      12
      Potent Metabolic Sialylation Inhibitors Based on C-5-Modified Fluorinated Sialic Acids
      Heise, Torben; Pijnenborg, Johan F. A.; Buell, Christian; van Hilten, Niek; Kers-Rebel, Esther D.; Balneger, Natasja; Elferink, Hidde; Adema, Gosse J.; Boltje, Thomas J.
      Journal of Medicinal Chemistry (2019), 62 (2), 1014-1021CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Sialic acid sugars on mammalian cells regulate numerous biol. processes, while aberrant expression of sialic acid is assocd. with diseases such as cancer and pathogenic infection. Inhibition of the sialic acid biosynthesis may therefore hold considerable therapeutic potential. To effectively decrease the sialic acid expression, we synthesized C-5-modified 3-fluoro sialic acid sialyltransferase inhibitors. We found that C-5 carbamates significantly enhanced and prolonged the inhibitory activity in multiple mouse and human cell lines. As an underlying mechanism, we have identified that carbamate-modified 3-fluoro sialic acid inhibitors are more efficiently metabolized to their active cytidine monophosphate analogs, reaching higher effective inhibitor concns. inside cells.
    13. 13
      Bloemendal, V. R., Moons, S. J., Heming, J. J., Chayoua, M., Niesink, O., van Hest, J. C., Boltje, T. J., and Rutjes, F. P. (2019) Chemoenzymatic Synthesis of Sialic Acid Derivatives Using Immobilized N-Acetylneuraminate Lyase in a Continuous Flow Reactor. Adv. Synth. Catal. 361, 2443– 2447,  DOI: 10.1002/adsc.201900146
      13
      Chemoenzymatic Synthesis of Sialic Acid Derivatives Using Immobilized N-Acetylneuraminate Lyase in a Continuous Flow Reactor
      Bloemendal, Victor R. L. J.; Moons, Sam J.; Heming, Jurriaan J. A.; Chayoua, Mohamed; Niesink, Olaf; van Hest, Jan C. M.; Boltje, Thomas J.; Rutjes, Floris P. J. T.
      Advanced Synthesis & Catalysis (2019), 361 (11), 2443-2447CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The synthesis of N-acetylneuraminic acid (Neu5Ac) derivs. is drawing more and more attention in glycobiol. research because of the important role of sialic acids in e. g. cancer, bacterial, and healthy cells. Chem. prepn. of these carbohydrates typically relies on multistep synthetic procedures leading to low overall yields. Herein we report a continuous flow process involving N-acetylneuraminate lyase (NAL) immobilized on Immobead 150P (Immobead-NAL) to prep. Neu5Ac derivs. Batch expts. with Immobead-NAL showed equal activity as the native enzyme. Moreover, by using a fivefold excess of either N-acetyl-D-mannosamine (ManNAc) or pyruvate the conversion and isolated yield of Neu5Ac were significantly improved. To further increase the efficiency of the process, a flow setup was designed providing a chemoenzymic entry into a series of N-functionalized Neu5Ac derivs. in conversions of 48-82%, and showing excellent stability over 1 wk of continuous use.
    14. 14
      Saxon, E., Luchansky, S. J., Hang, H. C., Yu, C., Lee, S. C., and Bertozzi, C. R. (2002) Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation. J. Am. Chem. Soc. 124, 14893– 14902,  DOI: 10.1021/ja027748x
      14
      Investigating Cellular Metabolism of Synthetic Azidosugars with the Staudinger Ligation
      Saxon, Eliana; Luchansky, Sarah J.; Hang, Howard C.; Yu, Chong; Lee, Sandy C.; Bertozzi, Carolyn R.
      Journal of the American Chemical Society (2002), 124 (50), 14893-14902CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The structure of sialic acid on living cells can be modulated by metab. of unnatural biosynthetic precursors. Here we investigate the conversion of a panel of azide-functionalized mannosamine and glucosamine derivs. into cell-surface sialosides. A key tool in this study is the Staudinger ligation, a highly selective reaction between modified triarylphosphines and azides that produces an amide-linked product. A preliminary study of the mechanism of this reaction, and refined conditions for its in vivo execution, are reported. The reaction provided a means to label the glycoconjugate-bound azidosugars with biochem. probes. Finally, we demonstrate that the cell-surface Staudinger ligation is compatible with hydrazone formation from metabolically introduced ketones. These two strategies provide a means to selectively modify cell-surface glycans with exogenous probes.
    15. 15
      Fondy, T. P. and Emlich, C. A. (1981) Haloacetamido analogs of 2-amino-2-deoxy-D-mannose. Syntheses and effects on tumor-bearing mice. J. Med. Chem. 24, 848– 852,  DOI: 10.1021/jm00139a016
      15
      Haloacetamido analogs of 2-amino-2-deoxy-D-mannose. Syntheses and effects on tumor-bearing mice
      Fondy, Thomas P.; Emlich, Cheryl A.
      Journal of Medicinal Chemistry (1981), 24 (7), 848-52CODEN: JMCMAR; ISSN:0022-2623.
      Haloacetylation of D-mannosamine gave amides I (R = F, Cl, Br), which were acetylated to give the corresponding tetra-O-acetates (β-anomers). Data are given for I and their tetraacetates on the in vitro inhibition of thymidine incorporation into Lizio leukemia cells and on the in vivo antitumor activity in mice against Ehrlich tumor.
    16. 16
      Humphrey, A. J., Fremann, C., Critchley, P., Malykh, Y., Schauer, R., and Bugg, T. D. (2002) Biological properties of N-acyl and N-haloacetyl neuraminic acids: processing by enzymes of sialic acid metabolism, and interaction with influenza virus. Bioorg. Med. Chem. 10, 3175– 3185,  DOI: 10.1016/S0968-0896(02)00213-4
      16
      Biological Properties of N-Acyl and N-Haloacetyl Neuraminic Acids: Processing by Enzymes of Sialic Acid Metabolism, and Interaction with Influenza Virus
      Humphrey, Andrew J.; Fremann, Claire; Critchley, Peter; Malykh, Yanina; Schauer, Roland; Bugg, Timothy D. H.
      Bioorganic & Medicinal Chemistry (2002), 10 (10), 3175-3185CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)
      Several unnatural N-acyl neuraminic acids (N-propionyl, N-hexanoyl, N-benzoyl, N-trifluoroacetyl, N-chloroacetyl, N-difluoroacetyl) were prepd. enzymically using immobilized sialic acid aldolase. N-Trifluoroacetyl-, N-chloroacetyl- and N-difluoroacetyl neuraminic acids were shown to enhance up to 10-fold the rate of assocn. of influenza virus A to a sialoglycolipid neomembrane by surface plasmon resonance, and were found to act as weak inhibitors (Kiapp 0.45-2.0 mM) of influenza virus neuraminidase. The N-propionyl, N-chloroacetyl- and N-difluoroacetyl neuraminic acids were found to be substrates for recombinant Escherichia coli CMP sialate synthase, to give the corresponding CMP-N-acyl-neuraminic acids. CMP-N-propionyl neuraminic acid was found not to be a substrate for CMP-N-acetyl neuraminic acid hydroxylase from pig submandibular gland.
    17. 17
      Watts, A. G. and Withers, S. G. (2004) The synthesis of some mechanistic probes for sialic acid processing enzymes and the labeling of a sialidase from Trypanosoma rangeli. Can. J. Chem. 82, 1581– 1588,  DOI: 10.1139/v04-125
      17
      The synthesis of some mechanistic probes for sialic acid processing enzymes and the labeling of a sialidase from Trypanosoma rangeli
      Watts, Andrew G.; Withers, Stephen G.
      Canadian Journal of Chemistry (2004), 82 (11), 1581-1588CODEN: CJCHAG; ISSN:0008-4042. (National Research Council of Canada)
      Sialyl hydrolases, trans-sialidases, and sialyl transferases are biol. important enzymes that are responsible for the incorporation and removal of sialic acid residues, which decorate many cell surface glycoconjugates. Two fluorinated sialic acid derivs. have been synthesized as mechanism-based inactivators, to probe the catalytic mechanisms through which sialidases and trans-sialidases operate. Both compds. are known to be covalent inactivators of a trans-sialidase from Trypanosoma cruzi. Here, 3-fluoro-sialosyl fluoride has been found to covalently label the catalytic nucleophile of a sialidase from T. rangeli, and the residue involved is shown to be Tyr346 within the sequence DENSGYSSVL. This is the first demonstration that sialidases operate through a covalent glycosyl-enzyme intermediate, strongly suggesting a common catalytic mechanism amongst all members of the sialidase super-family. CMP-3-fluoro-sialic acid is a competitive inhibitor of sialyl transferases and was synthesized via a two-step enzymic process from com. available N-acetyl mannosamine, 3-fluoropyruvic acid, and cytidine triphosphate in around 84% yield.
    18. 18
      Büll, C., Heise, T., Beurskens, D. L. M., Riemersma, M., Ashikov, A., Rutjes, F. P., van Kuppevelt, T. H., Lefeber, D. J., den Brok, M. H., and Adema, G. J. (2015) Sialic acid glycoengineering using an unnatural sialic acid for the detection of sialoglycan biosynthesis defects and on-cell synthesis of siglec ligands. ACS Chem. Biol. 10, 2353– 2363,  DOI: 10.1021/acschembio.5b00501
      18
      Sialic Acid Glycoengineering Using an Unnatural Sialic Acid for the Detection of Sialoglycan Biosynthesis Defects and On-Cell Synthesis of Siglec Ligands
      Buell, Christian; Heise, Torben; Beurskens, Danielle M. H.; Riemersma, Moniek; Ashikov, Angel; Rutjes, Floris P. J. T.; van Kuppevelt, Toin H.; Lefeber, Dirk J.; den Brok, Martijn H.; Adema, Gosse J.; Boltje, Thomas J.
      ACS Chemical Biology (2015), 10 (10), 2353-2363CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Sialoglycans play a vital role in physiol., and aberrant sialoglycan expression is assocd. with a broad spectrum of diseases. Since biosynthesis of sialoglycans is only partially regulated at the genetic level, chem. tools are crucial to study their function. Here, the authors report the development of propargyloxycarbonyl sialic acid (Ac5NeuNPoc) as a powerful tool for sialic acid glycoengineering. Ac5NeuNPoc showed strongly increased labeling efficiency and exhibited less toxicity compared to those of widely used mannosamine analogs in vitro and was also more efficiently incorporated into sialoglycans in vivo. Unlike mannosamine analogs, Ac5NeuNPoc was exclusively used in the sialoglycan biosynthesis pathway, allowing a genetic defect in sialic acid biosynthesis to be specifically detected. Furthermore, Ac5NeuNPoc-based sialic acid glycoengineering enabled the on-cell synthesis of high-affinity Siglec-7 ligands and the identification of a novel Siglec-2 ligand. Thus, Ac5NeuNPoc glycoengineering is a highly efficient, nontoxic, and selective approach to study and modulate sialoglycan interactions on living cells.
    19. 19
      Ng, P. S., Day, C. J., Atack, J. M., Hartley-Tassell, L. E., Winter, L. E., Marshanski, T., Padler-Karavani, V., Varki, A., Barenkamp, S. J., and Apicella, M. A. (2019) Nontypeable Haemophilus influenzae has evolved preferential use of N-acetylneuraminic acid as a host adaptation. mBio 10, e00422-19,  DOI: 10.1128/mBio.00422-19
      There is no corresponding record for this reference.
    20. 20
      Rillahan, C. D., Antonopoulos, A., Lefort, C. T., Sonon, R., Azadi, P., Ley, K., Dell, A., Haslam, S. M., and Paulson, J. C. (2012) Global metabolic inhibitors of sialyl-and fucosyltransferases remodel the glycome. Nat. Chem. Biol. 8, 661– 668,  DOI: 10.1038/nchembio.999
      20
      Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome
      Rillahan, Cory D.; Antonopoulos, Aristotelis; Lefort, Craig T.; Sonon, Roberto; Azadi, Parastoo; Ley, Klaus; Dell, Anne; Haslam, Stuart M.; Paulson, James C.
      Nature Chemical Biology (2012), 8 (7), 661-668CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Despite the fundamental roles of sialyl- and fucosyltransferases in mammalian physiol., there are few pharmacol. tools to manipulate their function in a cellular setting. Although fluorinated analogs of the donor substrates are well-established transition state inhibitors of these enzymes, they are not membrane permeable. By exploiting promiscuous monosaccharide salvage pathways, it is shown that fluorinated analogs of sialic acid and fucose can be taken up and metabolized to the desired donor substrate-based inhibitors inside the cell. Because of the existence of metabolic feedback loops, they also act to prevent the de novo synthesis of the natural substrates, resulting in a global, family-wide shutdown of sialyl- and/or fucosyltransferases and remodeling of cell-surface glycans. As an example of the functional consequences, the inhibitors substantially reduce expression of the sialylated and fucosylated ligand sialyl Lewis X on myeloid cells, resulting in loss of selectin binding and impaired leukocyte rolling.
    21. 21
      van Scherpenzeel, M., Conte, F., Bull, C., Ashikov, A., Hermans, E., Willems, A., van Tol, W., Kragt, E., Moret, E., and Heise, T. (2020) Dynamic analysis of sugar metabolism reveals the mechanisms of action of synthetic sugar analogs. BioRxiv.https://www.biorxiv.org/content/10.1101/2020.09.15.288712v1.full (accessed 2021-05-25), DOI: 10.1101/2020.09.15.288712 .
      There is no corresponding record for this reference.
    22. 22
      Angata, T. and Varki, A. (2002) Chemical diversity in the sialic acids and related α-keto acids: an evolutionary perspective. Chem. Rev. 102, 439– 470,  DOI: 10.1021/cr000407m
      22
      Chemical Diversity in the Sialic Acids and Related α-Keto Acids: An Evolutionary Perspective
      Angata, Takashi; Varki, Ajit
      Chemical Reviews (Washington, D. C.) (2002), 102 (2), 439-469CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review with refs. on the evolutionary perspective, occurrence, biosynthesis, and structural diversity of sialic acids in bacteria and natural proteins.
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      Schauer, R. and Kamerling, J. P. (2018) Exploration of the sialic acid world. Adv. Carbohydr. Chem. Biochem. 75, 1– 213,  DOI: 10.1016/bs.accb.2018.09.001
      23
      Exploration of the Sialic Acid World
      Schauer Roland; Kamerling Johannis P
      Advances in carbohydrate chemistry and biochemistry (2018), 75 (), 1-213 ISSN:.
      Sialic acids are cytoprotectors, mainly localized on the surface of cell membranes with multiple and outstanding cell biological functions. The history of their structural analysis, occurrence, and functions is fascinating and described in this review. Reports from different researchers on apparently similar substances from a variety of biological materials led to the identification of a 9-carbon monosaccharide, which in 1957 was designated "sialic acid." The most frequently occurring member of the sialic acid family is N-acetylneuraminic acid, followed by N-glycolylneuraminic acid and O-acetylated derivatives, and up to now over about 80 neuraminic acid derivatives have been described. They appeared first in the animal kingdom, ranging from echinoderms up to higher animals, in many microorganisms, and are also expressed in insects, but are absent in higher plants. Sialic acids are masks and ligands and play as such dual roles in biology. Their involvement in immunology and tumor biology, as well as in hereditary diseases, cannot be underestimated. N-Glycolylneuraminic acid is very special, as this sugar cannot be expressed by humans, but is a xenoantigen with pathogenetic potential. Sialidases (neuraminidases), which liberate sialic acids from cellular compounds, had been known from very early on from studies with influenza viruses. Sialyltransferases, which are responsible for the sialylation of glycans and elongation of polysialic acids, are studied because of their significance in development and, for instance, in cancer. As more information about the functions in health and disease is acquired, the use of sialic acids in the treatment of diseases is also envisaged.
    24. 24
      Malaprade, L. (1934) A study of the action of polyalcohols on periodic acid and alkaline periodates. Bull. Soc. Chim. Fr. 5, 833– 852
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      Burkart, M. D., Zhang, Z., Hung, S.-C., and Wong, C.-H. (1997) A new method for the synthesis of fluoro-carbohydrates and glycosides using selectfluor. J. Am. Chem. Soc. 119, 11743– 11746,  DOI: 10.1021/ja9723904
      25
      A New Method for the Synthesis of Fluoro-Carbohydrates and Glycosides Using Selectfluor
      Burkart, Michael D.; Zhang, Zhiyuan; Hung, Shang-Cheng; Wong, Chi-Huey
      Journal of the American Chemical Society (1997), 119 (49), 11743-11746CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      This paper describes a high-yield, one-step synthesis of 2-deoxy-2-fluoro sugars and their glycosides from glycals using the available electrophilic fluorination reagent 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (Selectfluor) in the presence of a nucleophile. The method was further expanded to the prepn. of glycosyl fluorides and glycosides from anomeric hydroxy or thio glycoside derivs.

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