An efficient synthesis of 1,6-anhydro-<i>N</i>-acetylmuramic acid from <i>N</i>-acetylglucosamine

Calvert, Matthew; Mayer, Christoph; Titz, Alexander

doi:10.3762/bjoc.13.261

Cited by 8 publications

(7 citation statements)

References 14 publications

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“…ATP, glucose (Glc), GlcNAc, glucosamine (GlcN), N -acetyl galactosamine (GalNAc) and GlcNAc-6P were obtained from Sigma–Aldrich (Taufkirchen, Germany), MurNAc was obtained from Bachem (Bubendorf, Switzerland). MurNAc-6P was available from a previous study ( Unsleber et al, 2017 ) and 1,6-anhydro-MurNAc (anhMurNAc) was synthesized according to a published protocol ( Calvert et al, 2017 ).…”

Section: Methodsmentioning

confidence: 99%

N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism

et al. 2018

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Tannerella forsythia is an anaerobic, Gram-negative oral pathogen that thrives in multispecies gingival biofilms associated with periodontitis. The bacterium is auxotrophic for the commonly essential bacterial cell wall sugar N- acetylmuramic acid (MurNAc) and, thus, strictly depends on an exogenous supply of MurNAc for growth and maintenance of cell morphology. A MurNAc transporter (Tf_MurT; Tanf_08375) and an ortholog of the Escherichia coli etherase MurQ (Tf_MurQ; Tanf_08385) converting MurNAc-6-phosphate to GlcNAc-6-phosphate were recently described for T. forsythia. In between the respective genes on the T. forsythia genome, a putative kinase gene is located. In this study, the putative kinase (Tf_MurK; Tanf_08380) was produced as a recombinant protein and biochemically characterized. Kinetic studies revealed Tf_MurK to be a 6-kinase with stringent substrate specificity for MurNAc exhibiting a 6 × 10 4 -fold higher catalytic efficiency ( k cat / K m ) for MurNAc than for N- acetylglucosamine (GlcNAc) with k cat values of 10.5 s -1 and 0.1 s -1 and K m values of 200 μM and 116 mM, respectively. The enzyme kinetic data suggest that Tf_MurK is subject to substrate inhibition ( K i[S] = 4.2 mM). To assess the role of Tf_MurK in the cell wall metabolism of T. forsythia , a kinase deletion mutant ( ΔTf_murK::erm ) was constructed. This mutant accumulated MurNAc intracellularly in the exponential phase, indicating the capability to take up MurNAc, but inability to catabolize MurNAc. In the stationary phase, the MurNAc level was reduced in the mutant, while the level of the peptidoglycan precursor UDP-MurNAc-pentapeptide was highly elevated. Further, according to scanning electron microscopy evidence, the ΔTf_murK::erm mutant was more tolerant toward low MurNAc concentration in the medium (below 0.5 μg/ml) before transition from healthy, rod-shaped to fusiform cells occurred, while the parent strain required > 1 μg/ml MurNAc for optimal growth. These data reveal that T. forsythia readily catabolizes exogenous MurNAc but simultaneously channels a proportion of the sugar into peptidoglycan biosynthesis. Deletion of Tf_murK blocks MurNAc catabolism and allows the direction of MurNAc solely to peptidoglycan biosynthesis, resulting in a growth advantage in MurNAc-depleted medium. This work increases our understanding of the T. forsythia cell wall metabolism and may pave new routes for lead finding in the treatment of periodontitis.

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Section: Methodsmentioning

confidence: 99%

N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism

et al. 2018

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“…Finally, anhMurNAc was chemically synthesized [ 29 ] and purified by HILIC, with an achieved overall yield of 5.4%. All analytical NMR data of anhMurNAc were in agreement with previously published data (Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To serve as a standard for the analysis of the different MurNAc sources investigated within the frame of this study and as a putative growth supplement for T. forsythia , anhMurNAc was chemically synthesized via a five-step synthesis scheme according to a published protocol [ 29 ], with minor modifications. For the introduction of the trityl group at position four during the third step, the formation of the reagent trityl triflate beforehand is necessary.…”

Section: Methodsmentioning

confidence: 99%

“…1 H NMR were referenced internally to the residual solvent signal (1.94 ppm). Care needed to be taken upon analysing anhMurNAc by NMR, as only the protonated form is soluble in acetonitrile, for which the shifts have been reported [ 29 ] and could be reproduced. The chemical shifts in other solvents such as MeOD differed greatly, even from fraction to fraction (this study).…”

Section: Methodsmentioning

confidence: 99%

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Utilization of different MurNAc sources by the oral pathogen Tannerella forsythia and role of the inner membrane transporter AmpG

et al. 2020

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Background The Gram-negative oral pathogen Tannerella forsythia strictly depends on the external supply of the essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) for survival because of the lack of the common MurNAc biosynthesis enzymes MurA/MurB. The bacterium thrives in a polymicrobial biofilm consortium and, thus, it is plausible that it procures MurNAc from MurNAc-containing peptidoglycan (PGN) fragments (muropeptides) released from cohabiting bacteria during natural PGN turnover or cell death. There is indirect evidence that in T. forsythia, an AmpG-like permease (Tanf_08365) is involved in cytoplasmic muropeptide uptake. In E. coli, AmpG is specific for the import of N-acetylglucosamine (GlcNAc)-anhydroMurNAc(−peptides) which are common PGN turnover products, with the disaccharide portion as a minimal requirement. Currently, it is unclear which natural, complex MurNAc sources T. forsythia can utilize and which role AmpG plays therein. Results We performed a screen of various putative MurNAc sources for T. forsythia mimicking the situation in the natural habitat and compared bacterial growth and cell morphology of the wild-type and a mutant lacking AmpG (T. forsythia ΔampG). We showed that supernatants of the oral biofilm bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, and of E. coli ΔampG, as well as isolated PGN and defined PGN fragments obtained after enzymatic digestion, namely GlcNAc-anhydroMurNAc(−peptides) and GlcNAc-MurNAc(−peptides), could sustain growth of T. forsythia wild-type, while T. forsythia ΔampG suffered from growth inhibition. In supernatants of T. forsythia ΔampG, the presence of GlcNAc-anhMurNAc and, unexpectedly, also GlcNAc-MurNAc was revealed by tandem mass spectrometry analysis, indicating that both disaccharides are substrates of AmpG. The importance of AmpG in the utilization of PGN fragments as MurNAc source was substantiated by a significant ampG upregulation in T. forsythia cells cultivated with PGN, as determined by quantitative real-time PCR. Further, our results indicate that PGN-degrading amidase, lytic transglycosylase and muramidase activities in a T. forsythia cell extract are involved in PGN scavenging. Conclusion T. forsythia metabolizes intact PGN as well as muropeptides released from various bacteria and the bacterium’s inner membrane transporter AmpG is essential for growth on these MurNAc sources, and, contrary to the situation in E. coli, imports both, GlcNAc-anhMurNAc and GlcNAc-MurNAc fragments.

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“…Moreover, it is the main precursor in the synthesis of inhibitors for N-acetylneuraminidases from different sources [42]. The ring opening of 5 was not trivial since the substrate had a sterically hindered quaternary anomeric center, unlike the tertiary anomeric center C-1 of 1,6-anhydro sugars [29][30][31][32][33]43]. Furthermore, SnCl 4 and Sn(OTf) 2 might have formed a coordination bond with the carbonyl oxygen atom of the C-5 NHAc group and the O-6 position of the glucose glucopyranosyl ring.…”

Section: Resultsmentioning

confidence: 99%

SnCl₄-catalyzed solvent-free acetolysis of 2,7-anhydrosialic acid derivatives

Asressu

Wang

2019

Beilstein J. Org. Chem.

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Sialic acid-containing glycans are found in different sialic acid forms and a variety of glycosidic linkages in biologically active glycoconjugates. Hence, the preparation of suitably protected sialyl building blocks requires high attention in order to access glycans in a pure form. In line with this, various C-5-substituted 2,7-anhydrosialic acid derivatives bearing both electron-donating and -withdrawing protecting groups were synthesized and subjected to different Lewis acid-catalyzed solvent-free ring-opening reactions at room temperature in the presence of acetic anhydride. Among the various Lewis acids tested, the desired acetolysis products were obtained in moderate yields under tin(IV) chloride catalysis. Our methodology could be extended to regioselective protecting group installations and manipulations towards a number of thiosialoside and halide donors. 2990

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An efficient synthesis of 1,6-anhydro-N-acetylmuramic acid from N-acetylglucosamine

Cited by 8 publications

References 14 publications

N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism

N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism

Utilization of different MurNAc sources by the oral pathogen Tannerella forsythia and role of the inner membrane transporter AmpG

SnCl₄-catalyzed solvent-free acetolysis of 2,7-anhydrosialic acid derivatives

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An efficient synthesis of 1,6-anhydro-N-acetylmuramic acid from N-acetylglucosamine

Cited by 8 publications

References 14 publications

N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism

N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism

Utilization of different MurNAc sources by the oral pathogen Tannerella forsythia and role of the inner membrane transporter AmpG

SnCl4-catalyzed solvent-free acetolysis of 2,7-anhydrosialic acid derivatives

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SnCl₄-catalyzed solvent-free acetolysis of 2,7-anhydrosialic acid derivatives