Metabolic Incorporation of N ‐Acetyl Muramic Acid Probes into Bacterial Peptidoglycan
Abstract
Abstract Bacterial cells utilize small carbohydrate building blocks to construct peptidoglycan (PG), a highly conserved mesh‐like polymer that serves as a protective coat for the cell. PG production has long been a target for antibiotics, and its breakdown is a source for human immune recognition. A key component of bacterial PG, N ‐acetyl muramic acid (NAM), is a vital element in many synthetically derived immunostimulatory compounds. However, the exact molecular details of these structures and how they are generated remain unknown due to a lack of chemical probes surrounding the NAM core. A robust synthetic strategy to generate bioorthogonally tagged NAM carbohydrate units is implemented. These molecules serve as precursors for PG biosynthesis and recycling. Escherichia coli cells are metabolically engineered to incorporate the bioorthogonal NAM probes into their PG network. The probes are subsequently modified using copper‐catalyzed azide‐alkyne cycloaddition to install fluorophores directly into the bacterial PG, as confirmed by super‐resolution microscopy and high‐resolution mass spectrometry. Here, synthetic notes for key elements of this process to generate the sugar probes as well as streamlined user‐friendly metabolic labeling strategies for both microbiology and immunological applications are described. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1 : Synthesismore »
- Authors:
-
- Department of Chemistry and Biochemistry University of Delaware Newark Delaware
- Department of Chemistry and Biochemistry University of Delaware Newark Delaware, Cutaneous Microbiome and Inflammation Section, Dermatology Branch National Institute of Arthritis and Musculoskeletal and Skin Diseases Bethesda Maryland
- Department of Chemistry and Biochemistry University of Delaware Newark Delaware, Center for the Study of Organic Compounds, CEDECOR‐UNLP‐CIC, Department of Chemistry, Faculty of Exact Sciences National University of La Plata Buenos Aires Argentina
- Department of Chemistry and Biochemistry University of Delaware Newark Delaware, Department of Biological Sciences University of Delaware Newark Delaware
- Publication Date:
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1570027
- Resource Type:
- Publisher's Accepted Manuscript
- Journal Name:
- Current Protocols in Chemical Biology
- Additional Journal Information:
- Journal Name: Current Protocols in Chemical Biology Journal Volume: 11 Journal Issue: 4; Journal ID: ISSN 2160-4762
- Publisher:
- Wiley Blackwell (John Wiley & Sons)
- Country of Publication:
- Country unknown/Code not available
- Language:
- English
Citation Formats
DeMeester, Kristen E., Liang, Hai, Zhou, Junhui, Wodzanowski, Kimberly A., Prather, Benjamin L., Santiago, Cintia C., and Grimes, Catherine L. Metabolic Incorporation of N ‐Acetyl Muramic Acid Probes into Bacterial Peptidoglycan. Country unknown/Code not available: N. p., 2019.
Web. doi:10.1002/cpch.74.
DeMeester, Kristen E., Liang, Hai, Zhou, Junhui, Wodzanowski, Kimberly A., Prather, Benjamin L., Santiago, Cintia C., & Grimes, Catherine L. Metabolic Incorporation of N ‐Acetyl Muramic Acid Probes into Bacterial Peptidoglycan. Country unknown/Code not available. https://doi.org/10.1002/cpch.74
DeMeester, Kristen E., Liang, Hai, Zhou, Junhui, Wodzanowski, Kimberly A., Prather, Benjamin L., Santiago, Cintia C., and Grimes, Catherine L. Thu .
"Metabolic Incorporation of N ‐Acetyl Muramic Acid Probes into Bacterial Peptidoglycan". Country unknown/Code not available. https://doi.org/10.1002/cpch.74.
@article{osti_1570027,
title = {Metabolic Incorporation of N ‐Acetyl Muramic Acid Probes into Bacterial Peptidoglycan},
author = {DeMeester, Kristen E. and Liang, Hai and Zhou, Junhui and Wodzanowski, Kimberly A. and Prather, Benjamin L. and Santiago, Cintia C. and Grimes, Catherine L.},
abstractNote = {Abstract Bacterial cells utilize small carbohydrate building blocks to construct peptidoglycan (PG), a highly conserved mesh‐like polymer that serves as a protective coat for the cell. PG production has long been a target for antibiotics, and its breakdown is a source for human immune recognition. A key component of bacterial PG, N ‐acetyl muramic acid (NAM), is a vital element in many synthetically derived immunostimulatory compounds. However, the exact molecular details of these structures and how they are generated remain unknown due to a lack of chemical probes surrounding the NAM core. A robust synthetic strategy to generate bioorthogonally tagged NAM carbohydrate units is implemented. These molecules serve as precursors for PG biosynthesis and recycling. Escherichia coli cells are metabolically engineered to incorporate the bioorthogonal NAM probes into their PG network. The probes are subsequently modified using copper‐catalyzed azide‐alkyne cycloaddition to install fluorophores directly into the bacterial PG, as confirmed by super‐resolution microscopy and high‐resolution mass spectrometry. Here, synthetic notes for key elements of this process to generate the sugar probes as well as streamlined user‐friendly metabolic labeling strategies for both microbiology and immunological applications are described. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1 : Synthesis of peracetylated 2‐azido glucosamine Basic Protocol 2 : Synthesis of 2‐azido and 2‐alkyne NAM Basic Protocol 3 : Synthesis of 3‐azido NAM methyl ester Basic Protocol 4 : Incorporation of NAM probes into bacterial peptidoglycan Basic Protocol 5 : Confirmation of bacterial cell wall remodeling by mass spectrometry},
doi = {10.1002/cpch.74},
journal = {Current Protocols in Chemical Biology},
number = 4,
volume = 11,
place = {Country unknown/Code not available},
year = {Thu Oct 10 00:00:00 EDT 2019},
month = {Thu Oct 10 00:00:00 EDT 2019}
}
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