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

Information of the chemical, mechanical, and electrical properties of materials can be obtained using force volume mapping (FVM), a measurement mode of scanning probe microscopy (SPM). Protocols have been developed with FVM for a broad range of materials, including polymers, organic films, inorganic materials, and biological samples. Multiple force measurements are acquired with the FVM mode within a defined 3D volume of the sample to map interactions (i.e., chemical, electrical, or physical) between the probe and the sample. Forces of adhesion, elasticity, stiffness, deformation, chemical binding interactions, viscoelasticity, and electrical properties have all been mapped at the nanoscale with FVM. Subsequently, force maps can be correlated with features of topographic images for identifying certain chemical groups presented at a sample interface. The SPM tip can be coated to investigate-specific reactions; for example, biological interactions can be probed when the tip is coated with biomolecules such as for recognition of ligand−receptor pairs or antigen−antibody interactions. This review highlights the versatility and diverse measurement protocols that have emerged for studies applying FVM for the analysis of material properties at the nanoscale.

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Peptidoglycan (PG) is the core structural motif of the bacterial cell wall. Fragments released from the PG serve as fundamental recognition elements for the immune system. The structure of the PG, however, encompasses a variety of chemical modifications among different bacterial species. Here, the applicability of organic synthetic methods to address this chemical diversity is explored, and the synthesis of cross-linked PG fragments, carrying biologically relevant amino acid modifications and peptide cross-linkages, is presented using solution and solid phase approaches.

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