Internationale Ausgabe: DOI: 10.1002/anie.201409927 Amino Acid Labeling Deutsche Ausgabe: DOI: 10.1002/ange.201409927 Metabolic Profiling of Bacteria by Unnatural C-terminated d-Amino Acids Sean E. Pidgeon, Jonathan M. Fura, William Leon, Morgan Birabaharan, Dmitri Vezenov, and Marcos M. Pires* Abstract: Bacterial peptidoglycan is a mesh-like network comprised of sugars and oligopeptides. Transpeptidases cross- link peptidoglycan oligopeptides to provide vital cell wall rigidity and structural support. It was recently discovered that the same transpeptidases catalyze the metabolic incorporation of exogenous d-amino acids onto bacterial cell surfaces with vast promiscuity for the side-chain identity. It is now shown that this enzymatic promiscuity is not exclusive to side chains, but that C-terminus variations can also be accommodated across a diverse range of bacteria. Atomic force microscopy analysis revealed that the incorporation of C-terminus ami- dated d-amino acids onto bacterial surfaces substantially reduced the cell wall stiffness. We exploited the promiscuity of bacterial transpeptidases to develop a novel assay for profiling different bacterial species. T remendous strides have been made in the treatment and prevention of bacterial infections. However, two major hurdles in diagnostics continue to impede further progress: identification of the type of bacteria and the level of drug resistance. These two time-sensitive components often dictate the course of treatment. Methods that improve our ability to address these needs may have significant clinical utility. We envisioned a peptidoglycan metabolic labeling strategy that could form the basis of a precise and facile diagnostic test to determine these two components in a single step. A large number of enzymatic transformations are neces- sary to properly assemble the peptidoglycan precursors. The lipid-anchored peptidoglycan precursors are then flipped to the outside of the cytoplasmic membrane where they are incorporated onto the growing peptidoglycan matrix. These covalent modifications are crucial for tuning the physical and mechanical properties of the peptidoglycan. The amount and nature of these modifications are inherently linked to the type of bacteria and may also be related to phenotypic differences within these species. Chemical modifications of the peptidoglycan can mainly be attributed to the enzymatic processes by penicillin binding proteins (PBPs). The transpeptidase domains of PBPs are responsible for the cross-linking of neighboring stem peptides, a function that endows the peptidoglycan with increased rigidity and strength. [1] The cross-linking of the peptidoglycan is vital to bacteria. Interference with this process via b-lactam and glycopeptide treatment is lethal to many bacteria. [2] Recently, an alternate reaction of transpeptidase was discov- ered whereby terminal d-alanines were “swapped” with d- Figure 1. a) Representation of the peptidoglycan processing by PBP transpeptidase. GlcNAc = N-acetylglucosamine, MurNAc = N-acetyl- muramic acid, m-DAP = meso-diaminopimelic acid. b) Basic unit of d-amino acid and derivatives that have been shown to be tolerated. c) Representation of the assay to assess d-amino acid incorporation. d) Chemical structures of d-amino acid derivatives that were synthe- sized and evaluated. [*] S. E. Pidgeon, J. M. Fura, W. Leon, M. Birabaharan, Dr. D. Vezenov, Dr. M. M. Pires Department of Chemistry, Lehigh University 6 E. Packer Ave., Bethlehem, PA 18015 (USA) E-mail: map311@lehigh.edu Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201409927. . Angewandte Zuschriften 6256 # 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2015, 127, 6256 –6260