Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Fluorogenic d-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays


Peptidoglycan is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions, which construct peptide crosslinkages between polysaccharide chains to endow mechanical strength. However, tracking the transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis and application of rotor-fluorogenic d-amino acids (RfDAAs), enabling real-time, continuous tracking of transpeptidation reactions. These probes allow peptidoglycan biosynthesis to be monitored in real time by visualizing transpeptidase reactions in live cells, as well as real-time activity assays of d,d- and l,d-transpeptidases and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into peptidoglycan provides a powerful new tool to study peptidoglycan biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of peptidoglycan biosynthesis.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Synthetic routes for preparation of the RfDAAs.
Fig. 2: Unlike FDAAs, RfDAAs allow wash-free imaging of bacterial cell walls.
Fig. 3: RfDAAs allow real-time imaging of PG synthesis in S. venezuelae.
Fig. 4: RfDAAs facilitate real-time in vitro transpeptidation assays.

Data availability

All data are available upon reasonable request from the corresponding authors.


  1. 1.

    Typas, A., Banzhaf, M., Gross, Ca & Vollmer, W. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat. Rev. Microbiol. 10, 123–136 (2012).

    CAS  Article  Google Scholar 

  2. 2.

    Egan, A. J. F., Cleverley, R. M., Peters, K., Lewis, R. J. & Vollmer, W. Regulation of bacterial cell wall growth. FEBS J. 284, 851–867 (2017).

    CAS  Article  Google Scholar 

  3. 3.

    Vollmer, W., Blanot, D. & de Pedro, M. A. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149–167 (2008).

    CAS  Article  Google Scholar 

  4. 4.

    Lupoli, T. J. et al. Transpeptidase-mediated incorporation of d-amino acids into bacterial peptidoglycan. J. Am. Chem. Soc. 133, 10748–10751 (2011).

    CAS  Article  Google Scholar 

  5. 5.

    Wilke, M. S., Lovering, A. L. & Strynadka, N. C. β-Lactam antibiotic resistance: a current structural perspective. Curr. Opin. Microbiol. 8, 525–533 (2005).

    CAS  Article  Google Scholar 

  6. 6.

    Waxman, D. J. & Strominger, J. L. Penicillin-binding proteins and the mechanism of action of beta-lactam antibiotics. Annu. Rev. Biochem. 52, 825–869 (1983).

    CAS  Article  Google Scholar 

  7. 7.

    Tipper, D. J. & Strominger, J. L. Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-d-alanyl-d-alanine. Proc. Natl Acad. Sci. USA 54, 1133–1141 (1965).

    CAS  Article  Google Scholar 

  8. 8.

    Sauvage, E., Kerff, F., Terrak, M., Ayala, J. A. & Charlier, P. The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol. Rev. 32, 234–258 (2008).

    CAS  Article  Google Scholar 

  9. 9.

    Mainardi, J.-L. et al. A novel peptidoglycan cross-linking enzyme for a beta-lactam-resistant transpeptidation pathway. J. Biol. Chem. 280, 38146–38152 (2005).

    CAS  Article  Google Scholar 

  10. 10.

    Mainardi, J.-L. et al. Novel mechanism of β-lactam resistance due to bypass of d d-transpeptidation in Enterococcus faecium. J. Biol. Chem. 275, 16490–16496 (2000).

    CAS  Article  Google Scholar 

  11. 11.

    Hugonnet, J.-E. et al. Factors essential for l,d-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli. eLife 5, e19469 (2016).

    Article  Google Scholar 

  12. 12.

    Mazmanian, S. K., Liu, G., Jensen, E. R., Lenoy, E. & Schneewind, O. Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections. Proc. Natl Acad. Sci. USA 97, 5510–5515 (2000).

    CAS  Article  Google Scholar 

  13. 13.

    Ton-That, H., Liu, G., Mazmanian, S. K., Faull, K. F. & Schneewind, O. Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc. Natl Acad. Sci. USA 96, 12424–12429 (1999).

    CAS  Article  Google Scholar 

  14. 14.

    Egan, A. J. F., Biboy, J., van’t Veer, I., Breukink, E. & Vollmer, W. Activities and regulation of peptidoglycan synthases. Philos. Trans. R. Soc. B Biol. Sci. 370, 20150031 (2015).

    Article  Google Scholar 

  15. 15.

    Kuru, E. et al. In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent d-amino acids. Angew. Chem. Int. Ed. 51, 12519–12523 (2012).

    CAS  Article  Google Scholar 

  16. 16.

    Kuru, E., Tekkam, S., Hall, E., Brun, Y. V. & Van Nieuwenhze, M. S. Synthesis of fluorescent d-amino acids and their use for probing peptidoglycan synthesis and bacterial growth in situ. Nat. Protoc. 10, 33–52 (2015).

    CAS  Article  Google Scholar 

  17. 17.

    Hsu, Y.-P. et al. Full color palette of fluorescent d-amino acids for in situ labeling of bacterial cell walls. Chem. Sci. 290, 30540–30550 (2017).

    Google Scholar 

  18. 18.

    Hsu, Y.-P., Meng, X. & VanNieuwenhze, M. S. S. in Imaging Bacterial Molecules, Structures and Cells. Methods in Microbiology Vol. 43 (eds Harwood, C. & Jensen, G. J.) Ch. 1, 3–48 (Elsevier, 2016).

  19. 19.

    Bisson-Filho, A. W. et al. Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division. Science 355, 739–743 (2017).

    CAS  Article  Google Scholar 

  20. 20.

    Haidekker, M. A. & Theodorakis, E. A. Environment-sensitive behavior of fluorescent molecular rotors. J. Biol. Eng. 4, 11 (2010).

    Article  Google Scholar 

  21. 21.

    Haidekker, M. A. et al. A ratiometric fluorescent viscosity sensor. J. Am. Chem. Soc. 128, 398–399 (2006).

    Article  Google Scholar 

  22. 22.

    Kuimova, M. K. Mapping viscosity in cells using molecular rotors. Phys. Chem. Chem. Phys. 14, 12671 (2012).

    CAS  Article  Google Scholar 

  23. 23.

    Hosny, N. A. et al. Mapping microbubble viscosity using fluorescence lifetime imaging of molecular rotors. Proc. Natl Acad. Sci. USA 110, 9225–9230 (2013).

    CAS  Article  Google Scholar 

  24. 24.

    Goh, W. L. et al. Molecular rotors as conditionally fluorescent labels for rapid detection of biomolecular interactions. J. Am. Chem. Soc. 136, 6159–6162 (2014).

    CAS  Article  Google Scholar 

  25. 25.

    Yu, W.-T., Wu, T.-W., Huang, C.-L., Chen, I.-C. & Tan, K.-T. Protein sensing in living cells by molecular rotor-based fluorescence-switchable chemical probes. Chem. Sci. 7, 301–307 (2016).

    CAS  Article  Google Scholar 

  26. 26.

    Dziuba, D., Jurkiewicz, P., Cebecauer, M., Hof, M. & Hocek, M. A rotational BODIPY nucleotide: an environment-sensitive fluorescence-lifetime probe for DNA interactions and applications in live-cell microscopy. Angew. Chem. Int. Ed. 128, 182–186 (2016).

    Article  Google Scholar 

  27. 27.

    Nadler, A. & Schultz, C. The power of fluorogenic probes. Angew. Chem. Int. Ed. 52, 2408–2410 (2013).

    CAS  Article  Google Scholar 

  28. 28.

    Kamariza, M. et al. Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe. Sci. Transl. Med. 10, eaam6310 (2018).

    Article  Google Scholar 

  29. 29.

    Haidekker, M. A. et al. New fluorescent probes for the measurement of cell membrane viscosity. Chem. Biol. 8, 123–131 (2001).

    CAS  Article  Google Scholar 

  30. 30.

    Sawada, S., Iio, T., Hayashi, Y. & Takahashi, S. Fluorescent rotors and their applications to the study of G–F transformation of actin. Anal. Biochem. 204, 110–117 (1992).

    CAS  Article  Google Scholar 

  31. 31.

    De, K., Legros, J., Crousse, B. & Bonnet-Delpon, D. Solvent-promoted and -controlled aza-Michael reaction with aromatic amines. J. Org. Chem. 74, 6260–6265 (2009).

    CAS  Article  Google Scholar 

  32. 32.

    Shao, J. et al. Thiophene-inserted aryl-dicyanovinyl compounds: the second generation of fluorescent molecular rotors with significantly redshifted emission and large Stokes shift. Eur. J. Org. Chem. 2011, 6100–6109 (2011).

    CAS  Article  Google Scholar 

  33. 33.

    Monnereau, C., Blart, E. & Odobel, F. A cheap and efficient method for selective para-iodination of aniline derivatives. Tetrahedron Lett. 46, 5421–5423 (2005).

    CAS  Article  Google Scholar 

  34. 34.

    Suzuki, A. Recent advances in the cross-coupling reactions of organoboron derivatives with organic electrophiles, 1995–1998. J. Organomet. Chem. 576, 147–168 (1999).

    CAS  Article  Google Scholar 

  35. 35.

    Delcour, A. H. Outer membrane permeability and antibiotic resistance. Biochim. Biophys. Acta 1794, 808–816 (2009).

    CAS  Article  Google Scholar 

  36. 36.

    Sampson, B. A., Misra, R. & Benson, S. A. Identification and characterization of a new gene of Escherichia coli K-12 involved in outer membrane permeability. Genetics 122, 491–501 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Zhou, F. et al. Molecular rotors as fluorescent viscosity sensors: molecular design, polarity sensitivity, dipole moments changes, screening solvents and deactivation channel of the excited states. Eur. J. Org. Chem. 2011, 4773–4787 (2011).

    CAS  Article  Google Scholar 

  38. 38.

    Haidekker, M. A. et al. Molecular rotors—fluorescent biosensors for viscosity and flow. Org. Biomol. Chem. 5, 1669–1678 (2007).

    CAS  Article  Google Scholar 

  39. 39.

    Randich, A. M. & Brun, Y. V. Molecular mechanisms for the evolution of bacterial morphologies and growth modes. Front. Microbiol. 6, 580 (2015).

    Article  Google Scholar 

  40. 40.

    Lupoli, T. J. et al. Lipoprotein activators stimulate Escherichia coli penicillin-binding proteins by different mechanisms. J. Am. Chem. Soc. 136, 52–55 (2014).

    CAS  Article  Google Scholar 

  41. 41.

    Bertsche, U., Breukink, E., Kast, T. & Vollmer, W. In vitro murein (peptidoglycan) synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli. J. Biol. Chem. 280, 38096–38101 (2005).

    CAS  Article  Google Scholar 

  42. 42.

    Born, P., Breukink, E. & Vollmer, W. In vitro synthesis of cross-linked murein and its attachment to sacculi by PBP1A from Escherichia coli. J. Biol. Chem. 281, 26985–26993 (2006).

    CAS  Article  Google Scholar 

  43. 43.

    Qiao, Y. et al. Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams. Nat. Chem. Biol. 13, 793–798 (2017).

    CAS  Article  Google Scholar 

  44. 44.

    Navratna, V. et al. Molecular basis for the role of Staphylococcus aureus penicillin binding protein 4 in antimicrobial resistance. J. Bacteriol. 192, 134–144 (2010).

    CAS  Article  Google Scholar 

  45. 45.

    Wyke, A. W., Ward, J. B., Hayes, M. V. & Curtis, N. A. A role in vivo for penicillin-binding protein-4 of Staphylococcus aureus. Eur. J. Biochem. 119, 389–393 (1981).

    CAS  Article  Google Scholar 

  46. 46.

    Memmi, G., Filipe, S. R., Pinho, M. G., Fu, Z. & Cheung, A. Staphylococcus aureus PBP4 is essential for β-lactam resistance in community-acquired methicillin-resistant strains. Antimicrob. Agents Chemother. 52, 3955–3966 (2008).

    CAS  Article  Google Scholar 

  47. 47.

    Swenson, J. M. et al. Correlation of cefoxitin MICs with the presence of mecA in Staphylococcus spp. J. Clin. Microbiol. 47, 1902–1905 (2009).

    CAS  Article  Google Scholar 

  48. 48.

    Kocaoglu, O. & Carlson, E. E. Profiling of β-lactam selectivity for penicillin-binding proteins in Escherichia coli strain DC2. Antimicrob. Agents Chemother. 59, 2785–2790 (2015).

    CAS  Article  Google Scholar 

  49. 49.

    Wehrli, R., von Graevenitz, A. & Lüthy, R. Susceptibility and tolerance of β-lactamase-producing, methicillin-sensitive strains of Staphylococcus aureus towards seven broad-spectrum penicillins. Infection 11, 322–325 (1983).

    CAS  Article  Google Scholar 

  50. 50.

    Uri, J. V. Detection of beta-lactamase activity with nitrocefin of multiple strains of various microbial genera. Acta Microbiol. Hung. 32, 133–145 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Pitkälä, A., Salmikivi, L., Bredbacka, P., Myllyniemi, A.-L. & Koskinen, M. T. Comparison of tests for detection of beta-lactamase-producing staphylococci. J. Clin. Microbiol. 45, 2031–2033 (2007).

    Article  Google Scholar 

  52. 52.

    Cava, F., de Pedro, M. A., Lam, H., Davis, B. M. & Waldor, M. K. Distinct pathways for modification of the bacterial cell wall by non-canonical d-amino acids. EMBO J. 30, 3442–3453 (2011).

    CAS  Article  Google Scholar 

  53. 53.

    Guo, Y., Cai, S., Gu, G., Guo, Z. & Long, Z. Recent progress in the development of sortase A inhibitors as novel anti-bacterial virulence agents. RSC Adv. 5, 49880–49889 (2015).

    CAS  Article  Google Scholar 

  54. 54.

    Oh, K.-B. et al. Discovery of diarylacrylonitriles as a novel series of small molecule sortase A inhibitors. J. Med. Chem. 47, 2418–2421 (2004).

    CAS  Article  Google Scholar 

  55. 55.

    Lorand, L. & Graham, R. M. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat. Rev. Mol. Cell Biol. 4, 140–156 (2003).

    CAS  Article  Google Scholar 

  56. 56.

    Kunjapur, A. M. et al. Engineering posttranslational proofreading to discriminate nonstandard amino acids. Proc. Natl Acad. Sci. USA 115, 619–624 (2018).

    CAS  Article  Google Scholar 

Download references


The authors thank K. C. Huang for providing strain E. coli imp4213, S. Walker for providing S. aureus PBP4 plasmid, D. Kysela for help with image processing and analysis and J. Rittichier for providing the enzyme substrates used in the in vitro assays and his advice on RfDAA synthesis. This study is supported by NIH grants 5R01GM113172 to M.S.V. and Y.V.B. and R35GM122556 to Y.V.B., and by a Canada 150 research Chair in Bacterial Cell Biology to Y.V.B.

Author information




E.H., E.K. and Y.-P.H. designed RfDAAs. Y.-P.H., E.H., B.M., C.M. and J.Y. synthesized RfDAAs. Y.-P.H. characterized RfDAAs. Y.-P.H. and E.K. performed cell labelling and microscopy experiments. G.B., A.D.R., L.A. and F.C. prepared and performed the in vitro assays. Y.-P.H., A.D.R., E.K., Y.V.B. and M.S.V. wrote the paper. All authors were involved in the design of this work.

Corresponding authors

Correspondence to Yves V. Brun, Erkin Kuru or Michael S. VanNieuwenhze.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

The protocol for RfDAA synthesis, characterization data and bacteria labelling and imaging experiments. The file also includes supplementary discussions, figures, tables and NMR spectra of the probes.

Supplementary Movie 1.mp4

The time-lapse microscopy of PG synthesis labelled by Rf470DL in S. venezuelae.

Supplementary Movie 2.mp4

The time-lapse microscopy of PG synthesis labelled by Rf470DL in B. subtilis.

Supplementary Movie 3.mp4

The time-lapse microscopy of PG synthesis labelled by Rf470DL in B. subtilis. The effect of RfDAA labeling in dead cells was highlighted.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hsu, YP., Hall, E., Booher, G. et al. Fluorogenic d-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays. Nat. Chem. 11, 335–341 (2019).

Download citation

Further reading


Quick links