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Fluorescent amino acids as versatile building blocks for chemical biology

Nature Reviews Chemistry volume 4, pages 275–290 (2020)Cite this article

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Abstract

Fluorophores have transformed the way we study biological systems, enabling non-invasive studies in cells and intact organisms, which increase our understanding of complex processes at the molecular level. Fluorescent amino acids have become an essential chemical tool because they can be used to construct fluorescent macromolecules, such as peptides and proteins, without disrupting their native biomolecular properties. Fluorescent and fluorogenic amino acids with unique photophysical properties have been designed for tracking protein–protein interactions in situ or imaging nanoscopic events in real time with high spatial resolution. In this Review, we discuss advances in the design and synthesis of fluorescent amino acids and how they have contributed to the field of chemical biology in the past 10 years. Important areas of research that we review include novel methodologies to synthesize building blocks with tunable spectral properties, their integration into peptide and protein scaffolds using site-specific genetic encoding and bioorthogonal approaches, and their application to design novel artificial proteins, as well as to investigate biological processes in cells by means of optical imaging.

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Fig. 1: Applications of FlAAs in chemical biology.
Fig. 2: Representative non-natural FlAAs developed in the past decade.
Fig. 3: Synthetic schemes for the de novo preparation of non-natural FlAAs.
Fig. 4: Bioactive fluorescent peptides for structural studies and optical imaging.
Fig. 5: Fluorescent d-amino acids for studying bacterial growth.
Fig. 6: Genetically encoded site-specific incorporation of FlAAs in live cells.

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References

  1. Chan, J., Dodani, S. C. & Chang, C. J. Reaction-based small-molecule fluorescent probes for chemoselective bioimaging. Nat. Chem. 4, 973–984 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Lavis, L. D. & Raines, R. T. Bright building blocks for chemical biology. ACS Chem. Biol. 9, 855–866 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Klymchenko, A. S. Solvatochromic and fluorogenic dyes as environment-sensitive probes: design and biological applications. Acc. Chem. Res. 50, 366–375 (2017).

    Article  CAS  PubMed  Google Scholar 

  4. Park, S. J. et al. Mechanistic elements and critical factors of cellular reprogramming revealed by stepwise global gene expression analyses. Stem Cell Res. 12, 730–741 (2014).

    Article  CAS  PubMed  Google Scholar 

  5. Shimomura, O., Johnson, F. H. & Saiga, Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol. 59, 223–239 (1962).

    Article  CAS  PubMed  Google Scholar 

  6. Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  PubMed  Google Scholar 

  7. Heim, R., Cubitt, A. B. & Tsien, R. Y. Improved green fluorescence. Nature 373, 663–664 (1995).

    Article  CAS  PubMed  Google Scholar 

  8. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Griffin, B. A., Adams, S. R. & Tsien, R. Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    Article  CAS  PubMed  Google Scholar 

  12. Martin, B. R., Giepmans, B. N. G., Adams, S. R. & Tsien, R. Y. Mammalian cell–based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat. Biotechnol. 23, 1308–1314 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Krueger, A. T. & Imperiali, B. Fluorescent amino acids: modular building blocks for the assembly of new tools for chemical biology. ChemBioChem 14, 788–799 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Nitz, M., Mezo, A. R., Ali, M. H. & Imperiali, B. Enantioselective synthesis and application of the highly fluorescent and environment-sensitive amino acid 6-(2-dimethylaminonaphthoyl) alanine (DANA). Chem. Commun. 17, 912–1913 (2002).

    Google Scholar 

  15. Vázquez, M. E., Blanco, J. B. & Imperiali, B. Photophysics and biological applications of the environment-sensitive fluorophore 6-N,N-dimethylamino-2,3-naphthalimide. J. Am. Chem. Soc. 127, 1300–1306 (2005).

    Article  PubMed  CAS  Google Scholar 

  16. Socher, E. & Imperiali, B. FRET-Capture: a sensitive method for the detection of dynamic protein interactions. ChemBioChem 14, 53–57 (2013).

    Article  CAS  PubMed  Google Scholar 

  17. Shults, M. D. & Imperiali, B. Versatile fluorescence probes of protein kinase activity. J. Am. Chem. Soc. 125, 14248–14249 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Venkatraman, P. et al. Fluorogenic probes for monitoring peptide binding to class II MHC proteins in living cells. Nat. Chem. Biol. 3, 222–228 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Vázquez, M. E., Rothman, D. M. & Imperiali, B. A new environment-sensitive fluorescent amino acid for Fmoc-based solid phase peptide synthesis. Org. Biomol. Chem. 2, 1965–1966 (2004).

    Article  Google Scholar 

  20. Wang, J., Xie, J. & Schultz, P. G. A genetically encoded fluorescent amino acid. J. Am. Chem. Soc. 128, 8738–8739 (2006).

    Article  CAS  PubMed  Google Scholar 

  21. Kielland, N., Vendrell, M., Lavilla, R. & Chang, Y.-T. Imaging histamine in live basophils and macrophages with a fluorescent mesoionic acid fluoride. Chem. Commun. 48, 7401–7403 (2012).

    Article  CAS  Google Scholar 

  22. Weiss, J. T. et al. Extracellular palladium-catalysed dealkylation of 5-fluoro-1-propargyl-uracil as a bioorthogonally activated prodrug approach. Nat. Commun. 5, 3277–3285 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Ramil, C. P. & Lin, Q. Photoclick chemistry: a fluorogenic light-triggered in vivo ligation reaction. Curr. Opin. Chem. Biol. 21, 89–95 (2014).

    Article  CAS  PubMed  Google Scholar 

  24. Devaraj, N. K. The future of bioorthogonal chemistry. ACS Cent. Sci. 4, 952–959 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. de Moliner, F., Kielland, N., Lavilla, R. & Vendrell, M. Modern synthetic avenues for the preparation of functional fluorophores. Angew. Chem. Int. Ed. 56, 3758–3769 (2017).

    Article  CAS  Google Scholar 

  26. Teale, F. W. & Weber, G. Ultraviolet fluorescence of the aromatic amino acids. Biochem. J. 65, 476–482 (1957).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ghisaidoobe, A. B. T. & Chung, S. J. Intrinsic tryptophan fluorescence in the detection and analysis of proteins: a focus on Förster resonance energy transfer techniques. Int. J. Mol. Sci. 15, 22518–22538 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Merkel, L., Hoesl, M. G., Albrecht, M., Schmidt, A. & Budisa, N. Blue fluorescent amino acids as in vivo building blocks for proteins. ChemBioChem 11, 305–314 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. Talukder, P. et al. Cyanotryptophans as novel fluorescent probes for studying protein conformational changes and DNA–protein interaction. Biochemistry 54, 7457–7469 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Hilaire, M. R. et al. Blue fluorescent amino acid for biological spectroscopy and microscopy. Proc. Natl Acad. Sci. USA 114, 6005–6009 (2017). Small analogue of a naturally occurring amino acid that behaves as a non-disruptive building block for the preparation of fluorescent peptides.

    Article  CAS  PubMed  Google Scholar 

  31. Zhang, K. et al. Synthesis and application of the blue fluorescent amino acid l-4-cyanotryptophan to assess peptide–membrane interactions. Chem. Commun. 55, 5095–5098 (2019).

    Article  CAS  Google Scholar 

  32. Winn, M., Francis, D. & Micklefield, J. De novo biosynthesis of “non-natural” thaxtomin phytotoxins. Angew. Chem. Int. Ed. 57, 6830–6833 (2018).

    Article  CAS  Google Scholar 

  33. Boville, C. E., Romney, D. K., Almhjell, P. J., Sieben, M. & Arnold, F. H. Improved synthesis of 4-cyanotryptophan and other tryptophan analogues in aqueous solvent using variants of TrpB from Thermotoga maritima. J. Org. Chem. 83, 7447–7452 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Talukder, P., Chen, S., Arce, P. M. & Hecht, S. M. Efficient asymmetric synthesis of tryptophan analogues having useful photophysical properties. Org. Lett. 16, 556–559 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wen, J. et al. Highly N2-selective coupling of 1,2,3-triazoles with indole and pyrrole. Chem. Eur. J. 20, 974–978 (2014).

    Article  CAS  PubMed  Google Scholar 

  36. Williams, T. J., Reay, A. J., Whitwood, A. C. & Fairlamb, I. J. S. A mild and selective Pd-mediated methodology for the synthesis of highly fluorescent 2-arylated tryptophans and tryptophan-containing peptides: a catalytic role for Pd0 nanoparticles? Chem. Commun. 50, 3052–3054 (2014).

    Article  CAS  Google Scholar 

  37. Bartoccini, F., Bartolucci, S., Mari, M. & Piersanti, G. A simple, modular synthesis of C4-substituted tryptophan derivatives. Org. Biomol. Chem. 14, 10095–10100 (2016).

    Article  CAS  PubMed  Google Scholar 

  38. Talukder, P. et al. Tryptophan-based fluorophores for studying protein conformational changes. Bioorg. Med. Chem. 22, 5924–5934 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chen, S. et al. Fluorescent biphenyl derivatives of phenylalanine suitable for protein modification. Biochemistry 52, 8580–8589 (2013).

    Article  CAS  PubMed  Google Scholar 

  40. Chen, S. et al. Detection of dihydrofolate reductase conformational change by FRET using two fluorescent amino acids. J. Am. Chem. Soc. 135, 12924–12927 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Maity, J., Honcharenko, D. & Strömberg, R. Synthesis of fluorescent d-amino acids with 4-acetamidobiphenyl and 4-N,N-dimethylamino-1,8-naphthalimido containing side chains. Tetrahedron Lett. 56, 4780–4783 (2015).

    Article  CAS  Google Scholar 

  42. Cheruku, P. et al. Tyrosine-derived stimuli responsive, fluorescent amino acids. Chem. Sci. 6, 1150–1158 (2015). A toolbox of tyrosine-based FlAAs with tunable emission and reversible pH and redox responses, showing potential for biosensing applications.

    Article  CAS  PubMed  Google Scholar 

  43. Bylińska, I., Guzow, K., Wójcik, J. & Wiczk, W. New non-protienogenic fluorescent amino acids: benzoxazol-5-yl-alanine derivatives containing acetylene unit. Synthesis, spectral and photophysical properties. J. Photochem. Photobiol. A Chem. 364, 679–685 (2018).

    Article  CAS  Google Scholar 

  44. Hoppmann, C., Alexiev, U., Irran, E. & Rück-Braun, K. Synthesis and fluorescence of xanthone amino acids. Tetrahedron Lett. 54, 4585–4587 (2013).

    Article  CAS  Google Scholar 

  45. Speight, L. C. et al. Efficient synthesis and in vivo incorporation of acridon-2-ylalanine, a fluorescent amino acid for lifetime and Förster resonance energy transfer/luminescence resonance energy transfer studies. J. Am. Chem. Soc. 135, 18806–18814 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Mendive-Tapia, L. et al. Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging. Nat. Protoc. 12, 1588–1619 (2017).

    Article  CAS  PubMed  Google Scholar 

  47. Subiros-Funosas, R. et al. Fluorogenic Trp(redBODIPY) cyclopeptide targeting keratin 1 for imaging of aggressive carcinomas. Chem. Sci. 11, 1368–1374 (2020). Development of an optically enhanced Trp-redBODIPY and validation in cyclic peptides for imaging aggressive carcinomas.

    Article  CAS  Google Scholar 

  48. Terrey, M. J., Holmes, A., Perry, C. C. & Cross, W. B. C–H olefination of tryptophan residues in peptides: control of residue selectivity and peptide–amino acid cross-linking. Org. Lett. 21, 7902–7907 (2019).

    Article  CAS  PubMed  Google Scholar 

  49. Wang, W., Lorion, M. M., Martinazzoli, O. & Ackermann, L. BODIPY peptide labeling by late-stage C(sp3)–H activation. Angew. Chem. Int. Ed. 57, 10554–10558 (2018).

    Article  CAS  Google Scholar 

  50. Schischko, A. et al. Late-stage peptide C–H alkylation for bioorthogonal C–H activation featuring solid phase peptide synthesis. Nat. Commun. 10, 3553 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Pereira, G., Vilaça, H. & Ferreira, P. M. T. Synthesis of new β-amidodehydroaminobutyric acid derivatives and of new tyrosine derivatives using copper catalyzed C–N and C–O coupling reactions. Amino Acids 44, 335–344 (2013).

    Article  CAS  PubMed  Google Scholar 

  52. Bag, S. S., Jana, S. & Pradhan, M. K. Synthesis, photophysical properties of triazolyl-donor/acceptor chromophores decorated unnatural amino acids: Incorporation of a pair into Leu-enkephalin peptide and application of triazolylperylene amino acid in sensing BSA. Bioorg. Med. Chem. 24, 3579–3595 (2016).

    Article  CAS  PubMed  Google Scholar 

  53. Benedetti, E., Veliz, A. B. E., Charpenay, M., Kocsis, L. S. & Brummond, K. M. Attachable solvatochromic fluorophores and bioconjugation studies. Org. Lett. 15, 2578–2581 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Li, C. et al. Click chemistry to fluorescent amino esters: synthesis and spectroscopic studies. Eur. J. Org. Chem. 2010, 2395–2405 (2010).

    Article  CAS  Google Scholar 

  55. Ferreira, P. M. T., Monteiro, L. S., Pereira, G., Castanheira, E. M. S. & Frost, C. G. Synthesis of fluorescent alanines by a rhodium-catalysed conjugate addition of arylboronic acids to dehydroalanine derivatives. Eur. J. Org. Chem. 2013, 550–556 (2013).

    Article  CAS  Google Scholar 

  56. Hsu, Y.-P. et al. Full color palette of fluorescent d-amino acids for in situ labeling of bacterial cell walls. Chem. Sci. 8, 6313–6321 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Häußler, D. & Gütschow, M. Fluorescently labeled amino acids as building blocks for bioactive molecules. Synthesis 48, 245–255 (2016).

    Google Scholar 

  58. 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).

    Article  CAS  PubMed  Google Scholar 

  59. Katritzky, A. R., Ozcan, S. & Todadze, E. Synthesis and fluorescence of the new environment-sensitive fluorophore 6-chloro-2,3-naphthalimide derivative. Org. Biomol. Chem. 8, 1296–1300 (2010).

    Article  CAS  PubMed  Google Scholar 

  60. Esteves, C. I. C., Silva, A. M. F., Raposo, M. M. M. & Costa, S. P. G. Unnatural benz-X-azolyl asparagine derivatives as novel fluorescent amino acids: synthesis and photophysical characterization. Tetrahedron 65, 9373–9377 (2009).

    Article  CAS  Google Scholar 

  61. Yokoo, H., Kagechika, H., Ohsaki, A. & Hirano, T. A polarity-sensitive fluorescent amino acid and its incorporation into peptides for the ratiometric detection of biomolecular interactions. ChemPlusChem 84, 1716–1719 (2019).

    Article  CAS  PubMed  Google Scholar 

  62. Shukla, L., Moodie, L. W. K., Kindahl, T. & Hedberg, C. Synthesis and spectroscopic properties of fluorinated coumarin lysine derivatives. J. Org. Chem. 83, 4792–4799 (2018).

    Article  CAS  PubMed  Google Scholar 

  63. Bag, S. S. & De, S. Isothiocyanyl alanine as a synthetic intermediate for the synthesis of thioureayl alanines and subsequent aminotetrazolyl alanines. J. Org. Chem. 82, 12276–12285 (2017).

    Article  CAS  PubMed  Google Scholar 

  64. Mohite, A. R. & Bhat, R. G. Enantiopure synthesis of side chain-modified α-amino acids and 5-cis-alkylprolines. J. Org. Chem. 77, 5423–5428 (2012).

    Article  CAS  PubMed  Google Scholar 

  65. Fowler, L. S., Ellis, D. & Sutherland, A. Synthesis of fluorescent enone derived α-amino acids. Org. Biomol. Chem. 7, 4309–4316 (2009).

    Article  CAS  PubMed  Google Scholar 

  66. Navo, C. D. et al. Cell-penetrating peptides containing fluorescent d-cysteines. Chem. Eur. J. 24, 7991–8000 (2018).

    Article  CAS  PubMed  Google Scholar 

  67. Wörner, S., Rönicke, F., Ulrich, A. S. & Wagenknecht, H.-A. 4-Aminophthalimide amino acids as small and environment-sensitive fluorescent probes for transmembrane peptides. ChemBioChem 21, 618–622 (2020).

    Article  PubMed  CAS  Google Scholar 

  68. Xiang, Z. & Wang, L. Enantiospecific synthesis of genetically encodable fluorescent unnatural amino acid l-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid. J. Org. Chem. 76, 6367–6371 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Zhao, Y., Pirrung, M. C. & Liao, J. A fluorescent amino acid probe to monitor efficiency of peptide conjugation to glass surfaces for high density microarrays. Mol. BioSyst. 8, 879–887 (2012).

    Article  CAS  PubMed  Google Scholar 

  70. Arribat, M., Rémond, E., Clément, S., Lee, A. V. D. & Cavelier, F. Phospholyl(borane) amino acids and peptides: stereoselective synthesis and fluorescent properties with large Stokes shift. J. Am. Chem. Soc. 140, 1028–1034 (2018).

    Article  CAS  PubMed  Google Scholar 

  71. Strizhak, A. V. et al. Two-color fluorescent l-amino acid mimic of tryptophan for probing peptide–nucleic acid complexes. Bioconjugate Chem. 23, 2434–2443 (2012). First report of a tyrosine-based FlAA exhibiting excited-state intramolecular proton transfer and hydration-sensitive dual emission.

    Article  CAS  Google Scholar 

  72. Postupalenko, V. Y. et al. Dual-fluorescence l-amino acid reports insertion and orientation of melittin peptide in cell membranes. Bioconjugate Chem. 24, 1998–2007 (2013).

    Article  CAS  Google Scholar 

  73. Sholokh, M. et al. Fluorescent amino acid undergoing excited state intramolecular proton transfer for site-specific probing and imaging of peptide interactions. J. Phys. Chem. B 119, 2585–2595 (2015).

    Article  CAS  PubMed  Google Scholar 

  74. Taki, M., Yamazaki, Y., Suzuki, Y. & Sisido, M. Introduction of a highly photodurable and common-laser excitable fluorescent amino acid into a peptide as a FRET acceptor for protease cleavage detection. Chem. Lett. 39, 818–819 (2010).

    Article  CAS  Google Scholar 

  75. Bell, J. D. et al. Synthesis and photophysical properties of benzotriazole-derived unnatural α-amino acids. J. Org. Chem. 84, 10436–10448 (2019).

    Article  CAS  PubMed  Google Scholar 

  76. Gilfillan, L., Artschwager, R., Harkiss, A. H., Liskamp, R. M. J. & Sutherland, A. Synthesis of pyrazole containing α-amino acids via a highly regioselective condensation/aza-Michael reaction of β-aryl α,β-unsaturated ketones. Org. Biomol. Chem. 13, 4514–4523 (2015).

    Article  CAS  PubMed  Google Scholar 

  77. Harkiss, A. H., Bell, J. D., Knuhtsen, A., Jamieson, A. G. & Sutherland, A. Synthesis and fluorescent properties of β-pyridyl α-amino acids. J. Org. Chem. 84, 2879–2890 (2019).

    Article  CAS  PubMed  Google Scholar 

  78. Bell, J. D. et al. Conformationally rigid pyrazoloquinazoline α-amino acids: one- and two-photon induced fluorescence. Chem. Commun. 56, 1887–1890 (2020).

    Article  CAS  Google Scholar 

  79. Häußler, D. & Gütschow, M. Synthesis of a fluorescent-labeled bisbenzamidine containing the central (6,7-dimethoxy-4-coumaryl)alanine building block. Heteroat. Chem. 26, 367–373 (2015).

    Article  CAS  Google Scholar 

  80. Koopmans, T., van Haren, M., van Ufford, L. Q., Beekman, J. M. & Martin, N. I. A concise preparation of the fluorescent amino acid l-(7-hydroxycoumarin-4-yl) ethylglycine and extension of its utility in solid phase peptide synthesis. Bioorg. Med. Chem. 21, 553–559 (2013).

    Article  CAS  PubMed  Google Scholar 

  81. Moodie, L. W. K., Chammaa, S., Kindahl, T. & Hedberg, C. Palladium-mediated approach to coumarin-functionalized amino acids. Org. Lett. 19, 2797–2800 (2017).

    Article  CAS  PubMed  Google Scholar 

  82. Fernandez, A., Thompson, E. J., Pollard, J. W., Kitamura, T. & Vendrell, M. A fluorescent activatable AND-gate chemokine CCL2 enables in vivo detection of metastasis-associated macrophages. Angew. Chem. Int. Ed. 58, 16894–16898 (2019).

    Article  CAS  Google Scholar 

  83. Joshi, P. N. & Rai, V. Single-site labeling of histidine in proteins, on-demand reversibility, and traceless metal-free protein purification. Chem. Commun. 55, 1100–1103 (2019).

    Article  CAS  Google Scholar 

  84. Cheng, M. H. Y., Savoie, H., Bryden, F. & Boyle, R. W. A convenient method for multicolour labelling of proteins with BODIPY fluorophores via tyrosine residues. Photochem. Photobiol. Sci. 16, 1260–1267 (2017).

    Article  CAS  PubMed  Google Scholar 

  85. Zhao, C. et al. Searching for the optimal fluorophore to label antimicrobial peptides. ACS Comb. Sci. 18, 689–696 (2016).

    Article  CAS  PubMed  Google Scholar 

  86. Vendrell, M. et al. Biotin ergopeptide probes for dopamine receptors. J. Med. Chem. 54, 1080–1090 (2011).

    Article  CAS  PubMed  Google Scholar 

  87. Palomo, J. M. Solid-phase peptide synthesis: an overview focused on the preparation of biologically relevant peptides. RSC Adv. 4, 32658–32672 (2014).

    Article  CAS  Google Scholar 

  88. Sainlos, M., Iskenderian, W. S. & Imperiali, B. A general screening strategy for peptide-based fluorogenic ligands: probes for dynamic studies of PDZ domain-mediated interactions. J. Am. Chem. Soc. 131, 6680–6682 (2009). Pioneering work on solvatochromic phthalimide amino acids and their integration into peptide structures to study dynamic protein–protein interactions.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Loving, G. & Imperiali, B. A versatile amino acid analogue of the solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide: a powerful tool for the study of dynamic protein interactions. J. Am. Chem. Soc. 130, 13630–13638 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Wang, W. et al. A fluorogenic peptide probe developed by in vitro selection using tRNA carrying a fluorogenic amino acid. Chem. Commun. 50, 2962–2964 (2014).

    Article  CAS  Google Scholar 

  91. Wang, W. et al. Fluorogenic enhancement of an in vitro-selected peptide ligand by replacement of a fluorescent group. Anal. Chem. 88, 7991–7997 (2016).

    Article  CAS  PubMed  Google Scholar 

  92. Heru, C., Jurgen, S., Tino, Z. & Horst, A. Fluorescent analogues of the insect neuropeptide helicokinin I: synthesis, photophysical characterization and biological activity. Prot. Pept. Lett. 17, 431–436 (2010).

    Article  Google Scholar 

  93. Manandhar, Y. et al. In vitro selection of a peptide aptamer that changes fluorescence in response to verotoxin. Biotechnol. Lett. 37, 619–625 (2015).

    Article  CAS  PubMed  Google Scholar 

  94. Newton, L. D., Pascu, S. I., Tyrrell, R. M. & Eggleston, I. M. Development of a peptide-based fluorescent probe for biological heme monitoring. Org. Biomol. Chem. 17, 467–471 (2019).

    Article  CAS  PubMed  Google Scholar 

  95. Zhao, C., Mendive-Tapia, L. & Vendrell, M. Fluorescent peptides for imaging of fungal cells. Arch. Biochem. Biophys. 661, 187–195 (2019).

    Article  CAS  PubMed  Google Scholar 

  96. Mendive-Tapia, L. et al. Spacer-free BODIPY fluorogens in antimicrobial peptides for direct imaging of fungal infection in human tissue. Nat. Commun. 7, 10940 (2016). First report of Trp-BODIPY as a fluorogenic amino acid to non-invasively label peptides for live-cell and ex vivo tissue imaging.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Akram, A. R. et al. Enhanced avidity from a multivalent fluorescent antimicrobial peptide enables pathogen detection in a human lung model. Sci. Rep. 9, 8422 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  98. Subiros-Funosas, R. et al. A Trp-BODIPY cyclic peptide for fluorescence labelling of apoptotic bodies. Chem. Commun. 53, 945–948 (2017).

    Article  CAS  Google Scholar 

  99. Ge, J., Li, L. & Yao, S. Q. A self-immobilizing and fluorogenic unnatural amino acid that mimics phosphotyrosine. Chem. Commun. 47, 10939–10941 (2011).

    Article  CAS  Google Scholar 

  100. TB, K. C. et al. Wash-free and selective imaging of epithelial cell adhesion molecule (EpCAM) expressing cells with fluorogenic peptide ligands. Biochem. Biophys. Res. Commun. 500, 283–287 (2018).

    Article  CAS  Google Scholar 

  101. Maeno, T. et al. Targeted delivery of fluorogenic peptide aptamers into live microalgae by femtosecond laser photoporation at single-cell resolution. Sci. Rep. 8, 8271 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  102. 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).

    Article  CAS  PubMed  Google Scholar 

  103. Dörr, T., Moynihan, P. J. & Mayer, C. Bacterial cell wall structure and dynamics. Front. Microbiol. 10, 2051 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  104. Radkov, A. D., Hsu, Y.-P., Booher, G. & VanNieuwenhze, M. S. Imaging bacterial cell wall biosynthesis. Annu. Rev. Biochem. 87, 991–1014 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Hsu, Y.-P., Booher, G., Egan, A., Vollmer, W. & VanNieuwenhze, M. S. d-Amino acid derivatives as in situ probes for visualizing bacterial peptidoglycan biosynthesis. Acc. Chem. Res. 52, 2713–2722 (2019).

    Article  CAS  PubMed  Google Scholar 

  106. de Pedro, M. A., Quintela, J. C., Höltje, J. V. & Schwarz, H. Murein segregation in Escherichia coli. J. Bacteriol. 179, 2823–2834 (1997).

    Article  PubMed  PubMed Central  Google Scholar 

  107. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Siegrist, M. S. et al. d-Amino acid chemical reporters reveal peptidoglycan dynamics of an intracellular pathogen. ACS Chem. Biol. 8, 500–505 (2013).

    Article  CAS  PubMed  Google Scholar 

  110. 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). Development of a toolbox of fluorescent d-amino acids for labelling peptidoglycans and monitoring bacterial cell-wall growth.

    Article  CAS  Google Scholar 

  111. Liechti, G. W. et al. A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis. Nature 506, 507–510 (2014).

    Article  CAS  PubMed  Google Scholar 

  112. Hudak, J. E., Alvarez, D., Skelly, A., von Andrian, U. H. & Kasper, D. L. Illuminating vital surface molecules of symbionts in health and disease. Nat. Microbiol. 2, 17099 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Wang, W. et al. Assessing the viability of transplanted gut microbiota by sequential tagging with d-amino acid-based metabolic probes. Nat. Commun. 10, 1317 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  114. Hu, F. et al. Visualization and in situ ablation of intracellular bacterial pathogens through metabolic labeling. Angew. Chem. Int. Ed. https://doi.org/10.1002/anie.201910187 (2019).

    Article  Google Scholar 

  115. Kuru, E. Mechanisms of incorporation for d-amino acid probes that target peptidoglycan biosynthesis. ACS Chem. Biol. 14, 2745–2756 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Kuru, E. et al. Fluorescent d-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation. Nat. Microbiol. 2, 1648–1657 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Morales Angeles, D. et al. Pentapeptide-rich peptidoglycan at the Bacillus subtilis cell-division site. Mol. Microbiol. 104, 319–333 (2017).

    Article  CAS  PubMed  Google Scholar 

  118. Baranowski, C. et al. Maturing Mycobacterium smegmatis peptidoglycan requires non-canonical crosslinks to maintain shape. eLife 7, e37516 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Yang, X. et al. GTPase activity–coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis. Science 355, 744–747 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Liechti, G. et al. Pathogenic Chlamydia lack a classical sacculus but synthesize a narrow, mid-cell peptidoglycan ring, regulated by MreB, for cell division. PLoS Pathog. 12, e1005590 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  122. Hsu, Y.-P. et al. Fluorogenic d-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays. Nat. Chem. 11, 335–341 (2019). Rotor-fluorogenic d-amino acids for real-time visualization of transpeptidase reactions and high-throughput screening of antibacterial drugs.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Rodriguez, E. A. et al. The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem. Sci. 42, 111–129 (2017).

    Article  CAS  PubMed  Google Scholar 

  124. Jing, C. & Cornish, V. W. Chemical tags for labeling proteins inside living cells. Acc. Chem. Res. 44, 784–792 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Spicer, C. D. & Davis, B. G. Selective chemical protein modification. Nat. Commun. 5, 4740 (2014).

    Article  CAS  PubMed  Google Scholar 

  126. Kajihara, D. et al. FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids. Nat. Methods 3, 923–929 (2006).

    Article  CAS  PubMed  Google Scholar 

  127. Goto, Y., Katoh, T. & Suga, H. Flexizymes for genetic code reprogramming. Nat. Protoc. 6, 779–790 (2011).

    Article  CAS  PubMed  Google Scholar 

  128. Lang, K. & Chin, J. W. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. 114, 4764–4806 (2014).

    Article  CAS  PubMed  Google Scholar 

  129. Young, D. D. & Schultz, P. G. Playing with the molecules of life. ACS Chem. Biol. 13, 854–870 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Chin, J. W. Expanding and reprogramming the genetic code of cells and animals. Annu. Rev. Biochem. 83, 379–408 (2014).

    Article  CAS  PubMed  Google Scholar 

  131. Chin, J. W. Expanding and reprogramming the genetic code. Nature 550, 53–60 (2017).

    Article  CAS  PubMed  Google Scholar 

  132. Dumas, A., Lercher, L., Spicer, C. D. & Davis, B. G. Designing logical codon reassignment – expanding the chemistry in biology. Chem. Sci. 6, 50–69 (2015).

    Article  CAS  PubMed  Google Scholar 

  133. Fredens, J. et al. Total synthesis of Escherichia coli with a recoded genome. Nature 569, 514–518 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Wang, K. et al. Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nat. Chem. 6, 393–403 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. Sachdeva, A., Wang, K., Elliott, T. & Chin, J. W. Concerted, rapid, quantitative, and site-specific dual labeling of proteins. J. Am. Chem. Soc. 136, 7785–7788 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Lampkowski, J. S., Uthappa, D. M. & Young, D. D. Site-specific incorporation of a fluorescent terphenyl unnatural amino acid. Bioorg. Med. Chem. Lett. 25, 5277–5280 (2015).

    Article  CAS  PubMed  Google Scholar 

  137. Kuhn, S. M., Rubini, M., Müller, M. A. & Skerra, A. Biosynthesis of a fluorescent protein with extreme pseudo-Stokes shift by introducing a genetically encoded non-natural amino acid outside the fluorophore. J. Am. Chem. Soc. 133, 3708–3711 (2011).

    Article  CAS  PubMed  Google Scholar 

  138. Lacey, V. K. et al. A fluorescent reporter of the phosphorylation status of the substrate protein STAT3. Angew. Chem. Int. Ed. 50, 8692–8696 (2011).

    Article  CAS  Google Scholar 

  139. Amaro, M. et al. Site-specific analysis of protein hydration based on unnatural amino acid fluorescence. J. Am. Chem. Soc. 137, 4988–4992 (2015).

    Article  CAS  PubMed  Google Scholar 

  140. Steinberg, X. et al. Conformational dynamics in TRPV1 channels reported by an encoded coumarin amino acid. eLife 6, e28626 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  141. Luo, J. et al. Genetically encoded optochemical probes for simultaneous fluorescence reporting and light activation of protein function with two-photon excitation. J. Am. Chem. Soc. 136, 15551–15558 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Wan, W., Tharp, J. M. & Liu, W. R. Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Biochim. Biophys. Acta 1844, 1059–1070 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Srinivasan, G., James, C. M. & Krzycki, J. A. Pyrrolysine encoded by UAG in archaea: charging of a UAG-decoding specialized tRNA. Science 296, 1459–1462 (2002).

    Article  CAS  PubMed  Google Scholar 

  144. Elliott, T. S. et al. Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal. Nat. Biotechnol. 32, 465–472 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Summerer, D. et al. A genetically encoded fluorescent amino acid. Proc. Natl Acad. Sci. USA 103, 9785–9789 (2006).

    Article  CAS  PubMed  Google Scholar 

  146. Lee, H. S., Guo, J., Lemke, E. A., Dimla, R. D. & Schultz, P. G. Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae. J. Am. Chem. Soc. 131, 12921–12923 (2009). Seminal work on the genetic encoding of the environmentally sensitive amino acid ANAP into protein structures.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Chatterjee, A., Guo, J., Lee, H. S. & Schultz, P. G. A genetically encoded fluorescent probe in mammalian cells. J. Am. Chem. Soc. 135, 12540–12543 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  148. Chen, R. F. Fluorescence of dansyl amino acids in organic solvents and protein solutions. Arch. Biochem. Biophys. 120, 609–620 (1967).

    Article  CAS  Google Scholar 

  149. Kalstrup, T. & Blunck, R. Dynamics of internal pore opening in Kv channels probed by a fluorescent unnatural amino acid. Proc. Natl Acad. Sci. USA 110, 8272–8277 (2013). The incorporation of ANAP into proteins enabled the investigation of the gating mechanism of voltage-gated potassium channels.

    Article  CAS  PubMed  Google Scholar 

  150. Sakata, S., Jinno, Y., Kawanabe, A. & Okamura, Y. Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid. Proc. Natl Acad. Sci. USA 113, 7521–7526 (2016).

    Article  CAS  PubMed  Google Scholar 

  151. Park, S.-H., Ko, W., Lee, H. S. & Shin, I. Analysis of protein–protein interaction in a single live cell by using a FRET system based on genetic code expansion technology. J. Am. Chem. Soc. 141, 4273–4281 (2019).

    Article  CAS  PubMed  Google Scholar 

  152. Preciado, S. et al. Synthesis and biological evaluation of a post-synthetically modified Trp-based diketopiperazine. MedChemComm 4, 1171–1174 (2013).

    Article  CAS  Google Scholar 

  153. Mendive-Tapia, L. et al. New peptide architectures through C–H activation stapling between tryptophan–phenylalanine/tyrosine residues. Nat. Commun. 6, 7160 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  154. Chen, X. et al. Histidine-specific peptide modification via visible-light-promoted C–H alkylation. J. Am. Chem. Soc. 141, 18230–18237 (2019).

    Article  CAS  PubMed  Google Scholar 

  155. Peciak, K., Laurine, E., Tommasi, R., Choi, J.-W. & Brocchini, S. Site-selective protein conjugation at histidine. Chem. Sci. 10, 427–439 (2019).

    Article  CAS  PubMed  Google Scholar 

  156. Ban, H., Gavrilyuk, J. & Barbas, C. F. Tyrosine bioconjugation through aqueous ene-type reactions: a click-like reaction for tyrosine. J. Am. Chem. Soc. 132, 1523–1525 (2010).

    Article  CAS  PubMed  Google Scholar 

  157. Tilley, S. D. & Francis, M. B. Tyrosine-selective protein alkylation using π-allylpalladium complexes. J. Am. Chem. Soc. 128, 1080–1081 (2006).

    Article  CAS  PubMed  Google Scholar 

  158. Benson, S. et al. SCOTfluors: small, conjugatable, orthogonal, and tunable fluorophores for in vivo imaging of cell metabolism. Angew. Chem. Int. Ed. 58, 6911–6915 (2019).

    Article  CAS  Google Scholar 

  159. Su, L. et al. Cu(I)-catalyzed 6-endo-dig cyclization of terminal alkynes, 2-bromoaryl ketones, and amides toward 1-naphthylamines: applications and photophysical properties. J. Am. Chem. Soc. 141, 2535–2544 (2019).

    Article  CAS  PubMed  Google Scholar 

  160. Mellanby, R. J. et al. Tricarbocyanine N-triazoles: the scaffold-of-choice for long-term near-infrared imaging of immune cells in vivo. Chem. Sci. 9, 7261–7270 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  161. Cosco, E. D. et al. Flavylium polymethine fluorophores for near- and shortwave infrared imaging. Angew. Chem. Int. Ed. 56, 13126–13129 (2017).

    Article  CAS  Google Scholar 

  162. Tang, J. et al. Single-atom fluorescence switch: a general approach toward visible-light-activated dyes for biological imaging. J. Am. Chem. Soc. 141, 14699–14706 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  163. Zheng, Q. et al. Rational design of fluorogenic and spontaneously blinking labels for super-resolution imaging. ACS Cent. Sci. 5, 1602–1613 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  164. Raymo, F. M. Photoactivatable synthetic fluorophores. Phys. Chem. Chem. Phys. 15, 14840–14850 (2013).

    Article  CAS  PubMed  Google Scholar 

  165. Zhang, Y., Tang, S., Sansalone, L., Baker, J. D. & Raymo, F. M. A photoswitchable fluorophore for the real-time monitoring of dynamic events in living organisms. Chem. Eur. J. 22, 15027–15034 (2016).

    Article  CAS  PubMed  Google Scholar 

  166. Hendricks, J. A. et al. Synthesis of [18F]BODIPY: bifunctional reporter for hybrid optical/positron emission tomography imaging. Angew. Chem. Int. Ed. 51, 4603–4606 (2012).

    Article  CAS  Google Scholar 

  167. Zhou, E. Y., Knox, H. J., Liu, C., Zhao, W. & Chan, J. A conformationally restricted aza-BODIPY platform for stimulus-responsive probes with enhanced photoacoustic properties. J. Am. Chem. Soc. 141, 17601–17609 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  168. Onogi, S. et al. In situ real-time imaging of self-sorted supramolecular nanofibres. Nat. Chem. 8, 743–752 (2016).

    Article  CAS  PubMed  Google Scholar 

  169. Beesley, J. L. & Woolfson, D. N. The de novo design of α-helical peptides for supramolecular self-assembly. Curr. Opin. Biotechnol. 58, 175–182 (2019).

    Article  CAS  PubMed  Google Scholar 

  170. Davis, L. & Greiss, S. Genetic encoding of unnatural amino acids in C. elegans. Methods Mol. Biol. 1728, 389–408 (2018).

    Article  CAS  PubMed  Google Scholar 

  171. Bridge, T. et al. Site-specific encoding of photoactivity in antibodies enables light-mediated antibody–antigen binding on live cells. Angew. Chem. Int. Ed. 58, 17986–17993 (2019).

    Article  CAS  Google Scholar 

  172. Ren, T.-B. et al. A general method to increase Stokes shift by introducing alternating vibronic structures. J. Am. Chem. Soc. 140, 7716–7722 (2018).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

E.K. acknowledges funding of a fellowship from the Life Sciences Research Foundation. A.S. acknowledges funding from the Wellcome Trust (204593/Z/16/Z) and the Biotechnology and Biological Sciences Research Council (BB/R004692/1). M.V. acknowledges funding from an ERC Consolidator Grant (771443). The authors thank S. Shaikh for the useful comments and technical support with the graphical illustrations.

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Authors and Affiliations

  1. Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK

    Zhiming Cheng & Marc Vendrell

  2. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Erkin Kuru

  3. School of Chemistry, University of East Anglia, Norwich, UK

    Amit Sachdeva

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  1. Zhiming Cheng
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Cheng, Z., Kuru, E., Sachdeva, A. et al. Fluorescent amino acids as versatile building blocks for chemical biology. Nat Rev Chem 4, 275–290 (2020). https://doi.org/10.1038/s41570-020-0186-z

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