Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
, 18 (6), 486-501

Bio-orthogonal Fluorescent Labelling of Biopolymers Through Inverse-Electron-Demand Diels-Alder Reactions

Affiliations
Review

Bio-orthogonal Fluorescent Labelling of Biopolymers Through Inverse-Electron-Demand Diels-Alder Reactions

Eszter Kozma et al. Chembiochem.

Erratum in

Abstract

Bio-orthogonal labelling schemes based on inverse-electron-demand Diels-Alder (IEDDA) cycloaddition have attracted much attention in chemical biology recently. The appealing features of this reaction, such as the fast reaction kinetics, fully bio-orthogonal nature and high selectivity, have helped chemical biologists gain deeper understanding of biochemical processes at the molecular level. Listing the components and discussing the possibilities and limitations of these reagents, we provide a recent snapshot of the field of IEDDA-based biomolecular manipulation with special focus on fluorescent modulation approaches through the use of bio-orthogonalized building blocks. At the end, we discuss challenges that need to be addressed for further developments in order to overcome recent limitations and to enable researchers to answer biomolecular questions in more detail.

Keywords: bio-orthogonal building blocks; cycloaddition; dienophiles; fluorescence; inverse-electron-demand Diels-Alder reaction; tetrazines.

Figures

Scheme 1
Scheme 1
General scheme of IEDDA reactions.
Scheme 2
Scheme 2
Dienophiles, with second‐order rate constants (k 2 [m −1 s−1] in water) measured with tetrazines indicated in parentheses.
Scheme 3
Scheme 3
Some representative examples of synthetic small‐molecule fluorescent tetrazine probes. A) No spectral constraints between the fluorophore and the tetrazine linked together with a flexible linker. B) Fluorogenic FRET probes in which the spectrally matching fluorophore is linked to the tetrazine through a flexible linker. C) Fluorogenic TBET probes in which the fluorophore is linked to the tetrazine through a rigid linker.
Scheme 4
Scheme 4
Bio‐orthogonalized building blocks for chemical or enzymatic incorporation of strained dienophiles. Post‐synthetic modification of DNA and RNA by IEDDA cycloaddition with various tetrazines bearing fluorophores, biotin or boronic acid groups.
Scheme 5
Scheme 5
Vinyl‐type building blocks. VdU (12) was demonstrated to be an efficient metabolic probe for cellular DNA imaging. DNA modified with DvinA (11) was tagged with biotin‐tetrazine conjugate.
Scheme 6
Scheme 6
Building blocks from the toolbox of unnatural base pair genetic code expansion.
Scheme 7
Scheme 7
Tetrazine‐based nucleic acid building blocks.
Scheme 8
Scheme 8
Schematic and building blocks for template‐driven oligonucleotide labelling through modified DNA ligation, catalysed by hybridization against a template strand. Reprinted from ref. 42, with permission. Copyright: 2014, American Chemical Society.
Scheme 9
Scheme 9
Lipid building blocks for tagging phospholipids (left) or visualising the Golgi apparatus (right).
Figure 1
Figure 1
Super‐resolution imaging of the Golgi in live cells with the aid of Cer‐Sir. A) Confocal and STED images of the Golgi in live cells treated with Cer‐TCO and SiR‐3 a. Line traces through the Golgi (yellow) show the greatly improved resolving power of STED (right‐hand panel). B) Kymo‐graphs (line profile vs. time) of fixed cells imaged by STED in which the Golgi were labelled with Cer‐SiR or Rab6‐SNAP‐SiR (Rab6 is a Golgi‐targeted protein); note that the signal decays much more quickly when the protein is labelled with SiR. C) Time‐lapse STED of vesicle budding and trafficking out of the Golgi (green arrowhead). Reproduced from ref. 26b, with permission. Copyright: 2014, John Wiley & Sons, Inc.
Scheme 10
Scheme 10
Structures of noncanonical amino acids used as building blocks for fluorescent protein labelling in IEDDA.
Figure 2
Figure 2
Double labelling of calmodulin. A) Amber suppression is used to encode 40, whereas 25 is incorporated in response to a quadruplet codon. Double labelling is performed with dyes based on BCN and 1, respectively. B) Probing calmodulin dynamics by detecting FRET in the presence of increasing urea concentrations. Reprinted in part and with permission from ref. 62. Copyright: 2014, Nature Publishing Group.
Figure 3
Figure 3
Site‐specific fluorescent labelling of proteins through IEDDA cycloaddition enables super‐resolution microscopy. A) Cell‐membrane dual‐labelling of insulin receptors: schematic two‐step cell labelling protocol and GSDIM images of double‐labelled insulin receptors. B) Intracellular labelling of actin filaments and subsequent STORM imaging. Figure 3 A is reproduced in part and with permission from ref. 64. Copyright: 2014, John Wiley & Sons, Inc. Figure 3 B is reprinted with permission from ref. 65. Copyright: 2015, American Chemical Society.
Figure 4
Figure 4
Washout assay of ncAAs: experimental demonstration that nonspecific background in intracellular labelling is directly linked to the hydrophilicity of the ncAA. A) Confocal microscopy images of non‐transfected COS7 cells washed for different times before labelling with TAMRA‐3. B) Quantitative analysis. Reproduced in part and with permission from ref. 17. Copyright: 2016, John Wiley & Sons, Inc.
Figure 5
Figure 5
Timelapse imaging of the fusion process of IFITM‐containing vesicles with dextran particles. IFITM3‐TCO*‐Lys was labelled with BODIPY‐FL‐3 a, and dextran particles were labelled with pHrodo Red. Reprinted with permission from ref. 66. Copyright: 2016, American Chemical Society.
Scheme 11
Scheme 11
Structures of bio‐orthogonalized monosaccharides for fluorescent glycan labelling through IEDDA cycloaddition enabled by metabolic oligosaccharide engineering.
Figure 6
Figure 6
Double labelling of cell‐surface glycans through IEDDA and SPAAC. A) Labelling strategy, based on metabolic incorporation of Ac4ManCCp and Ac4GlcNAz and subsequent labelling with Cy3‐6 and AF488‐DIBO. B) Confocal microscopy images of doubly labelled cell‐surface glycans on HEK293T cells. Reprinted with permission from ref. 74a. Copyright: 2014, American Chemical Society.
Figure 7
Figure 7
Metabolic incorporation of BCNSia into zebrafish embryos and subsequent labelling with fluorogenic Oregon green tetrazine dye. A) Schematic representation of expected metabolic pathway for the incorporation of BCNSia into cell‐surface glycans and subsequent labelling. CMAS: cytidine monophosphate N‐acetylneuraminic acid. CMP: cytidine monophosphate. B) Schematic representation of labelling protocol. Zebrafish embryos were injected with BCNSia and then with fluorogenic tetrazine in the caudal vein. C) Projection images of 48 hpf (hours post‐fertilisation) embryos treated with BCNSia or vehicle; fluorogenic tetrazine was coinjected with AF647‐NH2 to map the vasculature. Scale bar: 200 μm. Reproduced in part, with permission, from ref. 77. Copyright: 2015, John Wiley & Sons, Inc.

Similar articles

See all similar articles

Cited by 10 articles

See all "Cited by" articles

References

    1. Saxon E., Bertozzi C. R., Science 2000, 287, 2007–2010. - PubMed
    1. None
    1. Sletten E. M., Bertozzi C. R., Angew. Chem. Int. Ed. 2009, 48, 6974–6998; - PMC - PubMed
    2. Angew. Chem. 2009, 121, 7108–7133;
    1. Patterson D. M., Nazarova L. A., Prescher J. A., ACS Chem. Biol. 2014, 9, 592–605; - PubMed
    1. Ramil C. P., Lin Q., Chem. Commun. 2013, 49, 11007–11022. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback