Online citations, reference lists, and bibliographies.
← Back to Search

Photoinducible Bioorthogonal Chemistry: A Spatiotemporally Controllable Tool To Visualize And Perturb Proteins In Live Cells.

Reyna K. V. Lim, Q. Lin
Published 2011 · Chemistry, Medicine

Cite This
Download PDF
Analyze on Scholarcy
Share
Visualization in biology has been greatly facilitated by the use of fluorescent proteins as in-cell probes. The genes coding for these wavelength-tunable proteins can be readily fused with the DNA coding for a protein of interest, which enables direct monitoring of natural proteins in real time inside living cells. Despite their success, however, fluorescent proteins have limitations that have only begun to be addressed in the past decade through the development of bioorthogonal chemistry. In this approach, a very small bioorthogonal tag is embedded within the basic building blocks of the cell, and then a variety of external molecules can be selectively conjugated to these pretagged biomolecules. The result is a veritable palette of biophysical probes for the researcher to choose from. In this Account, we review our progress in developing a photoinducible, bioorthogonal tetrazole-alkene cycloaddition reaction ("photoclick chemistry") and applying it to probe protein dynamics and function in live cells. The work described here summarizes the synthesis, structure, and reactivity studies of tetrazoles, including their optimization for applications in biology. Building on key insights from earlier reports, our initial studies of the reaction have revealed full water compatibility, high photoactivation quantum yield, tunable photoactivation wavelength, and broad substrate scope; an added benefit is the formation of fluorescent cycloadducts. Subsequent studies have shown fast reaction kinetics (up to 11.0 M(-1) s(-1)), with the rate depending on the HOMO energy of the nitrile imine dipole as well as the LUMO energy of the alkene dipolarophile. Moreover, through the use of photocrystallography, we have observed that the photogenerated nitrile imine adopts a bent geometry in the solid state. This observation has led to the synthesis of reactive, macrocyclic tetrazoles that contain a short "bridge" between two flanking phenyl rings. This photoclick chemistry has been used to label proteins rapidly (within ∼1 min) both in vitro and in E. coli . To create an effective interface with biology, we have identified both a metabolically incorporable alkene amino acid, homoallylglycine, and a genetically encodable tetrazole amino acid, p-(2-tetrazole)phenylalanine. We demonstrate the utility of these two moieties, respectively, in spatiotemporally controlled imaging of newly synthesized proteins and in site-specific labeling of proteins. Additionally, we demonstrate the use of the photoclick chemistry to perturb the localization of a fluorescent protein in mammalian cells.
This paper references
10.1002/(SICI)1521-3773(19981217)37:23<3281::AID-ANIE3281>3.0.CO;2-V
Highly Efficient Synthesis of Covalently Cross-Linked Peptide Helices by Ring-Closing Metathesis.
H. Blackwell (1998)
10.1002/anie.200901916
Constructing and exploiting the fluorescent protein paintbox (Nobel Lecture).
R. Tsien (2009)
10.1021/ja9094523
Direct observation of a photoinduced nonstabilized nitrile imine structure in the solid state.
Shao-Liang Zheng (2009)
10.1002/1521-3773(20020802)41:15<2840::AID-ANIE2840>3.0.CO;2-#
The selective incorporation of alkenes into proteins in Escherichia coli.
Z. Zhang (2002)
10.1021/jo800142s
Synthesis and conformational analysis of a cyclic peptide obtained via i to i+4 intramolecular side-chain to side-chain azide-alkyne 1,3-dipolar cycloaddition.
S. Cantel (2008)
10.1002/ANIE.197601231
Intramolecular 1,3‐Dipolar Cycloaddition Reactions
A. Padwa (1976)
10.1073/PNAS.0601637103
Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT).
D. Dieterich (2006)
10.1002/CHIN.201115177
Synthesis of Macrocyclic Tetrazoles for Rapid Photoinduced Bioorthogonal 1,3‐Dipolar Cycloaddition Reactions.
Z. Yu (2011)
10.1016/j.bmcl.2011.01.004
Synthesis of cell-permeable stapled peptide dual inhibitors of the p53-Mdm2/Mdmx interactions via photoinduced cycloaddition.
Michael M. Madden (2011)
10.1016/j.chembiol.2008.10.004
Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification.
Tatsuo Yanagisawa (2008)
10.1002/anie.200900942
Bioorthogonal chemistry: fishing for selectivity in a sea of functionality.
Ellen M. Sletten (2009)
10.1038/nrm1105
Ras proteins: different signals from different locations
J. Hancock (2003)
10.1039/B005567K
Nitrilimine cycloadditions in aqueous media
G. Molteni (2000)
10.1002/POLA.1994.080320907
Photomodification of a poly(acrylonitrile‐co‐butadiene‐co‐styrene) containing diaryltetrazolyl groups
R. Darkow (1994)
1,3-Dipolar cycloadditions. XXVII. Addition of diphenylnitrilimine to nonconjugate alkenes and alkynes. Steric course of reaction, orientation, and effect of substituents
R. Huisgen (1967)
10.1021/ja104350y
A biosynthetic route to photoclick chemistry on proteins.
J. Wang (2010)
10.1021/JO00403A009
Photochemical transformations of small ring heterocyclic compounds. 9. Intramolecular 1,3-dipolar cycloaddition reactions of alkenyl-subituted nitrile imines
A. Padwa (1978)
10.1021/ja803598e
Selective functionalization of a genetically encoded alkene-containing protein via "photoclick chemistry" in bacterial cells.
W. Song (2008)
10.1021/ja9003624
Reactivity and regioselectivity in 1,3-dipolar cycloadditions of azides to strained alkynes and alkenes: a computational study.
F. Schoenebeck (2009)
10.1021/cb100193h
A metabolic alkene reporter for spatiotemporally controlled imaging of newly synthesized proteins in Mammalian cells.
W. Song (2010)
10.1021/JA000563A
An All-Hydrocarbon Cross-Linking System for Enhancing the Helicity and Metabolic Stability of Peptides
C. Schafmeister (2000)
10.1021/JA0734086
Distortion/interaction energy control of 1,3-dipolar cycloaddition reactivity.
D. Ess (2007)
10.1038/nn.2580
In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons
D. Dieterich (2010)
10.1074/JBC.M101476200
A Functional Rhodopsin-Green Fluorescent Protein Fusion Protein Localizes Correctly in Transgenic Xenopus laevis Retinal Rods and Is Expressed in a Time-dependent Pattern*
O. Moritz (2001)
Photochemistry of diarylsubstituted 2H-tetrazole. VI. Quantum yields of the photolysis of diarylsubstituted 2H-tetrazoles
V. Lohse (1988)
10.1021/JA992749J
Efficient incorporation of unsaturated methionine analogues into proteins in vivo
J. V. Hest (2000)
10.1002/CHIN.198547097
Intramolecular 1,3-Dipolar Cycloadditions of Diaryl-nitrile-imines Generated from 2,5-Diaryl-tetrazoles.
H. Meier (1985)
10.1039/b912094g
Facile synthesis of stapled, structurally reinforced peptide helices via a photoinduced intramolecular 1,3-dipolar cycloaddition reaction.
Michael M. Madden (2009)
10.1021/JA00731A056
Photocycloaddition of arylazirenes with electron-deficient olefins
A. Padwa (1971)
10.1039/c003470c
A bioorthogonal chemistry strategy for probing protein lipidation in live cells.
W. Song (2010)
10.1002/CHIN.197642267
A FACILE SYNTHESIS OF 2,5-DISUBSTITUTED TETRAZOLES BY THE REACTION OF PHENYLSULFONYLHYDRAZONES WITH ARENEDIAZONIUM SALTS
S. Ito (1976)
10.1021/OL7017328
Convenient synthesis of highly functionalized pyrazolines via mild, photoactivated 1,3-dipolar cycloaddition.
Yizhong Wang (2007)
10.1002/ANIE.200702305
A biosynthetic route to dehydroalanine-containing proteins.
J. Wang (2007)
10.1046/j.1471-4159.2001.00182.x
Distribution of an NMDA receptor:GFP fusion protein in sensory neurons is altered by a C‐terminal construct
D. Marsh (2001)
10.1021/ja800009z
Theory of 1,3-dipolar cycloadditions: distortion/interaction and frontier molecular orbital models.
D. Ess (2008)
10.1021/BI00176A034
Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers.
J. Silvius (1994)
10.1073/pnas.0707090104
Copper-free click chemistry for dynamic in vivo imaging
Jeremy M Baskin (2007)
10.1039/c0cc02863k
Azirine ligation: fast and selective protein conjugation via photoinduced azirine-alkene cycloaddition.
Reyna K. V. Lim (2010)
10.1021/ol901300h
Synthesis and evaluation of photoreactive tetrazole amino acids.
Yizhong Wang (2009)
10.1016/1010-6030(89)80017-6
Bis-2H-tetrazoles VII: Quantum yields of bis-2H-tetrazoles and studies of thermal consecutive reactions of bis-nitrile imines by time-resolved spectroscopy
P. Weinberg (1989)
10.1002/CBER.19671000108
1.3‐Dipolare Cycloadditionen, XXV. Der Nachweis des freien Diphenylnitrilimins als Zwischenstufe bei Cycloadditionen
J. S. Clovis (1967)
10.1002/anie.200705805
A photoinducible 1,3-dipolar cycloaddition reaction for rapid, selective modification of tetrazole-containing proteins.
W. Song (2008)
10.1002/anie.200905590
Genetically encoded alkenes in yeast.
Hui-wang Ai (2010)
Nitrilimines
N. H. Toubro (1980)
10.1021/JA00803A018
Origin of reactivity, regioselectivity, and periselectivity in 1,3-dipolar cycloadditions
K. Houk (1973)
10.1021/CB6003228
A comparative study of bioorthogonal reactions with azides.
Nicholas J. Agard (2006)
10.1038/nmeth.f.326
The promise of optogenetics in cell biology: interrogating molecular circuits in space and time
Jared E. Toettcher (2011)
10.1002/CBER.19671000525
1.3-Dipolare Cycloadditionen, XXVII. Zur Anlagerung des Diphenylnitrilimins an nichtkonjugierte Alkene und Alkine; Sterischer Ablauf, Orientierung un Substituenteneinfluß
R. Huisgen (1967)
10.1002/HLCA.19850680524
Intramolekulare 1,3‐dipolare Cycloadditionen von Diarylnitriliminen aus 2,5‐Diaryltetrazolen
H. Meier (1985)
10.1002/ANIE.199204391
A Straightforward Synthesis of Nitrilimines; X‐Ray Crystal Structure of a Nitrilimine Stabilized by Non‐Heteroatom Substituents
R. Réau (1992)
10.1021/ol801350r
Discovery of long-wavelength photoactivatable diaryltetrazoles for bioorthogonal 1,3-dipolar cycloaddition reactions.
Yizhong Wang (2008)
10.1002/anie.200901220
Fast alkene functionalization in vivo by Photoclick chemistry: HOMO lifting of nitrile imine dipoles.
Yizhong Wang (2009)
10.1039/b925931g
Bioorthogonal chemistry: recent progress and future directions.
Reyna K. V. Lim (2010)
10.1021/JA028266O
Tuning the photoinduced electron-transfer thermodynamics in 1,3,5-triaryl-2-pyrazoline fluorophores: X-ray structures, photophysical characterization, computational analysis, and in vivo evaluation.
C. J. Fahrni (2003)
10.1002/PRAC.19883300310
Photochemie diarylsubstituierter 2H‐Tetrazole. VI. Quantenausbeuten der Photolyse diarylsubstituierter 2H‐Tetrazole
Volkmar Lohse (1988)
10.1016/j.bmcl.2011.04.087
Discovery of new photoactivatable diaryltetrazoles for photoclick chemistry via 'scaffold hopping'.
Zhipeng Yu (2011)
10.1146/annurev.biochem.052308.105824
Adding new chemistries to the genetic code.
C. Liu (2010)
10.1126/SCIENCE.1105654
An Acylation Cycle Regulates Localization and Activity of Palmitoylated Ras Isoforms
Oliver Rocks (2005)



This paper is referenced by
10.1002/ANGE.201407874
Postsynthetische “Photoklick”‐Modifizierung von DNA
Stefanie Arndt (2014)
10.1021/JACS.6B08521
Photoclickable MicroRNA for the Intracellular Target Identification of MicroRNAs.
Jinbo Li (2016)
10.1039/c2ob07165g
Kinetics studies of rapid strain-promoted [3 + 2]-cycloadditions of nitrones with biaryl-aza-cyclooctynone.
Craig S. McKay (2012)
10.1002/anie.201502954
UV-Induced Tetrazole-Thiol Reaction for Polymer Conjugation and Surface Functionalization.
Wenqian Feng (2015)
10.2174/1385272811317060006
Application of Metal-Free Click Chemistry in Biological Studies
Pāvels Ostrovskis (2013)
10.1039/C4SC00753K
Fluorescent and chemico-fluorescent responsive polymers from dithiomaleimide and dibromomaleimide functional monomers
M. Robin (2014)
10.1107/S2414314616015704
Ammonium hydrogen bis­[4-(2-phenyl-2H-tetra­zol-5-yl)benzoate]
D. Janzen (2016)
10.1002/anie.201502403
Photoelectrocyclization as an activation mechanism for organelle-specific live-cell imaging probes.
Mai N. Tran (2015)
10.1039/c5cc10445a
Photo-induced coupling reactions of tetrazoles with carboxylic acids in aqueous solution: application in protein labelling.
Shan Zhao (2016)
10.1002/ADSC.201201022
Copper‐Catalyzed C‐Arylation and Denitrogenation of Tetrazoles: Domino Synthesis of 1,3‐Diaminoisoquinoline Derivatives
L. Shi (2013)
10.1002/anie.201303477
Expanding the genetic code for photoclick chemistry in E. coli, mammalian cells, and A. thaliana.
Fahui Li (2013)
10.1021/jm500531z
Development and Bioorthogonal Activation of Palladium-Labile Prodrugs of Gemcitabine
Jason T. Weiss (2014)
10.1021/MA501386V
“Click”-Inspired Chemistry in Macromolecular Science: Matching Recent Progress and User Expectations
Pieter Espeel (2015)
10.1002/ejoc.202000549
Design of Hydrazone‐Modified 1,8‐Naphthalimides as Fluorogenic Click Probes Based on Nitrile Imine‐Alkyne Cycloaddition
Xiang Li (2020)
10.1016/bs.mie.2020.04.038
Fluorescent bioorthogonal labeling of class B GPCRs in live cells.
Srikanth Kumar Gangam (2020)
10.1039/c7cc03328a
Photo-clickable microRNA for in situ fluorescence labeling and imaging of microRNA in living cells.
L. Huang (2017)
10.1002/anie.201705006
Ruthenium‐Catalyzed Azide–Thioalkyne Cycloadditions in Aqueous Media: A Mild, Orthogonal, and Biocompatible Chemical Ligation
Paolo Destito (2017)
10.1039/c7np00024c
Covalent modification of biological targets with natural products through Paal-Knorr pyrrole formation.
A. Kornienko (2017)
10.1016/j.cbpa.2014.05.024
Photoclick chemistry: a fluorogenic light-triggered in vivo ligation reaction.
Carlo P Ramil (2014)
10.1038/nchembio.2024
Development and application of bond cleavage reactions in bioorthogonal chemistry.
Jie Li (2016)
10.1002/chem.201301838
Norbornenes in inverse electron-demand Diels-Alder reactions.
M. Vrabel (2013)
10.1021/JACS.6B09706
Co-opting a Bioorthogonal Reaction for Oncometabolite Detection.
Thomas T Zengeya (2016)
10.1039/c4cs00018h
Metalloprotein design using genetic code expansion.
Cheng Hu (2014)
10.1039/c4cc01551g
A highly efficient oxidative condensation reaction for selective protein conjugation.
Ao Ji (2014)
10.1016/bs.mie.2016.06.005
Method for Enzyme Design with Genetically Encoded Unnatural Amino Acids.
C. Hu (2016)
10.1016/j.tet.2020.131063
Regioselective synthesis of carboxylic and fluoromethyl tetrazoles enabled by silver-catalyzed cycloaddition of diazoacetates and aryl diazonium salts
Ming-Yang Xiao (2020)
10.1016/bs.mie.2016.09.022
Combining Click Chemistry-Based Proteomics With Dox-Inducible Gene Expression.
J. Gebert (2017)
10.1039/c8ob01404c
Sterically shielded tetrazoles for a fluorogenic photoclick reaction: tuning cycloaddition rate and product fluorescence.
Peng An (2018)
Development of multiplexable biosensors to quantify the spatiotemporal dynamics of Rho GTPases and protein kinases in the same living cell
Chia-Wen Hsu (2012)
10.1039/C7QO00054E
Cyclopropenes: a new tool for the study of biological systems
João M J M Ravasco (2017)
10.1039/c7ob01548h
Enzyme-instructed self-assembly with photo-responses for the photo-regulation of cancer cells.
Z. Zhou (2017)
10.1002/chem.201904059
Photochemical reactions in the synthesis of protein-drug conjugates.
J. Holland (2019)
See more
Semantic Scholar Logo Some data provided by SemanticScholar