Click Imaging of Biochemical Processes inLiving SystemsValery V. Fokin*Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037

C hemists and biologists have learnedreasonably well to study even themost complex biological molecules

when they are isolated, but how could oneget a molecular-level glimpse of the chemi-cal processes as they unfold in living sys-tems? After all, understanding the functionof specific proteins, glyco- and lipo-conju-gates, and their metabolites requires observ-ing the dynamics of their biosynthesis, distri-bution, and metabolism in their native envi-ronment (i.e., in as close to real-time livingconditions as possible) rather than their be-havior in the isolated (read dead) samples.Clearly, dissecting the intricate chemical net-works of processes that occur in living organ-isms—or at least living cells—is not a trivialtask. Enter bioorthogonal reporters. Theseare small and biologically inert functionalgroups that are incorporated in the targetbiomolecule at the genetic or post-trans-lational level by the cell’s native biochemi-cal pathways. They remain invisible and canbe carried unnoticed through multiple bio-synthetic steps until they are revealed via aselective chemical transformation. Becauseof their stability and very narrow reactivityprofiles, organic azides are perhaps bestsuited to function as small chemical report-ers. In a recent paper, Bertozzi and cowork-ers (1) describe a new reagent, 3,3-difluoro-cyclooctyne (DIFO), for addressing organicazides metabolically incorporated in the cellsurface glycans. DIFO is stable to biologicalnucleophiles and reacts with organic azidesrapidly, irreversibly, and selectively withouta catalyst.

Although methods for introduction ofbiopolymer-based reporter tags (GFP is aprime example) into proteins at the geneticlevel have been developed and used prolifi-cally for cellular imaging and protein track-ing, the repertoire of chemical transforma-tions that allow selective labeling ofproteins, nucleic acids, and glyco- and lipo-conjugates with small-molecule-based tagshas remained quite limited. This is not sur-prising given the aqueous milieu and the di-versity of chemical functionality present inliving organisms. To be useful for studies ofbiological systems at the molecular level,functional groups used for tagging (i) haveto be small and readily incorporated into thetarget of interest without significantly per-turbing its function; (ii) have to remain“invisible” to the cellular biochemical ma-chinery and unreactive with water and ubiq-uitously present endogenous nucleophiles,such as amines and thiols; and (iii) shouldbe addressable in a rapid and selectivefashion so they can be revealed, when re-quired, by using a selective and irreversiblechemical reaction with a visualization labelcontaining a complementary reactive center.Organic azides, because of their uniquelynarrow reactivity profiles, are among themost useful chemical reporters.

Although they have been used in synthe-sis for �100 years, the utility of organicazides has been often limited to the facileintroduction of the amino group into organicmolecules. Other facets of their unique reac-tivity, especially in biological environments,remained largely unexplored until relatively

*Corresponding author,fokin@scripps.edu.

Published online December 21, 2007

10.1021/cb700254v CCC: $37.00

© 2007 American Chemical Society

ABSTRACT Understanding the function ofbiomolecules is crucially dependent on observingthe dynamics of their biosynthesis, distribution,and metabolism in their native environment at thecellular and organismal levels rather than their be-havior in the isolated samples. Small bioorthogo-nal functional groups that cause minimal, if any,perturbations of the native structure and functionof the target molecule under physiological condi-tions and react selectively with an appropriatelyderivatized probe could function as chemicalhandles that allow selective visualization of thetarget molecule in the complex biological milieu.Small and generally unreactive, organic azides areideally suited for this task: they can be carried un-noticed through multiple biosynthetic steps onlyto be revealed, when desired, by action of a suit-ably derivatized visualization label.

Point ofVIEW

www.acschemicalbiology.org VOL.2 NO.12 • ACS CHEMICAL BIOLOGY 775

recent years. In the pioneering workon the development of small-molecule chemical reporters, Ber-tozzi recognized their nearlybioorthogonal properties and theease of introduction into the biologi-cal molecules and elegantly ex-ploited their reaction with phos-phines (the modified Staudingerligation, Figure 1, panel a) in meta-bolic oligosaccharide engineeringstudies (2, 3). Around the same time,Sharpless and colleagues (4) de-fined “click chemistry” as a set of“near-perfect” bond-forming reac-tions useful for rapid assembly ofmolecules with desired function.These transformations are easy toperform, give rise to their intendedproducts in very high yields with littleor no byproducts, work well undermany conditions (usually especiallywell in water), and are unaffected bythe nature of the groups being connectedto each other. The “click” moniker is meantto signify that with the use of these meth-ods, joining molecular fragments is as easyas “clicking” together the two pieces of abuckle. The buckle works no matter what isattached to it, as long as its two pieces canreach each other, and the components ofthe buckle can make a connection only witheach other. The potential of organic azidesas highly energetic yet very selective func-tional groups in organic synthesis was high-lighted, and their dipolar cycloaddition withalkynes was placed among the reactions ful-filling the “click” criteria.

The fundamental thermal reaction, involv-ing terminal or internal alkynes (Figure 1,panel b, 1,3-dipolar azide-alkyne cycloaddi-tion, AAC), has been known for more than acentury (the first 1,2,3-triazole was synthe-sized by Michael from phenyl azide and di-ethyl acetylene-dicarboxylate in 1893) (5)and has been most thoroughly investigatedby Huisgen and others in the 1950s–1970sin the course of their studies of the family

of 1,3-dipolar cycloaddition reactions (6, 7).The process is strongly thermodynamicallyfavored (�Ho � �45to �55 kcal/mol) be-cause of the high-energy nature of the tworeaction components, but it has a relativelyhigh kinetic barrier (�26 kcal/mol formethyl azide and propyne (8)), thus render-ing the reaction very slow at RT for unacti-vated reactants. Copper catalysts (Figure 1,panel c) accelerate the reaction of azideswith terminal alkynes (9, 10) by �107 rela-tive to the uncatalyzed version (8), making itconveniently fast at and even below RT.The reaction is unaffected by water and bymost organic and inorganic functionalgroups, therefore all but eliminating theneed for protecting group chemistry (11).The 1,2,3-triazole heterocycle has the ad-vantageous properties of high chemical sta-bility (in general, being inert to severe hydro-lytic, oxidizing, and reducing conditions,even at high temperature), strong dipolemoment (4.8–5.6 D), aromatic character,and hydrogen bond accepting ability (12,13). Thus, it can interact productively in sev-

eral ways with biological molecules andcan serve as a hydrolytically stable replace-ment for the amide linkage. Compatibility ofthe copper-catalyzed azide-alkyne cycload-dition (CuAAC) reaction with a broad rangeof functional groups and reaction conditions(14–17) has made it broadly useful acrossthe chemical disciplines, and its applica-tions include the synthesis of biologicallyactive compounds, the preparation of conju-gates to proteins and polynucleotides, thesynthesis of dyes, the elaboration of knownpolymers and the synthesis of new ones, thecreation of responsive materials, and the co-valent attachment of desired structures tosurfaces (18–22). CuAAC has become themost widely used click reaction discoveredso far (unsurprisingly, it is often called “clickchemistry”, whereas it is in fact only a repre-sentative of the “click” family of reactions).

Although CuAAC has been used in arange of bioconjugation applications formodification of virus particles (14) andwhole Escherichia coli cells (15, 16, 23)and labeling of proteins from murine tissue

N3P

O

OCH3

PhN

H2O

P O

O

NH

Ph PhPhP

O

OCH3

Ph Ph

+

+ NNN

NN

N

The Staudinger ligation

Copper catalyzed azide-alkyne cycloaddition

Strain-promoted azide-alkyne cycloaddition

[Cu]

R1 N3+ R2 R3

>100 °C

hours−daysN N

NN N

N+

Reactions are faster when R2,R3 are electron-withdrawing groups

1,3 Dipolar cycloaddition of azides and alkynes

−CH3OH

a

+ −

b

R1

R2

R3 R1 R2

R3

c

N3

d

+ N3

H2O

Figure 1. Chemical transformations used to address azides in biological systems. a) ModifiedStaudinger ligation takes advantage of the selective reactivity of phosphines with azides. The reactionhas been used prolifically in labeling of glycans and proteins, both in vitro and in vivo. However, it re-quires a large excess of phosphine to attain useful reaction rates, and phosphine reagents are prone tooxidation. b�d) 1,3-Dipolar cycloaddition of azides and alkynes. Although exquisitely selective, thethermal reaction (b) is very slow and cannot be used in bioconjugations. c) CuAAC is very selective andefficient, but it requires addition of a copper catalyst, which may not be compatible with live cells. d)Strain-promoted cycloaddition does not require a catalyst, but its rate is not sufficient for tracking fastbiological processes.

776 VOL.2 NO.12 • 775–778 • 2007 www.acschemicalbiology.orgFOKIN

lysates (17, 24), it has not been employeddirectly in living cells because of the cytotox-icity and attendant bioregulation of copper.In 2004, Bertozzi and coworkers demon-strated the utility of cyclooctyne-containingprobes (Figure 1, panel d) in catalyst-freebioconjugations (25). Cyclooctyne is suffi-ciently stable but is known to react withazides rapidly because of the ring strain(26). However, the rate of the reactions of cy-clooctyne probes with azides is similar tothat of the Staudinger ligation and is lowerthan the rate of CuAAC (27) to make it use-ful for studies of fast processes. Now, thesame researchers boosted the reactivityof the cyclooctyne by incorporating twostrongly electron-withdrawing fluorine at-oms adjacent to the triple bond. As reportedin the Bertozzi paper (1), the reactions ofDIFO probes with aliphatic azides were 17–63-fold faster than the correspondingStaudinger ligations, allowing efficient label-ing of the azide-containing proteins in�1 h ([DIFO] � 25 �M).

The authors then turned to labeling oflive mammalian cells that contained azido-sialic acid residues in their cell surface gly-cans (Figure 2). The cell’s own biosyntheticpathways are used to display azidosialo-sides at the cell surface using the now well-established metabolic engineering proce-dure, whereupon cells are fed a suitablylabeled azidosugar precursor, such as per-acetylated N-azidoacetyl mannosamine(28). Short of the synthesis of the DIFO la-bel itself, the subsequent labeling protocoldeveloped by Bertozzi and her team is ex-perimentally simple and involves incubationof azide-labeled cells with 25–100 �M ofthe biotinylated DIFO label for 1 h. Flow cy-tometry analysis indicated excellent labelingefficiency, superior to both Staudinger liga-tion with phosphine probes and nonfluori-nated cyclooctyne labels. Furthermore, theDIFO-based labels also performed well inthe time-lapse imaging studies of glycanstrafficking inside the cells, easily the mostdemanding test of this new labeling meth-

odology. To be useful for probing fast bio-logical processes, such as trafficking ofglycans between cell membrane and intra-cellular compartments, the labeling reaction

has to be exceptionally selective, biochemi-cally benign, and fast. DIFO-based reagentsappear to meet these criteria, acting asstealthy, yet exquisitely reactive probes ca-

F

F

O

ONH

Dye

= Alexa Fluor 488, Alexa Fluor 568 or biotin

OAcO

AcO

HN

OAc

AcO

O

N3

O

CO2−

OHO

OHOH

HNHO

O

Sialic acidderivatives

N3

O OHO

OHOH

HNHO

ONN

N

FF

OO

NH

Dye

CO2−

Dye

Figure 2. DIFO probes incorporate two activating features in the alkyne component, ringstrain and electron-withdrawing fluorine substituents, making them much more reactivethan their nonfluorinated parent cyclooctyne and allowing fast labeling of cell surface gly-cans containing azide groups.

www.acschemicalbiology.org VOL.2 NO.12 • 775–778 • 2007 777

Point ofVIEW

pable of tracking the fate of azide-containing conjugates. After incubation ofthe cells with Alexa Fluor 488-DIFO conju-gate for only 1 min, sufficient incorporationof the label occurred and surface glycanscould be visualized by fluorescence micros-copy. Images taken at 15 min intervals re-vealed the subtleties of the glycan transportfrom the cell membrane to the intracellularcompartments, showing that internalizationof these particular cell-surface messengerswas complete in �30 min.

The new bioconjugation protocol de-vised by Bertozzi and coworkers is an im-pressive addition to the still small family ofchemical transformations that can be usedto selectively address the azide functionalityin biological systems. DIFO-based labelingis noticeably more efficient than the widelyused Staudinger ligation and is devoid ofthe problems associated with phosphine la-bels. It also does not require a copper cata-lyst, which may limit the utility of the CuAACwhen following the dynamics of biomole-cules in the living cells and whole organ-isms by a covalent attachment of a track-able label is the goal. Clearly, it is not limitedto studies of glycans and should find imme-diate use in bioconjugation experiments in-volving proteins, nucleic acids, lipoconju-gates, and their metabolites.

REFERENCES1. Baskin, J. M., Prescher, J. A., Laughlin, S. T., Agard,

N. J., Chang, P. V., Miller, I. A., Lo, A., Codelli, J. A.,and Bertozzi, C. R. (2007) Copper-free click chemis-try for dynamic in vivo imaging, Proc. Natl. Acad.Sci. U.S.A. 104, 16793–16797.

2. Saxon, E., and Bertozzi, C. R. (2000) Cell surface en-gineering by a modified Staudinger reaction, Sci-ence 287, 2007–2010.

3. Hang, H. C., Yu, C., Kato, D. L., and Bertozzi, C. R.(2003) A metabolic labeling approach toward pro-teomic analysis of mucin-type O-linked glycosyla-tion, Proc. Natl. Acad. Sci. U.S.A. 100, 14846–14851.

4. Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001)Click chemistry: diverse chemical function from afew good reactions, Angew. Chem. Int. Ed. 40,2004–2021.

5. Michael, A. (1893) J. Prakt. Chem. 48, 94.6. Huisgen, R. (1989) Kinetics and reaction mecha-

nisms: selected examples from the experience offorty years, Pure Appl. Chem. 61, 613–628.

7. Huisgen, R. (1984) 1,3-Dipolar cycloaddition—intro-duction, survey, mechanism, in 1,3-Dipolar Cycload-dition Chemistry (Padwa, A., Ed.), pp 1–176, Wiley,New York.

8. Himo, F., Lovell, T., Hilgraf, R., Rostovtsev, V. V.,Noodleman, L., Sharpless, K. B., and Fokin, V. V.(2005) Copper(I)-catalyzed synthesis of azoles. DFTstudy predicts unprecedented reactivity and inter-mediates, J. Am. Chem. Soc. 127, 210–216.

9. Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Shar-pless, K. B. (2002) A stepwise Huisgen cycloaddi-tion process: copper(I)-catalyzed regioselective “li-gation” of azides and terminal alkynes, Angew.Chem., Int. Ed. 41, 2596–2599.

10. Tornøe, C. W., Christensen, C., and Meldal, M.(2002) Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides,J. Org. Chem. 67, 3057–3062.

11. Wu, P., and Fokin, V. V. (2007) Catalytic azide-alkyne cycloaddition: reactivity and applications,Aldrichimica Acta 40, 7–17.

12. Tome, A. C. (2004) Product class 13: 1,2,3-triazoles,in Science of Synthesis (Storr, R. C. , and Gilchrist,T. L. , Eds.), pp 415–601, Thieme, Stuttgart and NewYork.

13. Krivopalov, V. P., and Shkurko, O. P. (2005) 1,2,3-Triazole and its derivatives. Development of meth-ods for the formation of the triazole ring, Russ. Chem.Rev. 74, 339–379.

14. Wang, Q., Chan, T. R., Hilgraf, R., Fokin, V. V., Sharp-less, K. B., and Finn, M. G. (2003) Bioconjugation bycopper(I)-catalyzed azide-alkyne [3�2] cycloaddi-tion, J. Am. Chem. Soc. 125, 3192–3193.

15. Link, A. J., and Tirrell, D. A. (2003) Cell surface label-ing of Escherichia coli via copper(I)-catalyzed [3�2]cycloaddition, J. Am. Chem. Soc. 125, 11164–11165.

16. Link, A. J., Vink, M. K. S., and Tirrell, D. A. (2004) Pre-sentation and detection of azide functionality in bac-terial cell surface proteins, J. Am. Chem. Soc. 126,10598–10602.

17. Speers, A. E., Adam, G. C., and Cravatt, B. F. (2003)Activity-based protein profiling in vivo using acopper(I)-catalyzed azide-alkyne [3�2] cycloaddi-tion, J. Am. Chem. Soc. 125, 4686–4687.

18. Bock, V. D., Hiemstra, H., and van Maarseveen, J. H.(2006) Cu(I)-catalyzed alkyne-azide click cycloaddi-tions from a mechanistic and synthetic perspec-tive, Eur. J. Org. Chem. 51–68.

19. Goodall, G. W., and Hayes, W. (2006) Advances incycloaddition polymerizations, Chem. Soc. Rev. 35,280–312.

20. Graham, D., and Enright, A. (2006) Cycloadditionsas a method for oligonucleotide conjugation, Curr.Org. Synth. 3, 9–17.

21. Kolb, H. C., and Sharpless, K. B. (2003) The growingimpact of click chemistry on drug discovery, DrugDiscovery Today 8, 1128–1137.

22. Wang, Q., Chittaboina, S., and Barnhill, H. N. (2005)Advances in 1,3-dipolar cycloaddition reaction ofazides and alkynes—a prototype of “click” chemis-try, Lett. Org. Chem. 2, 293–301.

23. Beatty, K. E., Xie, F., Wang, Q., and Tirrell, D. A.(2005) Selective dye-labeling of newly synthesizedproteins in bacterial cells, J. Am. Chem. Soc. 127,14150–14151.

24. Speers, A. E., and Cravatt, B. F. (2004) Chemicalstrategies for activity-based proteomics, ChemBio-Chem 5, 41–47.

25. Agard, N. J., Prescher, J. A., and Bertozzi, C. R. (2004)A strain-promoted [3�2] azide-alkyne cycloadditionfor covalent modification of biomolecules in livingsystems, J. Am. Chem. Soc. 126, 15046–15047.

26. Wittig, G., and Krebs, A. (1961) Chem. Ber. 94,3260–3275.

27. Agard, N. J., Baskin, J. M., Prescher, J. A., Lo, A., andBertozzi, C. R. (2006) A comparative study ofbioorthogonal reactions with azides, ACS Chem.Biol. 1, 644–648.

28. Saxon, E., Luchansky, S. J., Hang, H. C., Yu, C., Lee,S. C., and Bertozzi, C. R. (2002) Investigating cellu-lar metabolism of synthetic azidosugars with theStaudinger ligation, J. Am. Chem. Soc. 124,14893–14902.

778 VOL.2 NO.12 • 775–778 • 2007 www.acschemicalbiology.orgFOKIN