Modern Synthetic Avenues for the Preparation of Functional Fluorophores

Abstract Biomedical research relies on the fast and accurate profiling of specific biomolecules and cells in a non‐invasive manner. Functional fluorophores are powerful tools for such studies. As these sophisticated structures are often difficult to access through conventional synthetic strategies, new chemical processes have been developed in the past few years. In this Minireview, we describe the most recent advances in the design, preparation, and fine‐tuning of fluorophores by means of multicomponent reactions, C−H activation processes, cycloadditions, and biomolecule‐based chemical transformations.


Introduction
Thesearch for functional molecules is apivotal process in many areas of chemistry where structures with well-defined reactivity and selectivity profiles are needed. [1] In this context, functional fluorophores are useful tools to interrogate biological processes by targeting very diverse analytes (from small ions to large macromolecules) in complex environments and under physiological conditions.A lthough standard synthetic approaches with classical reactions have been successful in many cases,t he preparation of sophisticated fluorophores cannot always be achieved through well-established reactions. [2] Herein, we review the modern strategies that have been developed during the past five years to synthesize functional fluorophores by multicomponent reactions (MCRs), metal-catalyzed CÀHa ctivation, cycloadditions, and biomolecule-based methods.T hese strategies have not only accelerated the preparation of unique fluorescent compounds but also enabled the exploration of chemotypes that are inaccessible through conventional approaches (Figure 1).

Multicomponent Reactions
MCRs constitute afavorable approach for the synthesis of complex molecules given their convergent character,modular features,a nd access to novel chemotypes. [3] Müller and Levi recently reviewed the preparation of chromogenic structures by MCRs, [4] and distinguished between two main strategies, Biomedical researchr elies on the fast and accurate profiling of specific biomolecules and cells in an on-invasive manner.F unctional fluorophores are powerful tools for such studies.Asthese sophisticated structures are often difficult to access through conventional synthetic strategies,new chemical processes have been developed in the past few years.Inthis Minireview,wedescribe the most recent advances in the design, preparation, and fine-tuning of fluorophores by means of multicomponent reactions,C À Ha ctivation processes,c ycloadditions, and biomolecule-based chemical transformations. namely 1) the "scaffold approach", where one of the reactants contains ac hromophore,a nd 2) the "chromophore approach", where the MCR generates ac hromophore from non-fluorescent materials.
Several examples of the chromophore approach have been reported. Theg roup of Pischel and Gois developed at hree-component sequential condensation reaction of ab oronic acid, as alicylaldehyde,a nd an amino substrate to obtain fluorescent boron-containing heterocycles with polarity-dependent fluorescence emission (Figure 2A). [5] These structures were suitable for the imaging of dendritic cells as well as cancer cells.A sarepresentative example of fluorophores obtained from more conventional condensation reactions,P alumbo Piccionello and co-workers described at hree-component reaction  of an imidazole-substituted dicarbonyl compound with aldehydes and an ammonia source to form 4,4'-bis(imidazole)s as selective fluorescent probes for heavy metals ( Figure 2B). [6] Cheng and co-workers exploited N-heterocyclic carbene chemistry to prepare fluorophores in MCRs ( Figure 2C). [7] Ther eaction of imidazopyridinium salts as carbene precursors,p hthalaldehydes,a nd dimethyl acetylenedicarboxylate( DMAD) afforded benzofuroazepines with emission wavelengths in the visible range (ca. 500 nm) and high fluorescence quantum yields (up to 81 %).
Metal-catalyzed MCRs represent al arge portion of the reactions applied in the chromophore approach. [8] In this respect, ab road range of fluorophores can be accessed through sequential Müller-type processes,w hich involve ap alladium-catalyzed Sonogashira coupling in combination with cascade cyclizations,c ycloadditions,a nd/or additional condensation reactions.L ibraries of structurally diverse merocyanines,i midazoles,i ndolones,f urans,o xazoles,t hiophenes,quinolones,and other heterocyclic systems have been prepared, providing an excellent chemical platform to finetune the photophysical and biological properties of fluorescent compounds.
Isocyanide-based MCRs are the most exploited family of multicomponent condensations.T hese reactions rely on the versatility of isocyanide species,w hich can engage with multiple counterparts.A ni nteresting example towards highthroughput chemistry was reported by Balakirev and coworkers,w ho made use of the Groebke-Blackburn-Bien-aymØ reaction between heterocyclic amidines,aldehydes,and isocyanides to generate al ibrary of 1600 compounds in droplet arrays ( Figure 2D). [9] Subsequent analysis identified fluorophores with emission wavelengths ranging from 485 nm to 627 nm, and selected fluorophores were used to image the mitochondrial benzodiazepine receptor TSPO in PC3 prostate cancer cells,a mong others.
TheU gi 4-CR, which combines isocyanides with aldehydes,a mines,a nd carboxylic acids,i saparticularly suitable method for the synthesis of fluorescent structures through the scaffold approach. Neto,d aSilva, and co-workers used ac oumarin carboxylic acid to prepare ac ollection of fluorescent adducts with high affinity for mitochondria (Figure 2E). [10] Likewise,W estermann and co-workers described asophisticated family of fluorescent tags for protein profiling from an Ugi 4-CR with rhodamine Ba st he carboxylic acid fluorophore ( Figure 2F). [11] TheU gi approach has also been successfully applied to polymeric structures.M ultifunctional fluorescent polymers for avidin and bovine serum albumin conjugation were prepared through an Ugi MCR in combination with ac ontrolled reversible addition-fragmentation chain-transfer polymerization ( Figure 2G). [12] An example of the applicability of Ugi 4-CRs to functionalize fluorophores for optical imaging was reported by our group. [13] We described the preparation of af luorescent isocyanide-BOD-IPY core and its derivatization using different MCRs. Subsequent biological analysis identified PhagoGreen as ap H-sensitive fluorophore for imaging phagocytic macrophages in vivo ( Figure 2H). An extension of this approach has been reported by PeÇa-Cabrera and co-workers with the derivatization of aldehyde-functionalized BODIPYs in aPasserini MCR. [14] With regard to the chromophore approach, Riva, Müller, and co-workers recently described an Ugi MCR in which the initial adducts were converted into furo [2,3c]isoquinolines in ap alladium-catalyzed insertion/alkynylation/cycloisomerization cascade. [15] Ther esulting isoquinolines displayed strong fluorescence,w ith emission maxima ranging from 396 nm to 443 nm and tunable quantum yields depending on the substituents ( Figure 2I).
Occasionally,unusual reactivity patterns in reactions with isocyanides can lead to unprecedented structures.A ni nter-esting example is the preparation of blue-fluorescent mesoionic acid fluorides from isocyanides,a zines,a nd fluorinated anhydrides. [16] These mesoionic acid fluorides were found to be remarkably stable to hydrolysis,a nd were employed for imaging histamine in live cells [17] as well as for labeling oligonucleotides [18] (Figure 3Aand B, respectively). Recently, this approach has been extended to isoquinoline-substituted BODIPY structures.T he resulting mesoionic BODIPY compounds were used for the activation-free labeling of bioactive amines,a nd af luorescent analogue of the antimycotic agent natamycin was developed for imaging fungal cells (Figure 3C). [19] Finally,Liand co-workers described adipolar isocyanidebased MCR to produce complex pyrrolophenanthrolines under solvent-free conditions in excellent yields from isocyanides,a ldehydes,m alononitriles,a nd phenanthrolines. [20] Thea dducts showed as elective increase in fluorescence emission upon incubation with Cu 2+ ,showing potential for the detection of metal ions in biological assays.

Metal-Catalyzed C À HA ctivation Reactions
Metal-catalyzed couplings,s uch as Suzuki-Miyaura reactions,a re the most common approach to prepare biaryl compounds.H owever,t he need for two functionalized sub- strates,s uch as ab oronic acid and an aryl halide,o ften represents al imitation owing to the restricted availability of substituted boronic acid derivatives.T hese limitations can be overcome with C À Hactivation processes that directly connect aryl halides to (hetero)arenes by metal-promoted activation of aC ÀHb ond in the latter compound. [21] In this context, we have recently described the straightforward synthesis of af luorogenic tryptophan (Trp) based amino acid as ak ey building block for the preparation of peptide-based fluorophores. [22] Thea mino acid was prepared in as ingle step and in good yields by coupling metaiodophenyl-substituted BODIPY and Fmoc-Trp-OH in the presence of Pd(OAc) 2 under microwave irradiation (Figure 4, top). Afterwards,the Tr p-BODIPY amino acid was incorporated into antimicrobial peptides to label the fungal pathogen Aspergillus fumigatus in ex vivo human tissue (Figure 4, bottom). Notably,t he peptide labeling did not compromise their activity and selectivity,creating numerous opportunities for the development of novel peptide-based imaging probes.
Ackermann and co-workers have developed amethod for the arylation of short peptides that is based on the use of hypervalent iodoaromatic species in palladium-catalyzed C À Ha ctivation processes. [23] Hansen and co-workers described the gold-catalyzed chemoselective ethynylation of Tr p-containing peptides and proteins [24] for subsequent fluorophore conjugation by click chemistry.
CÀHa ctivation has also been successfully employed for the functionalization of other heterocycles,a se xemplified in the recent work of Delcamp,H ammer,a nd co-workers. [25] Fluorescent thienopyrazine-based donor-acceptor-donor compounds were prepared by double CÀHa rylation of the thiophene moiety.T he resulting adducts displayed large Stokes shifts with emission in the near-infrared (NIR) region ( Figure 5A). Another modular CÀHa ctivation strategy was described for the preparation of highly substituted pyrazoles.  [17] (A) and the AmericanC hemical Society (C). [19] . Reproducedw ith permission from Springer Nature. [22] Four sequential palladium-catalyzed direct arylations enable the synthesis of fluorescent tetraaryl pyrazoles with emission maxima between 389 nm and 439 nm ( Figure 5B). [26] CÀH activation approaches have also been used to fine-tune the photophysical properties of functional fluorophores.Park and co-workers reported the preparation of ac ollection of pyrroloindolizinones (Seoul-Fluors,F igure 5C); the fluorescence quantum yields were systematically explored by derivatizing the central scaffold with different aryl groups in palladium-catalyzed couplings. [27] Furthermore,t hese results were exploited to develop new fluorophores for imaging reactive oxygen species in human cancer cells. [27] Aside from accelerating the diversification and optimization of fluorophores,C À Hactivation can be also employed to generate complex fluorescent structures from very simple precursors.Glorius and co-workers achieved the synthesis of polycyclic frameworks in as ingle step through ar hodium-(III)-catalyzed coupling between an aryl pyridine and ap yr-  Figure 5D). [28] Notably, the pyridyl moiety plays ad ual role;d uring the synthesis,i t stabilizes the intermediates and coordinates the catalyst while it later functions as af luorescent reporter by enabling the detection of metal ions.S pectroscopic analysis of these structures revealed ablue shift in the absorption and emission maxima in the presence of Cu 2+ or Zn 2+ ions,m aking them potentially useful for the detection of metal ions.
Another interesting strategy to construct fluorophores by means of C À Ha ctivation is to incorporate such activation processes into domino pathways,w here they take place alongside other bond-forming reactions.P erumal and Nandakumar recently reported at wo-step,o ne-pot palladiumcatalyzed carbopalladation/CÀHa ctivation method for the generation of xanthene derivatives featuring atetrasubstituted olefin in high yields ( Figure 5E). [29] These fluorophores are non-emissive in organic solvents,b ut exhibit pronounced green to yellow fluorescence with large Stokes shifts in water and thus constitute an example of aggregation-induced emission. Tietze and co-workers prepared as ophisticated fluorescent polyheteroaromatic scaffold through apalladiumcatalyzed cascade process encompassing one Sonogashira coupling followed by double carbometalation of triple bonds and afinal C À Harylation ( Figure 5F). [30] On the other hand, Dehaen and co-workers have extensively derivatized the fluorescent BODIPY core by CÀHa ctivation reactionsmaking use of either radical chemistry or palladium catalysis-to produce collections of BODIPY fluorophores (Figure 5G). [31] Thea pplicability of C À Ha ctivation can be extended by including cross-dehydrogenative couplings (CDCs), oxidative transformations linking two substrates through double CÀH activation processes that do not require any functionalized precursors. [32] These approaches are limited by the ubiquity of C À Hbonds but the recent discovery of new selectivity rules is enabling the rapid expansion of the field. Forinstance,CDCs have been used by Youa nd co-workers to tune donoracceptor dyads by linking electron-rich (e.g., furans,t hiophenes) and electron-poor heterocycles (e.g.,i ndazoles) in regioselective oxidative couplings ( Figure 5H). [33] Theresulting bis(heteroaryl) dyes (Indazofluors) display full-colortunable emission (393-725 nm), high fluorescence quantum yields (up to 93 %inCH 2 Cl 2 ,relative to rhodamine B), [33] and could find applications as subcellular organelle markers.
Alkyne annulations are another type of CDCs in which carbon-carbon triple bonds and two atoms of as uitable partner react to form carbo-or heterocycles.F or instance, Cheng and co-workers described rhodium-catalyzed annulations of 2-aryl pyridines and alkynes under O 2 atmosphere to prepare fluorescent pyridinium salts with potential applications in organic electronic devices. [34] Similarly,W ang and coworkers prepared polycyclic quinolinium cations in double CÀHactivation/annulation processes ( Figure 5I). [35] Hua and Zheng reported oxidative coupling reactions with [(RhCl 2 Cp*) 2 ]( Cp* = pentamethylcyclopentadienyl) as the catalyst to obtain complex heterofused phenanthroimidazoles in very good yields ( Figure 5J). [36] In this example,t he synthetic protocol was extended to CÀH/NÀHa ctivation of the heterocyclicinput, and led to new fluorescent ratiometric probes for Fe 3+ ions.F inally,p alladium-mediated CÀH/NÀH activation methods have been used by Kundu and co-workers for the synthesis of pyrido[1,2-a]indoles with high fluorescence quantum yields,t unable emission (478-588 nm), and properties suitable for cell imaging. [37]

Cycloaddition Reactions in Fluorescent Probe Development
Cycloaddition reactions are av aluable strategy to access highly functionalized structures owing to their experimental ease,g ood synthetic yields,a nd compatibility with multiple functional groups, [38] with the alkyne-azide 1,3-dipolar Huisgen cycloaddition [39] being one of the most widely used reactions in chemical biology.T his strategy was utilized by Fairfull-Smith and co-workers to conjugate azidocoumarin derivatives to an alkyne-containing isoindoline nitroxyl in ac opper-catalyzed azide-alkyne cycloaddition (CuAAC) process to generate fluorophores with high sensitivity to oxidative processes ( Figure 6A). [40] Another example is the preparation of highly decorated squaraine rotaxane dendrimers by Smith and co-workers. [41] In this case,asquaraine fluorescent core was encapsulated within am acrocycle containing four alkyne groups that were clicked to azido amines to achieve bright deep-red fluorophores with high photostability.T ogether with the Chang group,o ur group has also adapted CuAACr eactions for the synthesis of diversityoriented fluorescence libraries [42] to optimize the spectral properties of functional fluorophores [43] and their binding capabilities to specific biological targets. [44] Cycloaddition reactions employing heterocyclic or carbocyclic partners have also been described within the realm of fluorophore synthesis.T he use of didehydro-Diels-Alder reactions for the preparation of environmentally sensitive probes ( Figure 6B)w as recently summarized by Brummond and Kocsis. [45] Such processes can afford dihydronaphthalene and naphthalene fluorophores resembling the solvatochromic dye Prodan. Ishii and co-workers reported the synthesis of 1,4-diaryl-1-thio-1,3-butadienes with p-donor and p-acceptor groups by intramolecular [4+ +2] cycloadditions of 1-thioenynes linked to an anthracene ring. [46] These polycyclic frameworks fluoresced in the red and NIR regions and exhibited marked solvatochromism.
Thet etrazole-alkene photoclick reaction is an unusual [3+ +2] cycloaddition, taking place upon photoirradiation and producing pyrazolines via at ransient nitrile imine.T he preparation of profluorescent nitroxides by UV irradiation of isoindoline oxide tetrazoles and maleimides was recently reported by Barner-Kowollik, Blinco,a nd co-workers. [47] In these molecules,astable free radical moiety was covalently tethered to af luorophore so that the nitroxide radical quenched the fluorescence,a nd emission was only detected in the presence of radicals.A nother example was published by Lin and co-workers with the reaction between tetrazoles containing extended p-systems and dimethyl fumarate upon irradiation at 405 nm ( Figure 6C). [48] Theresulting pyrazoline cycloadducts displayed significant bathochromic shifts in organic solvents when compared to aqueous media, suggest-

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Minireviews ing their potential application as environmentally sensitive fluorophores.
Thei nvolvement of several reaction centers has been exploited to expand the versatility and complexity of fluorescent structures.F or instance,r hodium-catalyzed [2+ +2+ +2] cycloadditions of biaryl-linked diynes with alkynes,n itriles, and isocyanates can afford triphenylenes and azatriphenylenes with broad emission ranges (359-498 nm) and high quantum yields (up to 88 %) in as ingle operation. [49] Cycloadditions can be also combined with other reactions,s uch as oxidations,i no ne-pot transformations.A ni ntramolecular [2+ +2+ +2] strategy entailing the cyclization of bis(propargylphenyl)carbodiimides under rhodium catalysis followed by oxidative aromatization was reported by Saito and co-workers. [50] L-shaped penta-, hexa-, and heptacycles with apyrrolo-[1,2-a] [1,8]naphthyridine unit were prepared in ao ne-pot process as fluorophores with blue to orange emission. Alkynes have also been reacted with pyridoisoindoles and pyrrolopyridines to generate indolizines by intermolecular thermal cycloaddition and DDQ-promoted oxidation (DDQ = 2,3-dichloro-5,6-dicyano-1,4-benzoquinone). [51] Domino reactions are intrinsically useful as they allow to explore broad structural diversity in ahighly efficient manner. These processes can be used for the simultaneous formation of several bonds and, together with cycloaddition reactions, have enabled the assembly of synthetically challenging fluorophores.D iederich and co-workers reported the synthesis of complex tetracenes from cumulenes through a[2+ +2] cycloaddition of tetracyanoethylene to the central double bond of the cumulene structure,f ollowed by electrocyclization, dehydrogenation, and final copper-promoted thermal oxidation. [52] Notably,t he resulting structures showed fluorescence changes after binding to metal ions,such as Cu + and Ag + .A nother cascade-based approach was recently devel-oped by Wender and co-workers,w ith ad omino sequence rendering polycycliccompounds through [4+ +2] cycloaddition and elimination followed by as econd [4+ +2] cycloaddition. [53] This approach yielded solvatochromic tetracyclic dyes after two additional functionalization steps.
Cycloaddition reactions have been also adapted to bioorthogonal chemistry to prepare fluorogenic structures that undergo afluorescence enhancement upon reaction with their counterparts.B ertozzi and co-workers described the synthesis of "Calfluor" fluorogens with emission maxima covering the entire visible spectrum ( Figure 7A). [54] Conveniently,C alfluors are internally quenched by azide groups so that their fluorescence emission is turned on after ac lick reaction with suitable alkynes.T his feature enables their application for imaging under no-wash conditions in cells, tissues,a nd zebrafish. Strained cyclic alkynes have been developed to avoid the need for copper in cycloaddition reactions in biological systems.B oons and co-workers investigated cycloadditions of the dibenzocyclooctyne derivative FI-DIBO with different partners (e.g.,azides,nitrones,nitrile oxides,d iazo derivatives) under catalyst-free conditions,a nd obtained 1H-pyrazole fluorophores with 160-fold fluorescence enhancement over FI-DIBO. [55] Alternatively,t etrazines can be coupled to strained cyclic olefins,s uch as norbornenes or trans-cyclooctenes.W eissleder and co-workers reported the synthesis of non-fluorescent tetrazine-BODIPY dyes showing 1600-fold fluorescence enhancement after their reaction with trans-cyclooctenol ( Figure 7B). [56] Thea uthors validated the biological application of these fluorophores by visualizing intracellular and extracellular targets in both fixed and live cells.Finally,the groups of Houk and Murphy described the coupling of the mesoionic heterocycle sydnone to fluorophores amenable to cycloadditions with strained cyclooctynes. [57] In addition to displaying good Reproducedwith permission from the AmericanC hemical Society. [48] Angewandte Chemie Minireviews 3764 www.angewandte.org reactivity under physiological and catalyst-free conditions, these couplings proved to be orthogonal to the reactions between tetrazines and norbornenes ( Figure 7C).

Biomolecule-Based Chemical Transformations
Theneed for sophisticated probes in chemical biology has prompted the adaptation of new chemical strategies to interrogate biological systems under physiological conditions. In this Section, we review some recent examples of biologyoriented modern chemical transformations of functional fluorophores and their application at the interphase between chemistry and biology.

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Chemie localization in intracellular tubules upon reaction with activatable tetrazine-linked fluorophores. [59] Notably,the high rate of these reactions (ca. 1000 m À1 s À1 at 37 8 8C) renders them an optimal approach for identifying binding targets of tagged drugs in live cells.M ore recently,D eRose and co-workers modified the chemotherapeutic drug picoplatin with an azide group to identify and image its oligonucleotide binding targets upon conjugation with alkyne-derivatized dansyl fluorophores. [60] As imilar strategy was used by Wnuk and co-workers to modify nucleosides and nucleotides with azido groups and couple them to strained cyclooctynes for direct imaging in MCF-7 cancer cells. [61] Then ucleobase-triazole adducts proved to be suitable for fluorescence lifetime imaging of specific signaling events inside live cells.
Metabolic signaling is an important area in the life sciences,w hich has become much more accessible thanks to the development of bioorthogonal functional fluorophores. After the seminal work with metabolically compatible glycans, [62] Bertozzi and co-workers exploited the metabolic incorporation of UDP-4-azido-4-deoxyxylose (UDP = uridine diphosphate) to study the function of glycosaminoglycans (GAGs) in zebrafish embryogenesis. [63] Thei nvivo coupling of these sugars to fluorescent cyclooctynes revealed new links between GAGabundance and embryonic develop- and aGaussian fit (red). C) Two-step procedure for subcellular labeling of the Golgi apparatus in live cells;c ells are treated first with Cer-TCO, a trans-cyclooctene-containing ceramide lipid, and then reacted with the tetrazine fluorophore SiR-Tzf or 3D confocala nd stimulated emission depletion (STED) super-resolution microscopy.R eproducedw ith permission from Springer Nature [68] (A) and Wiley-VCH [74,77] (B, C).

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Chemie ment. Additional work has resulted in the adaptation of cycloaddition reactions for advanced imaging technologies, such as two-photon and fluorescence lifetime imaging, [64] and the ratiometric visualization of alkyne-modified metabolites in live cells. [65] Similar chemical transformations have been applied to peptide-based structures,a fter the pioneering work from the Schultz group on the genetic code expansion technology. [66] Lemke and co-workers described aset of strained dienophilic unnatural amino acids that could be incorporated into proteins through suppression of the amber stop codon. [67] Thec onjugation of these amino acids to tetrazine fluorophores enabled direct protein labeling in live cells in an orthogonal manner to cyclooctyne-azide chemistry.C hin et al. described the genetic encoding of norbornene amino acids in both E. coli and mammalian cells for exceptionally fast and site-specific protein labeling upon reaction with tetrazines ( Figure 8A). [68] This work has later been extended to other functional groups,s uch as the phenylsydnone 1,3dipole and bicyclononyne pair, for strain-promoted reactions under physiological conditions. [69] Kele and co-workers recently published strain-promoted azide-alkyne cycloadditions in peptide sequences. [70] Thea uthors synthesized aq uenched bis(azide) fluorogenic probe for two-point binding tagging of bis-cyclooctynylated short hexapeptides in the pursuit of self-labeling small peptide tag motifs.
Devaraj and co-workers described cycloaddition transformations on oligonucleotides to improve the detection of specific DNAo rR NA sequences in genomic analysis and diagnostics.The authors initially developed fluorescent DNA structures with quenched tetrazine fluorophores and methyl cyclopropenes that "clicked" only in the presence of complementary sequences. [71] More recently,t hese nucleic acid templated reactions between 7-azabenzonorbornadiene and fluorogenic tetrazines have been optimized to detect DNA and microRNAt emplates in picomolar concentrations. [72] Another area of biological research that has strongly benefited from new synthetic approaches towards functional fluorophores is super-resolution microscopy.The Lavis group has been am ajor contributor in this field and recently described the incorporation of four-membered azetidine rings into fluorescent scaffolds as as imple structural modification to improve the brightness and photostability of dyes. [73] Moreover,s ome recent work on rhodamine structures has led to caged Si-rhodamine fluorophores as photoactivatable labels for super-resolution imaging ( Figure 8B). [74] Such functional fluorophores have been prepared by means of cycloaddition reactions using the above-mentioned approaches. Fori nstance,C hin and co-workers recently reported superresolution stochastic optical reconstruction microscopy (STORM)i maging of cytoskeletal proteins (e.g., b-actin, vimentin) after introducing bicyclo[6.1.0]nonyne-functionalized lysine residues at specific sites and coupling them with tetrazine fluorophores. [75] Thee nhanced resolution achieved with these technologies has enabled the visualization of dynamic processes in specific subcellular compartments,such as single-molecule tracking of N-sialic acids and O-linked Nacetylgalactosamine in live cells, [76] and prolonged live-cell imaging of the Golgi apparatus by STED microscopy (Figure 8C). [77]

Summary and Outlook
Selective and non-invasive imaging of biologically relevant targets represents am ajor challenge in the life sciences. Probes that are able to meet these requirements tend to have sophisticated molecular frameworks,w hich are often at the limit of our synthetic capability.W ell-established reactions are robust and practical but might only provide access to ar estricted chemical space.T hese synthetic challenges have prompted the development of modern chemical methods to generate fluorescent structures with optimal properties. Synthetic methods such as C À Ha ctivation, multicomponent, or cycloaddition reactions have proven extremely useful to develop new functional fluorophores as well as to optimize their spectral features and integrate them into advanced imaging technologies,s uch as super-resolution microscopy. These approaches constitute an excellent synthetic platform and complement currently available methods to design the next generation of fluorophores for biomedical research.