Explorations into the Effect of meso‐Substituents in Tricarbocyanine Dyes: A Path to Diverse Biomolecular Probes and Materials

Abstract Polymethine cyanine dyes have been widely recognized as promising chemical tools for a range of life science and biomedical applications, such as fluorescent staining of DNA and proteins in gel electrophoresis, fluorescence guided surgery, or as ratiometric probes for probing biochemical pathways. The photophysical properties of such dyes can be tuned through the synthetic modification of the conjugated backbone, for example, by altering aromatic cores or by varying the length of the conjugated polymethine chain. Alternative routes to shaping the absorption, emission, and photostability of dyes of this family are centered around the chemical modifications on the polymethine chain. This Minireview aims to discuss strategies for the introduction of substituents in the meso‐position, their effect on the photophysical properties of these dyes and some structure–activity correlations which could help overcome common limitations in the state of the art in the synthesis.


Introduction
Polymethine cyanine dyes are important fluorescent building blocks commonly used for protein labeling,and cell imaging. [1] Thea dvantages of penta-and heptamethine dyes include that their absorption and emission maxima are found in the near-infrared window of the electromagnetic spectrum (650-1200 nm). In this region, absorption, autofluorescence, and scattering in biological tissue reach local minima. [2] This is especially important when in vivo experiments are performed alongside the imaging of single cells or cell colonies in preclinical investigations:i nt hese contexts,t he tissue penetration of excitation beams and emitted light are of paramount importance.F urthermore,s tructural modifications allowing for orthogonal functionalization may be carried out without significantly affecting their photophysical properties.T hus,b i-or tri-functional cyanine-based fluorescence emission probes can be prepared without significantly changing their quantum yields,e xtinction coefficients,o rn earinfrared emission. [3] Some derivatives have shown to selectively accumulate in cancer cells and are promising probes in fluorescence-guided surgery. [4] Am onoclonal antibody-tagged conjugate,b evacizumab-IR 800CW,used in the fluorescence-guided surgery of primary breast cancer, passed phase II clinical trials and led to high signal-to-background ratios,e ven at sub-micromolar concentrations. [5] Given the promising results of these highly fluorescent probes,u nderstanding the relationship between structure, pharmacokinetics,a nd photophysical properties is of great importance in order to fine-tune absorption/emission, photostability,a nd in vivo behavior. While early reports on heptamethine cyanine dyes,s uch as the clinically used indocyanine green (ICG), pointed out that these suffered from low fluorescence quantum yields,u nfavorable aggregation, and low photostability,t he rigidification of the polymethine chain by introduction of cyclic motifs led to remarkable advances.Anew generation of related probes with improved physicochemical properties in living cells thus emerged. [6] Theo bserved improvements were attributed to limiting the radiation-free loss of energy through rotation and planarization, as well as steric demand. Full conformational restraint of ah eptamethine backbone failed to produce any noticeable improvement in brightness,q uantum yield, or fluorescence lifetimes. [7] This suggested that photoisomerization of heptamethine cyaninesd oes not play as ignificant role in the excited-state behavior of these dyes. [7] Instead, the observed changes may be related to variations in bond angles or lengths, and insight into the structure-activity relationships is necessary.T he main strategies for the synthesis of chainsubstituted dyes are shown in Scheme 1. These routes led to structural rigidification through the inclusion of either acentral 5or 6-membered ring, or shielded dyes through bulky substituents,which in turn led to reduced rates of photobleaching and increased kinetic stability of these dyes.
Them ain synthetic strategy for the rigidification of the polymethine chain is the use of ac onjugated dialdehyde containing ac yclic motif.T hese are typically obtained from Polymethine cyanine dyes have been widely recognized as promising chemical tools for arange of life science and biomedical applications, such as fluorescent staining of DNAa nd proteins in gel electrophoresis,f luorescence guided surgery,o ra sratiometric probes for probing biochemical pathways.The photophysical properties of such dyes can be tuned through the synthetic modification of the conjugated backbone,f or example,b yaltering aromatic cores or by varying the length of the conjugated polymethine chain. Alternative routes to shaping the absorption, emission, and photostability of dyes of this family are centered around the chemical modifications on the polymethine chain. This Minireview aims to discuss strategies for the introduction of substituents in the meso-position, their effect on the photophysical properties of these dyes and some structure-activity correlations whichc ould help overcome common limitations in the state of the art in the synthesis.
ad ouble Vilsmeier-Haack-type reaction involving ac yclic ketone,s uch as cyclopentanone,c yclohexanone,o rc ycloheptanone. [6b, 8] In the course of this reaction, the ketone is 1,3bisformylated and the oxygen substituent is substituted by achloride.Using phosphoryl bromide,rather than phosphoryl chloride,yields the respective meso-Br derivatives. [8b] Another route to access the dialdehydes is by employing 1substituted cyclohexenes as the starting materials. [6b] A meso-Cl cyclohexene motif was reported to offer the highest molecular brightness,compared to the dyes featuring cyclopentene and cycloheptene motifs. [6b] Them olecular brightness for the cyclopentene derivative was found to be marginally lower and the cycloheptene derivative was found not to be fluorescent. While absorption maxima are similar for the cyclohexene and cycloheptened erivatives,c yclopentene derivatives display ab athochromic shift (ca. 20 nm). [6b, 8c] More importantly,t he nature of the mesoheteroatom at the center of the cyanine polymethine chains has been the focus of numerous studies investigating the routes to substitution and the effect of this substituent on the photophysical properties of the resulting dye.W hile most authors report on the synthetic modification of the mesosubstituent after the condensation of the aldehyde and the indoleine,itispossible to choose an appropriate precursor,or functionalize it before the condensation, as demonstrated by various authors who performed Suzuki-Miyaura couplings with the bis-imine. [9] The meso-chloride dyes are known to be reactive in most organic solvents,with the chloride acting both as anucleofuge and as al eaving group in the palladium-catalyzed Suzuki-Miyaura or Sonogashira coupling reactions. [11] This has resulted in ap lethora of easily accessible,f unctional dyes with new physicochemical properties.I nr ecent years,t he introduction and use of such functional molecules,e ither as ratiometric or as targeted probes has been investigated. Also, meso-substitution, especially with charged functional groups, drastically affects the pharmacokinetic behavior,distribution, excretion, and toxicity. [12] Discrepancies regarding the photophysical properties within the literature are as ignificant problem and only as mall number of systematic studies intercomparing the properties of various classes of mesosubstituted dyes were published. [11a, 13] This compelled us to explore the structural databases (see Section 2) aiming to ascertain ad eeper understanding of their structure-activity relationships.

Structural Insights
Whilst as earch on Scifinder of "aminocyanine", "ketopolymethine" and "meso cyanine" yielded 241 unique references (March 2020), we found that the nature of many of these compounds remains to be structurally elucidated. The earliest reports on meso-substituted dyes date back several decades and the focus of this Minireview will be on more recent publications,which detail the photophysical properties or biochemical applications additionally to their unequivocal molecular structure elucidation. Thecrystal structure mining in the CSD database (using ConQuest [14] CCDC software) employing as implified cyanine backbone ( Figure 1) yielded merely 35 hits.However,adeeper insight into their reported structural parameters revealed that many of these hits show extensive disorder of the cyanine cores and poor quality Rfactors.F igure 1d epicts selected hits and compares the structural parameters of the most representative mesosubstituted cyanine dyes.Interestingly,the notable exception is the meso-N-selenomorpholine-substitutedc ompound which exhibits as maller dihedral angle by ca. 138 8.However, the number of crystal structures is too low to allow for any statistical analysis and deeper data-mining approaches. [15] 3. Substitution of meso-Chloride Substituents Tw oroutes to dehalogenation of meso-Cl dyes have been described. Kçnig  diphenyl phosphine at elevated temperatures. [6c] Dehalogenated derivatives will not be discussed in this Minireview.I nstead, the focus will be on the introduction of carbon-, nitrogen-, oxygen-, and sulfur-based substituents in the meso-position. A wide variety of such derivatives can be accessed through nucleophilic substitution of the meso-Cl dyes (Scheme 2).
Such reactions probably proceed following an S RN 1m echanism. Due to their simplicity,a nd the often excellent yields,n umerous studies using this pathway have been published. [11b,17] Theo bserved changes in photophysical properties are remarkable and stretch from increased photostability over direct labeling of proteins to increased Stokes shifts and new functionalities. [11b, 18] While most of the work has been focusing on the synthesis of meso-nitrogen, -oxygen, and -sulfur derivatives,the reaction of cyanine dyes with carbon nucleophiles is,s of ar, rarely found in the literature.W hile some patents mention the synthesis of nitrile derivatives,f ull characterization or investigation of such derivatives has not been reported to date,t ot he best of our knowledge.W eh ypothesize that this might be due to the fact that side-reactions limit the accessibility of meso-nitrile derivatives. [19] Likewise, the characterization of other pseudo-halogenated derivatives has not been encountered in the literature to date,w ith the notable exception of the meso-azide derivative, [18a, 20] despite the use-

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Chemie ful reactivities they may offer.W hile the syntheses of mesoselenium derivatives [21] and meso-phosphine derivatives [22] have been described as probes for various oxidative species, they will not be discussed here,a st heir optical properties have not received ar igorous dissemination in the literature. Instead, the focus will be on design strategies of functional substituted probes based on meso-carbon, meso-nitrogen-, meso-oxygen-, and meso-thiol-substituted cyanine dyes. Numerous studies explore dyes obtained from palladiumcatalyzed cross-coupling reactions.C rystal structures of Pdsubstituted dyes have been published by Davydenko

Introduction of Carbon Substituents
While most carbon-based substituents are introduced via cross-coupling reactions,t here are three examples of readily available carbon nucleophiles which were successfully reacted with tricarbocyanine scaffolds.T he most noteworthy is the reaction with acetylacetone (acac) under basic conditions,a s shown by Mitra et al. [23] This modification introduces aligand into the original structure and was used as ap latinumcontaining prodrug for photodynamic therapy (Scheme 3). Thea bsorption maximum of the resulting dye 2 was blueshifted by ca. 5nm. Combined with an emission maximum, red-shifted by about 5nm, the Stokes shift is increased by ca. 10 nm. Mitra et al. also reported that, while the quantum yield of 2 is increased compared to 1,upon binding of diamagnetic Pt II ,apartial quenching of fluorescence was observed. [23] To our knowledge,n oa ttempts at utilizing the acetylacetone substituent in condensation reactions,s uch as the formation of pyrazoles or thiosemicarbazones,h ave been described thus far. Other compounds based on the same methodology have been reported by Pascal et al.,w ho used malononitrile as ac arbon nucleophile,a nd by Nagao et al., who used barbituric acid in as imilar way. [8b,c] Thef ourth carbon nucleophile that has been used, albeit only in patents which do not describe the synthesis or the photophysical properties,iscyanide. [19a] While it seems rather obvious to use this readily available,h ighly nucleophilic pseudo-halide,i ti s completely missing from the scientific literature and discus-sion. Ar eason for this may be as ide-reaction in which the cyanine attacks the bridgehead carbon at either end of the polymethine chain next to one of the nitrogen centers. [24] This reactivity has been exploited in the design of ratiometric probes for the quantification of cyanide. [19b,24] Š tackovµ et al. published a meso-nitrile derivative as one of the structures obtained through ring-opening of Zincke salts. [10a] Although their structures do not feature ar igidified polymethine chain, meso-CN substitution in their scaffold leads to ar ed-shift of absorption and emission maxima as compared to most other derivatives.
Alarger number of accounts describe the introduction of carbon-based substituents by means of cross-coupling methods. [11a,c, 18d, 25] Suzuki-Miyaura reactions typically employing palladium(0) complexes,s uch as Pd(PPh 3 ) 4 ,i nc onjunction with the appropriate boronic acid precursor have been described. [11c,25] Ther eaction is typically performed in water or aqueous solvent mixtures,s uch as water/dioxane mixtures. [11a,c, 18d, 25, 26] Apart from the mentioned stability improvements such meso-carbon dyes offer,they may also be used to produce probes which, dependent on the pH, reversibly form heterocyclic spiro derivatives (Scheme 4). At low pH, compound 3 is fluorescent and the rings are not fused, while at higher pH the nucleophilic ortho-substituent attacks the meso-position, forming anon-fluorescent spirane. [26] Alternatively,S onogashira cross-couplings may be performed to give meso-alkyne derivatives,which tend to be redshifted with respect to the parent dye. [11a] With simple aryl systems coupled at the meso-position, ahypsochromic shift of ca. 20 nm is observed. However,the Stokes shift of such dyes Scheme 2. Overviewo fcommon reactions to access structurally diverse cyanine imaging probes for biochemical species or processes and agents for photodynamic therapy.

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Minireviews 6234 www.angewandte.org was often reported to be slightly smaller (10-15 nm) than that of the parent dye.T he resulting dyes are reported to possess excellent chemical and optical stability in vitro. [18d] However, even in highly sulfonated cyanine dyes,s elf-aggregation is observed in meso-phenyl derivatives,potentially limiting their usefulness in vitro and in vivo. [11a] Thes ynthesis of as terically shielded non-rigid cyanine dye with remarkable properties through the ring-opening of aZ incke salt was also reported alongside its conjugation to cyclic-RGD peptide and (goat) IgG;t his demonstrated the effect of the steric shielding on the photophysical properties of the dye. [10b] Most noticeably,t he sterically shielded derivative did not show obvious signs of aggregation on the protein surface,u nlike other peptide or antibody-cyanine conjugates,w hich in many cases show hypsochromic shifts and signs of H-aggregate formation. [10b,27] It could be conceived that cyanine-dye-based rotaxanes [28] conjugated to antibodies may behave in as imilar fashion and limit aggregation and aggregation-caused quenching.

Introduction of Nitrogen Substituents
Thei ntroduction of nitrogen substituents in the mesoposition is astraightforward way to introduce new properties and functionalities. [6c] While only few examples of dyes with an unsubstituted amine in the meso-position exist, numerous accounts have been published on the synthesis of substituted amines.T oo btain a meso-NH 2 -functionalized cyanine dye, atwo-step procedure is necessary.Some patents claim to use phthalimide as an ucleophile and reduce the intermediate product. [29] Alternatively, meso-NH 2 dyes may be accessible through synthesis of the corresponding meso-azide derivative as an intermediate product and subsequent reduction with H 2 S, as reported by Yu et al. [20] While aminocyanine dyes obtained from the reaction with substituted amines were first prepared in the second half of the 20 th century,one of the first investigations into their peculiar properties was undertaken by . [1a,30] Fors everal years,o nly few publications concerning aminocyanine dyes appeared until Peng et al. synthesized two new derivatives in 2005 and noticed their large Stokes shift (Scheme 5, dyes 5 and 6). [18c] Amination with primary amines is typically achieved under mild conditions in polar, aprotic solvents. [17b, 18c, 31] Ther esulting aminocyanine dyes show an intense blue-shift (up to 180 nm) compared to their meso-chloride counterparts,a s well as asignificantly larger Stokes shift (in some cases larger than 100 nm). [8b,18c] In general, the changes observed due to amination can be understood as ap erturbation of the conjugated system due to the interaction of the lone pair of nitrogen with the conjugated polymethine system.
As such, ar esonance structure in which the central position can tautomerize,g iving either an enamine or an imine,i st he most straightforward way to visualize the electronic situation (Scheme 6). In primary amines this correlates to stronger blue-shifts in electron-rich structures and weaker blue-shifts in electron-poor substituents,w hile amino groups with two alkyl substituents lead to weaker blueshifts,presumably due to the increased steric demand. [8b,13b] If meso-amine dyes are treated with ab ase,s uch as potassium carbonate,a ne ven greater hypsochromic shift is observed, which would correspond to an even more pronounced perturbation of the conjugated system and the emergence of an actual imine bond (Scheme 6, dye 8). Scheme 4. Meso-spiro reactivity of atricarbocyanine dye. The authors described the use of 3 as an imaging agent for acidic microenvironments, often associated with inflammation.F rom left to right:control, LPS (lipopolysaccharides, provoke acute inflammatory response) with 3,a nd 3.I mages used with permission from Ref. [26].
It was suggested that the unexpectedly large Stokes shifts observed in these dyes were the result of an excited state intramolecular charge transfer (ICT). [18c] However,i ts eems that this is related to adrastic change of the overall electronic structure,w hich resembles an imine-type bonding in the ground and shifts towards an amine bond in the photoexcited state. [30,32] In addition to the character of the CÀNbond in the meso-position, symmetry breaking in the ground state was recently suggested to cause the broad absorption bands and large Stokes shifts. [33] While the meso-nitrogen becomes more electron rich, steric hindrance increases and inhibits an effective overlap between the polymethine p-system and the nitrogensl one pair, effectively leading to weaker blue-shifts than their counterparts with al ower degree of substitution.
According to Gray et al.,the isomerization of meso-amine cyanine dyes leads to low quantum yields due to the existence of both, anon-fluorescent and afluorescent form, depending on the orientation of the amine substituent. [30] Cao et al. investigated the influence of the position of the meso substituent on the photophysical properties in silico. [32] They found that an amino group at an odd chain position (as counted from the indole) acts as an electron donor in the excited state.H owever, if it is situated at an even position within the polymethine chain, it acts as an electron acceptor instead. Thee nergy barrier for rotation of the NH 2 group at an even position was found to be significantly larger (7.09 eV at the center of the chain, 0.1 and 0.06 eV at odd positions). [32] Considering these findings and the reports by Gray et al., the high quantum yields reported by some authors seem disproportionate and may be the result of using unsuitable fluorophores as reference. [18c, 31a, 34] Although some variability may be caused by using different cyanine backbones,s uch large differences are not to be expected. However,due to the large Stokes shift exhibited by aminocyanines (up to ca. 150 nm), relative measurements may lead to inaccurate determinations of fluorescence quantum yields.Furthermore, such al arge Stokes shift may lead to relative overestimation of quantum yield due to alack of reabsorption of the emitted light. This is especially troublesome if the standard used in the relative measurement possesses small Stokes shifts.Literature values for quantum yields range from low single-digit values to almost 50 %. [18c, 31a, 34] It has been reported that upon protonation of aminocyanine dyes featuring diamino substituents,s uch as piperazine,a ni ntense bathochromic shift has been observed:t his corresponded to the formation of an ammonium group.T his effectively halts the perturbation of the polymethine system [17b,d] and leaves the electron-withdrawing inductive effect, causing ah ypsochromic shift, relative to the meso-Cl dye,a s would be expected from ammonium substituents. [17b,d] There are accounts of blue-shifts upon protonation, especially in amine substituents derived from primary amines and monoamines. [18c,31a] This was suggested to occur as the result of an irreversible protonation of the polymethine system. [35] The effect of protonation on quantum yield has not yet been fully elucidated for meso-diamine substituents.Ithas been reported that the extent to which protonation affects the photo-physical characteristics,such as quantum yields,varies greatly within the series,thus further studies are necessary. [17b,d] Thef ormation of amide bonds from a meso-nitrogen substituent leads,m uch like protonation in some dyes,t oa n intense red-shift, which almost re-establishes the original optical properties of the dye (Scheme 7, dye 11). [17b,31a, 36] This is ar esult of the involvement of the nitrogensl one pair in ar esonance structure with the carbonyl. Likewise,a romatic amines in the meso-position lead to significantly smaller differences in terms of absorption maximum, due to the electron donation into the aryl system. [8b,13b] Dye 10 (Scheme 8) has the potential to act as aswitch-on probe for nitric oxide. [37] Upon addition of NO to the mesonitrogen, the imine-type resonance structure is suppressed, and the fluorescence intensity increases significantly (Scheme 8). Apart from these structurally simple examples, various aminocyanine dyes have been synthesized, bearing functional moieties,targeting molecules,orgroups for further conjugation in the meso-position. Examples include as phingosine derivative (an amino alcohol, and ap rimary part of sphingolipids,which make up parts of the cell membrane), [34] photocleavable prodrugs such as tamoxifen-like compounds, [38] (PEG)ylated derivatives such as those reported by Lu et al., [39] ratiometric probes for nitroreductase, [40] and agents for photodynamic therapy,asreported by Jiao et al. [41] Alternative strategies also include pH cleavable prodrugs,a s reported by Xing et al., [42] and pH activated agents for photodynamic therapy. [43] Alimitation in the use of aminocyanines may be their lack of chemical stability,a ss olutions in serum or cell culture medium deteriorate over time. [13a] Likewise,they are prone to photobleaching compared to their respective parent compounds or other meso-substituents.This is likely caused by the increased electron density in the backbone,e ffectively increasing reactivity towards reactive oxygen species. These dyes showed improved photophysical properties as compared to the parent dye,s uch as (slightly) larger Stokes shift, greater photostability,and higher molecular brightness. It has been suggested that the reason for this was the electron donation into the triazole,r ather than into the polymethine system.
In many cases,t he library of triazole-functionalized dyes possessed higher molecular brightness than the original meso-Cl dye.U tilizing an asymmetric derivative of the triazole-functionalized dye featuring acarboxylic acid moiety activated by formation of an NHS-ester,M ellanby et al. reported the labeling of T-cells and performed non-invasive fluorescence imaging in mice. [18a] More recently,anumber of bifunctional probes featuring three triazole handles was published. [3b] Using absolute quantum yield measurements,t he authors determined av alue of 3.22 %i nP BS for a( PEG)ylated derivative (as compared to 1.90 %f or ICG). [3b] Despite the significant amount of research results published regarding aminocyanine dyes, [17d, 18c, 30, 32,41] several fundamental considerations remain am atter of debate.F or example,the effect of the amine substituent on quantum yield remains to be definitively answered, ideally by measurements of fluorescence quantum yield with an integrating sphere.I f the amine is prevented from perturbing the conjugated system, it remains to be clarified whether the quantum yield increases or decreases.Further investigations into the role of solvents and concentration would likewise be of interest.

Introduction of Oxygen Substituents
Thei ntroduction of oxygen in the meso-position can be accomplished in two different ways.The meso-Cl fluorophore may be reacted with an ucleophilic oxygen compound (e.g. aphenol in the presence of base). [6c, 44] This reaction has been reported to not proceed well with simple,n on-aromatic alcohols,p resumably because the reaction proceeds via an S RN 1-type mechanism. [6c, 45] Nevertheless,non-aromatic mesoethers may be obtained through Smiles rearrangements. [45] Thel atter methodology has been applied to synthesize both, probes for imaging of the ureter [46] and antibody-dye conjugates to study the effect of charge localization on the in vivo properties of the resulting conjugates. [47] Alternatively, oxygen, in the form of an enol or ketone,may be introduced under oxidative conditions.T his may be accomplished by heating the meso-Cl dye in ab asic solution of dimethylformamide [48] or through oxidation with acatalytic amount of Nhydroxysuccinimide in alkaline solution. [49] Both reactions are mild oxidative methods and as such indicate the high reactivity of the C À Cl bond in these molecules.T he photophysical behavior of the probes obtained by these methods can best be described by assuming ak eto-enol equilibrium (not tautomerization) in protic solvents (Scheme 10, 18 and 19). [48,50] Thefact that the underlying isomerization process is not at automerization is evident due to the positive solvatochromism observed under pH-neutral conditions,s uggesting an uncharged ground state. [49a, 51] Similar to the situation described above for primary amine substituents,these simple oxocyanines show bathochromic shifts of ca. 200-300 nm and Stokes shifts approaching 100 nm in the keto form. [48,52] It is noteworthy that the Stokes shifts of these dyes are signifi-cantly larger (about 30 to 50 nm) in protic solvents,which may be related to hydrogen bonding between the ketone and polarized hydrogen atoms. [49a, 51] Additionally,t he fluorescence quantum yield in protic solvents is significantly higher. [51] Upon protonation, the dye is found in its enolic form. This change is accompanied by asignificant hypsochromic shift, as the conjugation between the nitrogen centers is re-established. Compared to their meso-Cl counterparts,the enol dye still shows as ignificant hypsochromic shift (ca. 50-100 nm). Unfortunately,f luorescence properties of these oxocyanines dyes have not been investigated in detail. Zheng et al. and Pascal et al. provided quantum yields. [49a,51] Their values are highly solvent-dependent, and significantly higher (in protic solvents) than the parent dyesquantum yield.
Much like aminocyanine dyes, meso-oxocyanines exhibit the expected reactivity,t hat is,t hey react as nucleophiles themselves,a nd can be reacted with acid halides [48,50,53] and sulfonyl halides. [54] Rates of photobleaching are highly dependent on the protonation state of the oxocyanines.I n the keto form, photobleaching occurs at am uch lower rate compared to the parent compound. In the enol form, photobleaching occurs at am uch higher rate. [49a] Reactions of oxocyanine dyes with phosphorous reagents have,t oo ur knowledge,o nly been reported by Zhang et al., who reacted dye 18 with phosphoryl chloride (Scheme 11). [55] It may be considered aproof of concept for the design of turnon ratiometric probes,a st he phosphorylated probesp hotophysical characteristics are similar to that of the parent dye, while upon dephosphorylation by alkaline phosphatase, ac hange back to the absorption characteristics of 18 is observed.
Some authors employed similar methodologies to establish fluorescent probes for the detection of hydrogen sulfide and hydrazine. [50,56] Li et al. synthesized af luorescent switch-on probe for the detection of nitroreductase (NTR) (Scheme 12), an enzyme which is overexpressed in many hypoxic tumors. [57] Ac aveat in using probe 21 is that slow side-reactions with cysteinea nd glutathione may occur. [50,58] Likewise,Huetal. demonstrated that esters in the mesoposition can be cleaved under reductive conditions. [53] They utilized this reactivity to prepare ratiometric probes for the detection of hydrazine.
Furthermore, meso-ethers,a nd especially meso-phenyl ethers,h ave been found to be kinetically unstable towards many endogenous thio-nucleophiles,i ncluding cysteine and glutathione. [44] Nevertheless,t he IR 800CW conjugates have been successfully used in vivo.T he extent to which the formation of ag lutathione adduct of the IR 800CW dye influences its photophysical properties in vivo remains amatter of investigation. [25] Scheme 11. Derivatization of oxocyanine dyes by reaction with phosphoryl chloride and subsequent cleavage of the phosphorousÀoxygen bond by alkaline phosphatase (ALP). Image adapted from Ref. [55] with permission.C ounterions omitted for clarity.
Scheme 12. Derivatization of oxocyanines dyes by reaction with acid chlorides.U pon enzymatic reduction of the p-nitro substituent of 21 by NTR, fluorescence intensity increased more than 100-fold (22). [57] Counterions omitted for clarity.
Scheme 10. Synthesis of oxygenated derivatives of rigid tricarbocyanine dyes and pH-induced keto-enol equilibrium. Counterions were omitted for clarity.

Introduction of Thio Substituents
Theformation and biological fate of thiocyanine dyes are of particular interest for many biochemical and medicinal applications,a sm any endogenous species contain highly nucleophilic thio substituents,a nd several enzymes catalyze the formation of thiols,o rr eactions involving them. Mesoarylethers may undergo nucleophilic substitution under physiological conditions and form meso-thio derivatives.A s fluorescent labels for proteins,these functional NIR dyes may allow for straightforward detection and quantification. In terms of photophysical properties,they typically show asmall bathochromic shift compared to the parent dye.Inthioethers with an aromatic substituent, the bathochromic shift is larger than in thioethers with non-aromatic substituents. [13b,59] Photostability is similar to the original meso-Cl derivatives,a nd mostly comparable to the meso-aryl and meso-alkyl ethers. [13b] Not much is known about their chemical stability.A lthough some authors suggested that thioethers may undergo further reactions or rearrangements, [44,45] several conjugates were found to be kinetically stable. [18b,45] Thes tability may be related to steric hindrance or other, yet unelucidated factors. Thequantum yield of aromatic thioethers seems to be smaller than those of arylethers,b ut larger than those of aryl amines. [13a] Their molar absorptivity and molecular brightness in PBS and FBS were reported to be lower than that of the respective parent dye or aryl ethers for both aliphatic and aromatic thioethers (Scheme 13). [13a] No turn-on/turn-off reactivity has been described to our knowledge,w hich is in stark contrast to the amino-and oxocyanine dyes discussed above.N evertheless,t hey may be useful ratiometric probes for certain biomarkers or enzyme activity.S everal such probes based on ICT-type mechanisms have been described, for instance for N-acetyltransferase [60] or nitroreductase. [57] Like the meso-arylethers, meso-Cl dyes were found to undergo nucleophilic substitution with arange of endogenous thiol-based nucleophiles. [11b,18b,27b,61] This reactivity may be used to directly label peptides and proteins and could, by extension, be used to directly label monoclonal antibodies,or fragments thereof.Ithas also been suggested to influence the pharmacokinetics and to be the reason for the observed tumor selectivity and retention of meso-Cl dyes,t hrough formation of covalent albumin conjugates. [27b] Thef ormation of dye-albumin conjugates appears to proceed in two steps.F irst, an on-covalent aggregate is formed, leading to abathochromic shift. Then, the dye reacts with af ree thiol, forming ac ovalent bond, which leads to as mall hypsochromic shift. [27b] Overall, the dye-albumin conjugate is red-shifted relative to the meso-Cl dye,consistent with the formation of athiocyanine.Ithas been suggested that the observed selectivity and strong retention of such tumorseeking meso-Cl dyes is caused by ac ombination of overexpression of albumin receptors on tumor cells and the EPR effect. [27b] Other fluorophore-albumin conjugates have been reported to behave similarly and have been investigated for their use as imaging agents in the treatment of various cancers. [62] Lin et al. used the reactivity of a meso-Cl dye to label vimentin (a structural protein) and several other proteins. [11b] It was found that amounts as low as 1ng substrate are detectable on ag el-electrophoresis imaging device.C anovas et al. applied an analogous approach in the labeling of peptides,a lbumin, and the antigen-binding fragment of pertuzumab (Fab,for the treatment of HER2 positive breast cancer). [18b] Figure 1s hows the structure of ac yanine sulfonyl derivative (CCDC-272535). Although the crystal structure has been deposited, no literature is available detailing its synthesis or optical properties.Apossible way could be the oxidation of the corresponding meso-thiophenyl ether with asuitable oxidizing agent like OXONE or periodate.

Conclusion and Outlook
In the development of heptamethine cyanine dyes,s ome classes of substituents have,h istorically,b een difficult to access.R ecently,n ew synthetic methodologies which may overcome these barriers were developed and may help resolve some of the current limitations.Even if these methods do not yield rigid dyes,t hey offer access to new mesosubstituents and substitution patterns.These could offer novel reactivities or improved photophysical characteristics.S ome derivatives that could be of interest and were previously not accessible,o rd ifficult to synthesize,i nclude meso-fluoride dyes,aswell as some pseudohalide derivatives,such as mesonitrile or meso-thiocyanates.T he photostability of some derivatives remains an issue,a nd additional work to further elucidate the causes of the observed differences is needed. Thed iscrepancies encountered in the literature call for the establishment of standardized procedures so the properties of these dyes can be compared and evaluated in ar ational and robust manner.T his would help pave the way towards the development of the full potential of tricarbocyanine dyes. More recently,v arious authors suggested and demonstrated the use of the off-peak tail emission of such dyes,oralbumin chaperoned dye aggregates,w hich extends into the second NIR window (NIR-II, 1000-1700 nm). [63] Theu se of compounds already established for NIR-I imaging could signifi-cantly ease the establishment of imaging in the NIR-II window. [63][64] Likewise,f urther development of cyanine dyes for applications such as PDT is of great interest, due to the penetration depth of light in the NIR window and their high molar absorptivity. [3b, 41, 43, 65] Notwithstanding the extensive synthetic advances achieved, and some of the commercial opportunities presented regarding the production of meso-substituted cyanine dyes, [66] ag eneral transition as everyday probes either in in vivo settings or in the clinic remains to be seen. This is in contrast to the promising photophysical properties,t he synthetic simplicity of the introduction of substituents in meso cyanine dyes,a nd the sheer amount of possible applications and probes that can be realized.
Thes uccess of IR 800CW,c urrently in clinical trials for the fluorescence-guided surgery of several cancers (e.g. of the brain, head and neck, esophageal, breast, lung,p ancreatic, kidney,a nd colorectal cancer), [67] is ag ood indicator of the progress that has been made and the general usefulness of such fluorescent probes.