Rhodols – synthesis, photophysical properties and applications as fluorescent probes

Yevgen M. Poronik , Kateryna V. Vygranenko , Dorota Gryko * and Daniel T. Gryko *
Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland. E-mail: dorota.gryko@icho.edu.pl; dtgryko@icho.edu.pl

Received 28th February 2019

First published on 24th September 2019

The formal replacement of one dialkylamino group in rhodamines with a hydroxyl group transforms them into rhodols. This apparently minor difference is not as small as one may think; rhodamines belong to the cyanine family whereas rhodols belong to merocyanines. Discovered in the late 19th century, rhodols have only very recently begun to gain momentum in the field of advanced fluorescence imaging. This is in part due to the increased understanding of their photophysical properties, and new methods of synthesis. Rationalization of how the nature and arrangement of polar substituents around the core affect the photophysical properties of rhodols is now possible. The emergence of so-called π-expanded and heteroatom-modified rhodols has also allowed their fluorescence to be bathochromically shifted into regions applicable for biological imaging. This review serves to outline applicable synthetic strategies for the synthesis of rhodols, and to highlight important structure–property relationships. In the first part of this Review, various synthetic methods leading to rhodols are presented, followed by structural considerations and an overview of photophysical properties. The second part of this review is entirely devoted to the applications of rhodols as fluorescent reporters in biological imaging.

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Yevgen M. Poronik

Dr Yevgen Poronik received his MSc (2001) from Department of Chemical Technology of Kyiv Technical University and PhD (2006) from Institute of Organic Chemistry, Ukrainian Academy of Sciences, under the supervision of Prof. Yuriy Kovtun. He worked as a post-doctoral researcher at Institute of Organic Chemistry of Polish Academy of Sciences in the group of Prof. Daniel Gryko. He is currently a research assistant in the same group, working on new donor–acceptor chromophoric systems, synthesis and application of functional organic dyes.

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Kateryna V. Vygranenko

Kateryna Vygranenko was born in Ukraine in 1992. She obtained her MSc from Taras Shevchenko National University of Kyiv in 2015. She is a PhD student at the Institute of Organic Chemistry Polish Academy of Sciences under the supervision of Prof. Daniel Gryko. Her research interest currently focuses on developing new organic dyes for STED-microscopy.

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Dorota Gryko

Dorota Gryko obtained a PhD from the Institute of Organic Chemistry at the Polish Academy of Sciences in 1997, under the supervision of Prof. J. Jurczak. After a post-doctoral stay with Prof. J. Lindsey in North Carolina State University (1998–2000), she started an independent career in Poland. In 2009 and 2018, she received the prestigious TEAM grants from the Foundation for Polish Science. Her current research interests are focused on light-induced processes with particular attention being paid to porphyrinoid catalysis as well as on vitamin B12 chemistry.

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Daniel T. Gryko

Daniel T. Gryko obtained his PhD from the Institute of Organic Chemistry of the Polish Academy of Sciences in 1997, under the supervision of Prof. J. Jurczak. After a post-doctoral stay with Prof. J. Lindsey at North Carolina State University (1998–2000), he started his independent career in Poland. He became Full Professor in 2008. The same year he received the Society of Porphyrins and Phthalocyanines Young Investigator Award and in 2017 the Foundation for Polish Science Award. His current research interests are focused on the synthesis of various functional dyes as well as on two-photon absorption, solvatofluorochromism, excited-state intramolecular proton transfer and fluorescence probes.

1. Introduction

Rhodols belong to the xanthene dye family and have recently become popular fluorescent scaffolds with many applications connected to fluorescence microscopy. Though these dyes have been known since the 19th century their appearance in the literature has remained limited, being largely overshadowed by the more popular xanthene chromophores – rhodamine and fluorescein. Since the title chromophore was first indirectly synthesized from fluorescein in 1889,1 it was named rhodol2 highlighting its similarity to rhodamine yet emphasizing the inclusion of a ‘phenol functionality’ (Fig. 1). Until 1990 rhodols remained largely forgotten and only a few reports appeared in the literature,3–17 presumably due to the fact that more convenient methods for the preparation of both rhodamines and fluoresceins were available.
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Fig. 1 Position numbering in the rhodol chromophore.

Nevertheless, since the last decade of the 20th century, the chemistry of rhodol dyes has been experiencing a renaissance due to the development of new fluorescent sensing and imaging techniques requiring specifically designed functional organic dyes. As a consequence, many of the scientific reports detailing the applications of rhodol dyes have a biological or medical focus. Many of these reviews only touch on rhodols (sometimes called rhodafluors),18–20 their focus mainly devoted to other research topics and describe rhodol chemistry in a fragmentary manner. This work comprehensively presents chemical advances related to the synthesis, properties, and applications of rhodols and their close analogues.

2. Introduction of rhodol electronic structure

Structural analysis of rhodols, like all xanthene chromophores, reveals their similarity to polymethine dyes. Indeed, xanthene dyes contain a polymethine-like conjugation chain located between polar terminal moieties. As the rhodamine chromophore possesses a positive charge delocalized along the conjugated system it bears a stronger resemblance to typical cyanine chromophores. Fluorescein chromophores, on the other hand, possess a negative charge delocalized along the conjugated system and belong to oxonole family. The rhodol system, in contrast, has an electronic structure represented by two limiting forms (neutral and dipolar) and is analogous to merocyanine chromophores (Fig. 2).21
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Fig. 2 Resonance structures of rhodol as a hybrid of fluorescein and rhodamine.

Although the oxygen bridge atom at the central position of the molecule is not a part of the polymethine chromophore it plays a dual role of increasing the rigidness of the chromophore and acting as a polar substituent. Chromophores of the same length without the oxygen bridge have been known since 195022,23 and demonstrate much stronger solvatochromism in comparison to rhodols. In nonpolar solvents unbridged analogues show blue-shifted absorption (<450 nm) but in polar solvents the absorption maxima can occur at longer than 570 nm. According to X-ray diffraction structural analysis the unbridged chromophore adopts a propeller-like shape,23 the dihedral angle and the charge distribution of which is affected by the nature of the solvent. Conversely, the oxygen bridge leads to more planar and rigid structures24 which show much stronger fluorescence responses and much weaker solvatochromism in both absorption and fluorescence compared with unbridged chromophores (Fig. 3).15,22,23

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Fig. 3 Single crystal X-ray diffraction structures of compounds 1 (CCDC 1413998) and 2 (CCDC 1564215).

The aryl substituent at the central position of rhodol (position 9, Fig. 1) is not a part of the xanthene chromophore. Indeed, in the S0 state the aryl substituents are oriented almost orthogonally (Fig. 3). In the S1 state the dihedral angle between the aryl substituent and the xanthene framework is reduced somewhat, however, the angle is still large enough to prevent meso-aromatic substituents from being significantly conjugated with the main chromophore.25

Moreover, the presence of an aryl substituent at the meso-position decreases the sensitivity of the rhodol chromophore to nucleophiles, e.g. unsubstituted rhodol reacts with the bisulfite anion resulting in the interruption of the conjugation chain.26

Computational studies based on TD-DFT calculations suggest that the HOMO displays a mainly neutral character as a large portion of the electron density localizes on the terminal amino group. Upon excitation, the electron density from the amino group redistributes towards the keto group, thus, the LUMO demonstrates charge transfer character. Indeed, the dipole moment in the S1 state is higher than that of S0, indicating a more polar excited state. Therefore polar solvents stimulate the charge separation thus decreasing an energy gap between HOMO and LUMO of rhodol and related chromophores.25,27

3. Synthesis

3.1. Rhodols

The most common method for the preparation of rhodols, like for other xanthene dyes, is based on the Friedel–Crafts acylation methodology.15,28 Typically the symmetry of xanthene dyes enables their synthesis in a one-pot procedure from phthalic anhydride and two equivalents of either a 3-dialkylaminophenol (for rhodamine) or a 1,3-dihydroxybenzene (for fluorescein). This is not the case for rhodols, which due to their asymmetry, must be synthesized in a stepwise manner from phthalic anhydride. The condensation of resorcinol with 4-dialkylamino-2-hydroxybenzophenones 5 and 6,17,29 resulting from the reaction of phthalic anhydride with corresponding 3-aminophenols 3 and 4, represents the most straightforward synthetic route to rhodols (Scheme 1).5–8,17–20,24,25,28–40 In contrast to the simple one-step rhodamine synthesis proceeding easily in approx. 30% yield,41 the two step rhodol preparation via a benzophenone intermediate, requires an excess of phthalic anhydride to suppress the formation of unwanted rhodamine, and leads generally to diminished yields. The application of unsymmetrically substituted phthalic anhydrides is also impractical for the synthesis of rhodols as the key intermediate forms as a mixture of regioisomers which are difficult to separate, lowering the yield even further.
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Scheme 1 The classical rhodol synthesis from phthalic anhydride, m-aminophenol and resorcinol.

Despite its two-step nature, the readily available starting materials and operationally simple synthetic procedure contribute to this method's popularity. The presence of the COOH group in the product's structure offers an additional advantage since it enables: (1) increased polarity and hence water solubility; (2) the formation of a leuco-lactone structure; (3) conjugation with various molecules. A modification of the above method, employing acetophenones 9, 1015,42 gives access to rhodols 11, 12 substituted with the methyl group at the meso-position (Scheme 2).43 Acetophenone 9 is synthesized via the Fries rearrangement of 3-acetoxy-N,N-dimethylaniline in 16% yield.15 A more convenient synthetic route to acetophenone 10 starts from 7-diethylamino-4-hydroxycoumarin and gives the product in 90% yield via ring opening and subsequent decarboxylation after treatment with mineral acid.43 The use of phosphoric acid instead of the commonly applied sulfuric acid for the condensation of acetophenone with resorcinol leads to rhodol 12 in 75% yield. The product can be easily isolated by precipitation of the xanthylium perchlorate salt from an aqueous solution.

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Scheme 2 The synthesis of 9-methylrhodols 11 and 12.

This modification enables a better control over the character of the substituent at position 9. At the same time, however, the availability of aromatic ketones with a substitution pattern similar to 9 remains problematic.

Monothioresorcinol has been reacted with benzophenone 13 in polyphosphoric acid resulting in compound 14 which incorporates a sulfur atom at the bridge position in place of oxygen (Scheme 3).15 This reaction is the only example of a rhodol analogue with an endocyclic sulfur atom reported. Curiously, the same reaction in sulfuric acid leads to the unsubstituted rhodol with an endocyclic oxygen atom. This is due to the conversion of thioresorcinol to resorcinol under the reaction conditions.

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Scheme 3 The synthesis of rhodol with sulfur in position 10.

Another classical rhodol preparation method6,44–59 is based on a reversed substrate pattern utilizing 2,4-dihydroxybenzophenones47 and 3-aminophenol derivatives as substrates (Scheme 4).

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Scheme 4 Rhodol formation from dihydroxybenzophenone and 3-aminophenols.

The roots of this synthetic protocol date back to the 19th century with Bayer's observation of the formation of rhodol from 2-carboxy-2′,4′-dihydroxybenzophenone, which was generated by an alkali-induced decomposition of fluorescein.1 In the original patent, 3-dimethylamino-, 3-diethylamino-, or 3-(phenylamino)phenols were condensed with dihydroxybenzoylbenzoic acid, as well as with its bromo- and dibromo-derivatives in the presence of ZnCl2 at 140–160 °C. This initial strategy was later utilized by Ghatak4 and Chen's groups.16 As an alternative to fluorescein hydrolysis, the benzophenone intermediate can also be generated by the reaction of phthalic anhydride with resorcinol derivatives in the presence of a Lewis acid. Although this ‘reverse’ methodology does not offer any substantial advantages over the classical one, in cases where the 3-aminophenol is more complex this particular strategy can minimize the use of the more expensive and less available substrate.

The unusual coupling of phthalimide with 3-aminophenols can afford rhodol derivatives in decent yields.60 The original idea behind this strategy was to improve the yield of rhodamine 110 by avoiding side reactions. Nevertheless, the treatment of phthalimide with an excess of 3-aminophenol in sulfuric acid leads to hydrolysis of the C–N bond to form rhodol 18 instead of rhodamine 110 (Scheme 5).

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Scheme 5 The synthesis of rhodol from phthalimide and 3-aminophenol.

Rhodols containing a highly functionalized 9-aryl group are typically prepared from phthalic anhydride derivatives as an isomeric mixture of 5- and 6-substituted derivatives which are non-trivial to separate. Chevalier and co-workers discovered that functionalized aldehydes, used in place of phthalic anhydride derivatives, give rise to an intermediate possessing a structure similar to monomethine dye 20 which then reacts with resorcinol to afford rhodol 21 in 50% yield (Scheme 6).61–63

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Scheme 6 The synthesis of rhodols from benzaldehydes developed by Chevalier and co-workers.

Even though this method solves the regioselectivity problem, it appears to be demanding, since RP-HPLC is exploited for analysis and purification. The chromatography fractions containing product were lyophilized to give rhodol as a TFA salt. Curiously, the authors did not supply information on the character of the oxidation, which occurs in the last step. The same synthetic protocol was employed with various m-aminophenols as phenolic partners to generate a range of unsymmetrical sulforhodamines with similar yields.

The Friedel–Crafts reaction of aldehydes with two equivalents of resorcinol or 3-dialkylaminophenol is much more often used for the synthesis of fluoresceins and rhodamines10 than it is for rhodols. The simple reason is that in the case of rhodols, the reaction must be somehow stopped upon reaching the methine salt (i.e. after first step), which is not easy since this intermediate is typically more reactive than the starting aldehyde.

The synthesis of fluoresceins and rhodamines through the preparation of xanthones and subsequent addition of organometallic reagents is currently broadly utilized. The same strategy has been employed for the synthesis of Singapore Green, a structural hybrid of rhodamine 110, and Tokyo Green. In attempts to find a fluorophore which can be employed for microarray-based protease substrates a synthetic protocol for the rhodol isomer Singapore Green with an amino-group on one side as a centre of peptide sequence conjugation was developed. The targeted molecule should also possess absorption and emission maxima in the visible range, and phenolic group on the other side providing solid-phase peptide synthesis and microarray immobilization.64 The conjugate is sensitive to protease activity, which causes amide bond cleavage, thus releasing the highly fluorescent dye. The synthetic protocol proceeds through the formation of asymmetric xanthone 24via the Ullmann-type coupling of 3-acetamidophenol (23) with 2-chloro-4-nitrobenzoic acid (22). After introduction of an alkoxy substituent in place of the amino group, compound 25 is reduced and protected to give derivative 27. Consecutive arylation and deprotection affords Singapore Green fluorophores 29 and 30 (Scheme 7).

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Scheme 7 The synthesis of Singapore Green 29 and 30.

A combination of substitution at the meso-position of xanthones with catalytic amination via a trifluoromethylsulfonate derivative is an alternative synthetic approach towards the rhodol skeleton.65,66 In bistrifluoromethylsulfonate derivatives, one triflate group is substituted with the amino group in the first step, which after a series of subsequent transformations, i.e., hydrolysis of the trifluoromethylsulfonate group, protection of hydroxyl group with TBDMSCl, arylation and deprotection, leads to O-rhodol 39, C-rhodol 40, Si-rhodol 41 and P-rhodol 4267 (Scheme 8).

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Scheme 8 The general synthetic route to O-, C-, Si- and P-rhodols.

The Buchwald–Hartwig amination can also be utilized for the synthesis of rhodols starting from fluoresceins. Trying to find an easy combinatorial method for the construction of a rhodol library, an efficient synthetic protocol was developed with a catalytic amination reaction as the key step.68,69

Monoprotected fluorescein68,70–74 in the spiroform can be transformed into trifluoromethyl sulfonate 43 which then undergoes catalytic amination and deprotection to yield a large variety of rhodol derivatives 44a–p (Scheme 9). In the original method mono-phenol protection by MOM was employed with subsequent triflation and catalytic amination of the other phenolic group. This procedure works well for fluoresceins in their spiroform, where the carboxyl group is effectively protected. For 5-carboxyfluorescein, the carboxylic group was transformed into a t-butyl ester and the phenolic group was protected with benzyl chloride.75 This approach allows for the deprotection of the phenol or carboxylic acid functional groups without affecting each other. Similar to previous approaches used to prepare rhodols from fluoresceins through triflation, conversion of fluoresceins to iodoarenes with subsequent catalytic amination gives rhodols.76

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Scheme 9 General method for rhodol preparation via Pd-catalyzed amination starting from fluorescein.

The synthesis of rhodol 47 in 2 steps with 51% yield from Pennsylvania Green 45 exemplifies the utility of this methodology (Scheme 10). The synthesized rhodol 47 and its analogues possess fluorine atoms at the 2- and 7-positions for improved photophysical properties, and a hydrophobic methyl substituent instead of a polar carboxyl group for better cellular membranes affinity.

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Scheme 10 The method for rhodol preparation from corresponding fluoresceins via Pd-catalyzed amination.

Over the years quite a few synthetic strategies leading to rhodols have been developed. All of these approaches can be roughly divided into two major methodologies: (a) condensation of aromatic 2-hydroxyketones with resorcinol, its derivatives and analogs, and (b) synthesis of xanthones, followed by addition of organometallic reagents. It seems that the first strategy has been exhausted over the last century and one cannot expect further synthetic developments in this field. At the same time it is at present the most often used as it possesses the two significant advantages of a low number of synthetic steps and large overall yields. Consequently, it can be recommended for newcomers in the field who would like to reach their target without significant time investment. The ‘reverse methodology’ utilizing 2,4-dihydroxybenzophenones and 3-aminophenols as substrates is surprisingly underdeveloped especially taking into consideration that aromatic 1,3-dihydroxyketones are easier to obtain than analogous compounds bearing an R2N group. There is some room for optimization and use here.

It seems that the methodology utilizing xanthones is the most promising in terms of modular approach, generality and the broadest range of potentially available functionalities. At the same time, it is the longest one when considering the number of steps and therefore the poor overall yields. The simplification of this strategy is therefore a viable target for future research. Undoubtedly the most recommended pathway towards rhodols possessing a 2′-carboxyphenyl substituent at position 9, starts from easily available fluorescein with Buchwald–Hartwig amination of the MOM-protected triflate 43 as the key step (Scheme 9). This method contains a reasonable number of steps with the ability to introduce a broad range of amino groups.

Some of the published methodologies such as condensation with monothioresorcinol or reaction of aldehyde with 8-hydroxyjulolidine remained an exotic curiosity, typically due to the combination of low yields, unavailability of the starting materials or narrow scope.

3.2. π-Expanded rhodols

In addition to the aforementioned rhodol representatives, there is a family of functional organic dyes analogous to rhodols with a π-expanded chromophore system. Commonly, π-expanded rhodol-like chromophores are divided into two groups depending on the conjugation type (Fig. 4). Cooperative conjugation defines systems where charge transfer between the donor and acceptor moieties exists in a similar manner to the parent rhodol. Non-cooperative conjugation defines systems where the π-expansion obstructs the transfer in charge due to a change in relative orientation between the amine and carbonyl moieties. Formally, representatives from this second group do not belong to the rhodol family in terms of the electronic structure, though, as these compounds are prepared in similar ways to the former group, they will be considered in this synthetic section.
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Fig. 4 π-Expanded rhodol-like chromophores.
3.2.1. π-Expanded rhodols (cooperative effect). Undoubtedly the most well-known π-expanded rhodol derivatives are seminaphthorhodafluors (SNARFs), featuring rhodol-like molecules expanded with a naphthalene fragment instead of one phenyl ring. The synthesis of SNARF-2 49, presented in Scheme 11, exemplifies the method for seminaphthorhodafluor preparation discovered by Haugland and co-workers in 1991.77 1,6-Naphthalenediol 51 used for SNARF synthesis possesses hydroxyl groups in the correct orientation to reinforce conjugation which is crucial to secure the rhodol-like chromophore. Indeed, any naphthalenediols with an analogous configuration of the substituents tend to form rhodol-like chromophores. Subsequently, the method for the seminaphthorhodafluor preparation has been developed by other groups.78,79 Although, the vast majority of known SNARFs80 are prepared using an analogous procedures to that of Haugland, another approach, based on a mixed condensation of aldehyde and two phenol derivatives, was reported (Scheme 12).61,81 Despite the original chromophore structure and promising properties, classic SNARFs are not common fluorophores due to the complexity of substrates required.81–83 Initially, the name seminaphthorhodafluor and the abbreviation SNARF concerned only fluorophores – products of the reaction with naphthalenediol 51,77 nevertheless, in later reports the authors used the same name to refer to analogous π-expanded rhodols derived from other naphthalenediols, though with hydroxyl groups at reinforcing positions.
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Scheme 11 The synthesis of seminaphthorhodafluors.

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Scheme 12 The mixed method for SNARF preparation.

Researchers from another group found that using a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 mixture of CH3SO3H and TFA improved the yield of Haugland's method significantly. Scheme 11 shows the synthesis of the SNARF spiroform 53 from naphthalenediol 52.84 The former underwent ring-opening and the seminaphthorhodafluor 54 was obtained as the ester form. The protonated form of SNARF 54 equilibrates with its rhodol-like form 55.

Seminaphthorhodafluors can also be obtained with naphthalenediol 56 to give spiroform 57, which after esterification is in equilibrium with the rhodol form 59.32,85

Expansion of the π-system on the amino side of the rhodol as opposed to the hydroxyl is much rarer and leads to seminaphthorhodafluors with the reversed ring configuration.51,84 Compound 70 was obtained in 69% yield via the condensation of benzophenone 15 with 1-hydroxy-6-piperazyno-naphthalene 69 in neat TFA at 125 °C (Scheme 13).

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Scheme 13 The synthesis of naphthorhodol 70.

A recent report presents the synthesis of two π-expanded rhodols which cannot be described as seminaphthorodafluors as the additional benzene or naphthalene moieties are annulated to the rhodol chromophore without its lengthening. The reaction of benzophenone 5 and dihydroxynaphthalene 60 in neat TFA leads to the formation of the carboxylic acid intermediate, which was then transformed into ester 61 in order to facilitate purification (Scheme 11).34 Given the reasonably good access to the necessary building blocks this approach was also utilized for the synthesis of analogs possessing an additional fused benzene ring.34

3.2.2. π-Expanded chromophores (non-cooperative effect). The situation when benzophenone 5 reacts with 1,4-dihydroxynaphthalene 62 differs from the aforementioned SNARF synthesis as the product 63 does not formally contain the rhodol chromophore as the phenol moiety is not conjugated with the amine (Scheme 11). Although, chromophores with such a configuration are not technically speaking π-expanded rhodols,39 their synthesis is mentioned due to the formal structural similarity.

An extension of this concept is the replacement of the naphthalenediol 62 with the benzocoumarin 65. The resulting product was obtained in reasonable yield in the lactone form 66 and further transformed to its open form 67via esterification.86

3.2.3. V-shaped bis-xanthene dyes. Since the aryl moiety at position 9 is orthogonal to the xanthene framework, it hardly influences the photophysical properties of rhodol dye. In order to expand the π-system in this direction, V-shaped xanthene dyes with a quinone moiety mutually shared by two rhodols were designed.87 Compared to simple fluorescein, the V-shaped dye 71 has significantly red-shifted absorption and emission maxima, but possess poor solubility and a low quantum yield (Scheme 14). In attempts to mitigate these disadvantages, the rhodamine and rhodol like V-shaped fluorophores comprising one to three piperidine moieties were prepared.
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Scheme 14 Synthesis of V-shaped rhodol analogues.

The dyes 74–76 can be considered as π-expanded rhodols, although the π-expansion here is not due to the presence of additional fused aromatic rings, but rather the fusing of the benzene ring at position 9 to the core xanthene unit. The synthetic route proceeds through the formation of derivatives 72 and 73, generated from V-shaped fluorescein 71 (Scheme 14).88 In the case of a large excess of piperidine the amination of compound 72 leads to V-shaped rhodol 75 as a major product (53%) along with the regioisomer 76 and overaminated derivative 77 as minor products (10% and 5% respectively). In the neutral form these products exhibit a considerable increase in the fluorescence quantum yield and bathochromically shifted absorption and emission maxima with respect to 71 in DMSO.

The synthesis of both types of π-expanded rhodols mostly relies on the classical approach i.e. condensation of aromatic o-hydroxyketones with electron-rich dihydroxynaphthalenes. Needless to say this strategy limits the control over the pattern of substituents. Moreover, the number of dihydroxynaphthalenes which are commercially available is rather limited and this substrate pool has been exhausted already. On the other hand the synthesis of both SNARFs and less popular π-expanded rhodols is a straightforward two-step process from commercially available materials, which makes it appealing for researchers aiming at imaging applications. More importantly, certain key scaffolds are not available using the so far developed methodologies. Prominent examples of these are 97a,b which are shown in Fig. 5. The strategies employing xanthene analogs have not been attempted in this sub-field so far.

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Fig. 5 Structures of not yet synthesized π-expanded rhodols.

Consequently, new, more general methods are waiting to be discovered in order to avoid the drawback of the limited variety of substituents and cores.

3.3. Heteroatom substituted rhodols

The replacement of the bridging oxygen atom in rhodol with carbon, silicon or phosphorus is an efficient strategy to modify the photophysical properties (Scheme 8). Two methods for the synthesis of silicon analogues from the fluorescein TokyoMagenta89 and from the respective xanthone have been described.90 The coupling of 2-bromo-4-methoxybenzyl chloride (78) with 8-bromojulolidine (79) catalyzed by AlCl3 followed by silylation with dichlorodimethylsilane and subsequent oxidation provides silaxanthone 81. Subsequent reaction with lithium derivative of indole 82 and deprotection affords Si-rhodol 83 (Scheme 15). The indolyl moiety was then functionalized to produce a reaction-based fluorescent probe for the detection and imaging of formaldehyde in living cells.
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Scheme 15 The synthesis of Si-rhodols.

Fluorescein-like derivatives lacking the carboxyl group in the meso-aryl moiety do not require MOM protection thus allow a somewhat shortened synthetic route to rhodols.69,91,92 Tokyo Magenta dye 84 is transformed into Si-rhodol 86, via the monotriflated intermediate 85, forming in quantitative yields (Scheme 16).89

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Scheme 16 The method for Si-rhodol preparation via Pd-catalyzed amination.

In terms of pure synthetic accessibility, carborhodols represent the class of rhodol analogues that are the most difficult to synthesize. Indeed, their synthesis requires introduction of a carbon bridge to the asymmetrically substituted substrate. An eleven-step synthetic route to carborhodols starts from the coupling of 3-bromoanisole and 4-nitrobenzoyl chloride in the presence of a Lewis acid and subsequent reduction of the nitro group affording ketone 89.93 Since ketal protection proved challenging, the keto-group was fully reduced after methylation of the amino group. Subsequently the six-step process of ring-closure and arylation produces the methylated rhodol molecule which, after amidation followed by demethylation, gives rise to carborhodol 96 with an overall yield of 1.6% (Scheme 17).

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Scheme 17 The synthesis for C-rhodols.

The method of rhodol preparation through rhodamine hydrolysis5,91 is now generally considered obsolete and fallen from usage since it requires long reaction times and yields low conversion. However, the replacement of the bridge fragment with more electron-withdrawing moieties facilitates the hydrolysis of an amino group in rhodamine-like chromophores thus increases the potential utility of this method. Hydrolysis of P[double bond, length as m-dash]O-bridged rhodamines 98–101 occurs much faster than in conventional rhodamines and provides rhodols 102–105 in good yields (Scheme 18).94 Steric hindrance of the 9-position is crucial since it prevents nucleophilic attack of the hydroxyl ion at this position leading to discoloration under exposure to basic conditions. This is exemplified by the fact that the 2-tolyl-substituted P[double bond, length as m-dash]O-bridged rhodamine does not yield the corresponding P[double bond, length as m-dash]O-bridged rhodol, whereas the 2,6-dimethoxyphenyl derivative 98 efficiently provides to P[double bond, length as m-dash]O-bridged rhodol 102. The hydrolysis of these P[double bond, length as m-dash]O-bridged rhodamines to P[double bond, length as m-dash]O-bridged rhodols proceeds selectively, because rhodols precipitate from the reaction mixture preventing further hydrolysis. Full conversion to P[double bond, length as m-dash]O-bridged fluoresceins is much slower demanding more concentrated alkali solutions and prolonged reaction times. An exception to this is rhodol 104 formed as a water soluble sodium salt, which undergoes further hydrolysis easily.

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Scheme 18 The synthesis of P-rhodols via the rhodamine hydrolysis.

The synthesis of rhodols with an endocyclic selenium atom95 using an earlier developed methodology for Se-rhodamines has been reported.96,97 3-Bromo-N,N-diethylaniline (106) was transformed into the diselenium bridged intermediate 107, followed by the condensation with allyloxybenzamide 108 to form selenoxanthone 109 (Scheme 19). Subsequent arylation and cleavage of the allylic moiety afforded the spiroform of Se-bridged rhodol 110 in 20% yield.

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Scheme 19 The synthesis of Se-rhodols.

While the photophysical properties of core-modified rhodols are in most cases better compared with their parent chromophore, their synthesis is more complex. All developed synthetic routes are long and low-yielding. There is strong motivation to find shorter and more efficient strategies, especially to Si-rhodols and C-rhodols, but the problem is challenging and requires an inventive approach.

Since the first review detailing the synthesis of rhodols in 1922,3 new approaches based on modern organic and organometallic chemistry for the synthesis of these dyes have been developed and thoroughly investigated. Over the last 10 years, seen by an increased number of publications, rhodols have been attracting much more attention due to their application in bio-imaging. Still, the number of synthetic routes to the rhodol skeleton is very limited and warrants further study. Moreover, many potentially interesting scaffolds are yet to be synthesized using any methodology. No single heterocyclic analogue of rhodol exists possessing any carbon atom replaced by nitrogen, or a benzene unit with a five-membered heterocyclic ring for example.

4. Photophysical properties

4.1. Linear optical properties

The solvent polarity governs the contribution of the two limiting forms of merocyanines, which affects the position of the absorption maxima (Fig. 2). In the case of equal contribution of the nonpolar and dipolar limiting forms the chromophore approaches an ideal state with no bond alternation98 and the excited state geometry closely resembles the Franck–Condon geometry. The absorption spectrum of the chromophore with ideal conjugation shows a narrow band with minimal or without a vibronic mode.99 Rhodol molecules, as with the majority of merocyanine dyes, show positive solvatochromism where an increase in solvent polarity leads to a red-shift of the absorption maxima.100 DFT calculations confirm that in nonpolar media model rhodol 111 has an electronic structure with low charge separation, while in the polar media it exhibits a polarized structure close to the ideally conjugated state (Fig. 6).14,15,94,100 The effect of solvent polarity on the absorption spectrum can be clearly seen for 112 in Fig. 7.
image file: c9cs00166b-f6.tif
Fig. 6 TD DFT calculation data of model rhodol 111 for toluene, acetonitrile and water. Reprinted with permission from Chem. – Eur. J., 2017, 23, 13028–13032. Copyright © 2017, John Wiley and Sons.

image file: c9cs00166b-f7.tif
Fig. 7 The absorption spectra of rhodol 112 in solvents of different polarity. Reprinted with permission from J. Org. Chem., 2013, 78, 11721–11732. Copyright © 2013, American Chemical Society.

The majority of rhodol derivatives obtained from the two-step condensation method with phthalic anhydride15 contain the carboxyl group at the 2′-position of the meso-aryl substituent necessitating an intrinsic equilibrium between spiro (A) and open dye (B) forms (Scheme 20).

image file: c9cs00166b-s20.tif
Scheme 20 The equilibrium between the dye and the spiroforms for rhodol, rhodamine 110 and fluorescein.

Fig. 8 shows that the spiro form of rhodamine in dioxane readily makes the dye form with the minimal addition of water. For rhodol, the equilibrium becomes significant in a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 dioxane–water mixture. On the other hand, fluorescein mainly exists in the spiro form even in pure water.11

image file: c9cs00166b-f8.tif
Fig. 8 The dependence of equilibrium between spiro and open forms for rhodamine 110 (1, R = H), rhodol (2, R = H) and fluorescein (3) in dioxane–water mixtures. Reprinted from Zh. Org. Khim., 1965, 1, 343–346.

In basic conditions rhodols exist in the dye form (D) as the formation of the rhodol salt prevents ring closure, while in acidic conditions depending on the substituents the equilibrium shifts to either isomeric spiroform (A) or protonated form (C) (Scheme 20).11,25,37

Fig. 9 shows how polar substituents affect the optical properties of rhodol dyes with regard to both their nature and position. Moreover the photophysical data of selected rhodol dyes are summarized in Table S1 (ESI).

image file: c9cs00166b-f9.tif
Fig. 9 The influence of polar substituents on optical properties of rhodol dyes.

Unsubstituted rhodol (44a) exhibits an absorption maximum at 493 nm in water with a strong fluorescence response (Scheme 9). The addition of one alkyl substituent at the nitrogen atom (44b) increases the electron-donor strength of the amino group and leads to a small red-shift (10–15 nm) in both absorption and fluorescence maxima. In the case of an analogue of 44b fused with one cyclohexane ring (113a) the bathochromic effect is somewhat stronger (about 25 nm) due to restricted rotation of the amino group (Fig. 10).

image file: c9cs00166b-f10.tif
Fig. 10 Substituted rhodols.

The presence of second alkyl group (44e) leads to an additional bathochromic shift in both absorption and fluorescence maxima by another 10–15 nm. What is more, the position of the absorption and the fluorescence maxima for the isoelectronic analogues of rhodol 44e with cyclic alkyl substituents on the nitrogen atom (44i,j) hardly differ from that of 44e (Scheme 9).

On the other hand, the introduction of two fused cyclohexane rings (113b) gives rise to an additional bathochromic shift of the absorption maximum (about 50 nm compared with 44a) as the fused alkyl chains increase the rigidity of the chromophore and make polar conjugation more efficient. In the context of fluorescence efficiency, rhodols substituted with one alkyl group at the nitrogen atom (44b, 113a) demonstrate the strongest fluorescence response, which is comparable with that of unsubstituted rhodol 44a, while dialkyl amino derivatives, either linear (44e), cyclic (44i,j) or fused (113b) exhibit much weakened fluorescence efficiency. In contrast to N-alkyl rhodols, all N-arylated derivatives exhibit very low fluorescence signals (44m,n).

Introducing an electron-withdrawing group at the nitrogen atom leads to a reversed effect compared with mono and dialkyl substitution. The acylation of the nitrogen atom weakens the electron donating properties of the amino group resulting in a blue-shift of the absorption and emission (44p).

There is a number of known rhodol molecules substituted at the position adjacent to the carbonyl group. This sort of substitution weakly affects the rhodol chromophore and leads to a small bathochromic shift (about 5 nm) on both absorption and fluorescence (113c,d, 114a,b).

The optical properties of rhodols unsubstituted at the meso-position (115) fit this trend and its photophyscial properties resemble that of its analogue 113b. Nevertheless, dye 115 is more sensitive toward nucleophiles, which hinders the measurement of its fluorescence quantum yield.

Annulation of the rhodol chromophore with benzene or naphthalene rings (61, 118) results in a bathochromic shift of both absorption and emission, however, a direct comparison with 44e is not possible as spectra were measured in different solvents (Fig. 11). Compound 49 represents the generic structure of SNARF chromophores, which are vinylogues of rhodol chromophores. Expanding the rhodol system by additional benzene ring leads to 60 nm bathochromic shift of the absorption maximum. In addition SNARF chromophores are characterized by higher Stokes shifts which can suggest bigger differences of the ground and excited state geometries. Therefore, SNARFs show emission maxima close to NIR spectral range (Fig. 11).77,81

image file: c9cs00166b-f11.tif
Fig. 11 The structure of π-expanded rhodol analogues.

Compound 70 is a vinylogue of rhodol with the conjugation chain lengthened by a phenyl ring. The comparison of the absorption properties of 70 with 44j shows almost no difference in the maximum position. This can suggest that compound 70 mainly adopts a nonpolar structure with no charge separation. There is no fluorescence efficiency data for compound 70, however, the Stokes shift is much larger than that for 44j which often suggests low fluorescence quantum yield.

Within a series of bis-xanthene dyes, compounds 74-76 consist of the rhodol framework expanded with an additional π-system so that they represent a cross-conjugation between fluorescein–rhodol (74), two rhodols (75) and rhodamine–rhodol chromophores (76). Nevertheless, Fig. 11 shows that compounds 74–76 demonstrate almost identical spectroscopic properties comparable with annulated rhodol 118.

Recently, the introduction of a second electron-donating amino group at the neighbouring position to the first has become a viable way to modulate the photophysical properties (116–117, Fig. 11).53,58 This modification leads to a strong increase in the Stokes shift (120–140 nm) resulting from red-shift of the emission maxima (to ca. 660 nm). The absorption maxima remain in the spectral range typical of substituted rhodols. The most probable reason for this effect is that the dye molecule after excitation forms a charge transfer state.

A number of rhodol congeners with a bridging atom other than oxygen have been reported within the last decade. The study on the influence of polar substituents on the conjugation chain, and hence absorption spectra, of polymethine dyes including rhodamines is rationalized by the Dewar–Knott rule.101,102

According to this rule, electron-donating groups at the meso- and bridge positions result in a hypsochromic shift of the absorption maxima, while electron-withdrawing groups show the reverse effect. On the basis of this rationale, rhodol analogues with a more electron withdrawing ‘bridge’ than oxygen atom should show a red-shift in both absorption and fluorescence maxima while electron donating moieties should show a hypsochromic shift (Fig. 12).

image file: c9cs00166b-f12.tif
Fig. 12 Rhodol 119,37 carborhodol 96,93 Si-rhodol 120,103 P-rhodol 10394 and Se-rhodol 121.95

The position of both absorption and fluorescence maxima correlate with the average polar effect of the substituent. The electron-donor effect of oxygen bridge of O-rhodol 119 results in the somewhat blue-shift of the absorption maximum in comparison with unbridged chromophore 1 (Fig. 3).22 The increase of electron-withdrawing properties of the bridge atom leads to the bathochromic shift of the absorption and fluorescence maxima. According to Fig. 13 carborhodol 9693 demonstrates a significant red-shift which is the result of the absence of the resonance effect occurring for the O-bridge. At first glance Si-rhodol derivatives89,90,104,105 may be expected to have their absorption maxima slightly blue-shifted to that of carborhodol as silicon is less electronegative than a carbon atom. The truth is, however, that although the silicon atom is less electronegative compared to a carbon atom, an interaction of the chromophore LUMO with the pseudo-π* of the silicon bridge106,107 (σ*–π* hyperconjugation) decreases the LUMO energy thus resulting in the red-shift of both absorption and emission for derivative 121103 (Fig. 13).

image file: c9cs00166b-f13.tif
Fig. 13 Optical properties diagram for rhodol (119), carborhodol (96), P-rhodol (103), Si-rhodol (120) and Se-rhodol (121) in PBS buffer.

P-rhodols tend to have the most bathochromically shifted absorption and fluorescence among other known rhodol-like molecules as the P-containing ‘bridge’ affects the π-system by means of electron withdrawing resonance interaction and σ*–π* hyperconjugation similarly to Si-rhodols. Both effects result in a bathochromic shift of the absorption maximum, so that for compound 103 it almost reaches the NIR spectral region.94,108

Oxygen and selenium, both belonging to group 16, affect the chromophore in the same manner. A bathochromic shift of the absorption band for Se-rhodol 12195 compared to rhodol 119 is the result of weakened σ*–π* hyperconjugation with the larger Se atom. The presence of a heavy atom in the Se-rhodol structure also causes the quenching of fluorescence (Fig. 13). The small Stokes shifts for the rhodol-like structures 96, 103, 119–121 in such a polar medium as PBS buffer is due to the chromophores closely resembling the ideal polymethine state.98

4.2. Non-linear optical properties

In contrast to both rhodamines and fluoresceins, the asymmetric, more polarized structure of rhodol displays more efficient two-photon (2P) absorption σ2 due to increased intramolecular charge transfer (ICT). Only few reported rhodol chromophores have been examined with respect to 2P absorption and include 112, 122 and 124 (Fig. 7, 14 and 16).69,73,100,109 Rhodols feature moderate 2P absorption cross-sections (up to 165 GM), although this is much higher than analogous rhodamines and fluoresceins. Additionally, rhodols show strong fluorescence efficiency which leads to the 2P brightness (σmax2·Φ) high enough for 2P excited fluorescence microscopy experiments (Fig. 14).
image file: c9cs00166b-f14.tif
Fig. 14 1P and 2P absorption and fluorescence spectra for rhodol 112 in DCM. Reprinted with permission from J. Org. Chem., 2013, 78, 11721–11732. Copyright © 2013, American Chemical Society.

In summary, assuming the same pattern of substituents, the absorption and emission of rhodols is slightly hypsochromically shifted versus that of rhodamines and bathochromically versus fluoresceins. The position of both maxima are less pH-sensitive than for the two more popular chromophores. Except for SNARFs, the π-expansion of rhodol chromophore is not a viable pathway for realizing a notable bathochromic shift of absorption and fluorescence. The replacement of the oxygen atom in the bridging position with C, Si, Se and P turned out to be a much better strategy, with P-rhodols reaching record values (λem = 695 nm). Replacement of the oxygen atom bridge comes at a cost, however, and fluorescence quantum yields are lowered to 0.3 or below which leaves sizable room for further improvement.

5. Fluorescent imaging

5.1. Fluorescent reporters

Given the renewed interest in the use of small molecules as fluorescent probes,110–112 it is perhaps not surprising that the rhodol chromophore has become a popular scaffold for the design and implementation of fluorescence probes for a range of diverse biological studies. This is due to the many possible ways of functionalizing the rhodol moiety for different research purposes and to tune the photophysical properties of rhodol containing probes. Prior to the 21st century there were few published reports of rhodols acting as fluorescent sensors5 possibly due to the plethora of classic and popular dyes such as cyanines, rhodamines and fluoresceins. Nevertheless, the use of rhodol chromophores has been progressively increasing in popularity over the last two decades, especially in more advanced biological studies.

The rhodol chromophore features certain structural elements commonly employed for modulation of the optical properties of the probe depending on its application (Fig. 15). The adjustment of the bridge atom and π-expansion allow the spectral range to be tuned. The dye/spiro form tautomerism governs fluorescence on/off signaling.40,48–50,60,113,114O-Substitutions enforce the colorless spiroform, while the cleavage of the O-terminal group bond resumes the equilibrium and turns on rhodol fluorescence (Scheme 20).115–117 A combined approach using polar substituents at position 2, which allow excited state intramolecular proton transfer (ESIPT) between the hydroxy group of the spiroform and the polar substituent, enables another type of fluorescent signaling that allows switching between fluorescence regimes.118–120 Moreover, the terminal amino group is a linking position to introduce functional groups necessary for selective sensing or targeting.52,121,122 A range of rhodol probes employing a photoinduced electron transfer (PET) approach using either a one-photon (1P) or two-photon (2P) excitation regimes have been applied in fluorescence microscopy experiments and ratiometric fluorescence sensing.47,55

image file: c9cs00166b-f15.tif
Fig. 15 Rhodol structural features used in probe design.

As the polarization of the rhodol chromophore is a function of the external electric field stimulus, the study of neuron activity with a specifically designed rhodol probe has been achieved (Fig. 16).69

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Fig. 16 Rhodol probe used to study neuron activity.

Probe 122 features a rhodol chromophore modified with two chlorine atoms and a linear polyene substituent at the meso-position with a terminal electron-donor group. It exhibits absorption and emission in the range typical of rhodol dyes (λabs = 520 nm, ε = 83[thin space (1/6-em)]000 cm−1 M−1, λem = 535 nm, Φ = 0.27; PBS, pH 7.4). The presence of the polyene chain makes this probe voltage sensitive as PET from the dimethylamino group to the rhodol chromophore causes fluorescence quenching. Indeed, probe 122 intercalated the plasma membrane where it is influenced by the membrane potential, which governs the strength of PET. The fluorescence efficiency decreases under hyperpolarized cellular conditions (typical resting neuronal membrane potential) as PET strengthens. In contrast, cellular depolarization weakens PET and leads to the increase of the fluorescence response thus enabling the detection of fast-spiking action potentials in neurons (Fig. 17).69

image file: c9cs00166b-f17.tif
Fig. 17 Probe 122 voltage sensing via PET using 1P or 2P excitation. (a) At hyperpolarized or resting potentials fluorescence is quenched, whereas at depolarized potentials PET is inhibited by the electric field across the membrane, enhancing fluorescence. (b) Fractional change in fluorescence (ΔF/F) vs. time in a patch-clamped HEK cells under 2P illumination (820 nm), held at −60 mV and then stepped to potentials ranging from −100 to +100 mV in 20 mV increments. (c) Assessing differences in spontaneous activity in WT and Tsc1 KO neurons with 122. Wide-field fluorescence image of RVF5 staining in DIV 14 cultured Tsc1fl/fl mouse hippocampal neurons infected with a virus encoding mCherry-Cre to knock out the Tsc1 gene (Tsc1 KO). (d) Representative optical recordings of spontaneous activity in pairs of wild-type (WT, upper, black trace) and Tsc1 KO cultures (Tsc1 KO, lower, red trace). Arrowheads (▼) indicate periods of ‘burst’ firing in the Tsc1 KO neuron. Reproduced from ref. 69Proc. Natl. Acad. Sci. U. S. A., 2017, 114, 2813–2818.

The voltage sensitivity of 122 is comparable under both 1P and 2P conditions. Nevertheless, the improved 2P brightness compared with fluorescein-based voltage sensitive probes123 allows the application of 122 in thick tissue or brain samples. In addition, the high photostability of 122 enables the probing of neuronal activity in a mouse model of the human genetic epilepsy disorder Tuberous Sclerosis Complex (TSC), both in cultured neurons and in brain slices.

A number of reports demonstrate the design and application of metal ion probes employing both photoinduced electron transfer and the equilibrium between spiro and open forms of rhodol (Fig. 18). A rhodol chromophore functionalized with a BAPTA moiety (123) was developed to monitor spontaneous activity in cultured neurons and other intracellular processes connected with Ca2+ exchange. The formation of Ca2+ complexes leads to growth in the fluorescence response of sensor 123 due to a decrease in PET. In addition to conventional (1P) fluorescence microscopy techniques, microscopy experiments with 2P excitation were carried out and showed higher resolution fluorescence imaging (Fig. 19).73

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Fig. 18 Cation sensing rhodol based probes.

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Fig. 19 Live-cell imaging of histamine-evoked Ca2+ fluctuations with rhodol Ca2+ sensor 123. (a) Confocal fluorescence microscopy images (1P) of HeLa cells incubated with ester form of rhodol Ca2+ sensor (1.7 μM). Scale bar is 20 μm. (b) Quantification of intracellular Ca2+ concentration fluctuations measured in response to stimulation with histamine (5 μM). (c) 2P laser scanning fluorescence microscopy images of HeLa cells incubated with the ester form of rhodol Ca2+ sensor (1.7 μM). Scale bar is 20 μm. (d) Quantification of intracellular Ca2+ concentration fluctuations measured in response to stimulation with histamine (5 μM). Reprinted with permission from Biochemistry, 2018, 57, 237–240. Copyright 2018 American Chemical Society.

Copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining body weight and energy stores.124 A group of probes for Cu+ built upon O-, carbo-, Si- and P-rhodols 39–42 containing an identical sensing podand fragment were designed (Fig. 18). The podand moiety selectively binds with Cu+ resulting in the growth of the fluorescence response. The probes showed absorption maxima in the range 510–570 nm depending on the bridging atom with weak fluorescence. By means of fluorescence microscopy these probes could detect changes in labile copper levels in living cells (Fig. 20).65,67

image file: c9cs00166b-f20.tif
Fig. 20 Fluorescence imaging of labile copper pools in mouse embryonic fibroblast (MEF) wildtype (WT) and Atp7a−/− knockout fibroblast cells with 40. (a) MEF WT cells and (b) MEF Atp7a−/− knockout cells were stained with 2 μM 40 for 10 min in DMEM, and their average fluorescence intensity was (c) quantified. Scale bars: 40 μm. Data were normalized to MEF atp7a wt cells and shown as average ± s.d. (40, n = 4). Adapted with permission from ACS Chem. Biol., 2018, 13, 1844–1852. Copyright 2018 American Chemical Society.

Sensor molecule 124 uses an azacrown ether as the receptor to probe sodium cation concentration as the presence of Na+ leads to a growth in the fluorescence response (Fig. 18). In addition, chelation of the sodium ion in the crown ether cavity leads to an increase in the two-photon absorption (2PA) cross-section. Experiments with the 2P excitation show a twelvefold increase in the two-photon brightness of probe 124 in the presence of Na+.109

For the construction of novel Zn2+ targeting ratiometric indicators the rhodol chromophore was modified with azacrown ether and dipicolylamine receptors. Both 12554 and 12657 exhibit visible absorption maxima at 514 nm and emission maxima around 540 nm, respectively (Fig. 18). Zinc coordination in 125 prompts a decrease in the absorption maximum and an increase of the fluorescence response from 0.36 to 0.56. Rhodol 126 responds to zinc coordination through a blue-shift in absorption to 495 nm and a blue-shift in emission to 523 nm combined with a subtle loss in fluorescence efficiency (from 0.62 to 0.52).

The Tl+ flux assay coupled with a cell-permeable fluorescent indicator is used for imaging K+ channel activity as it is compatible with kinetic imaging-based on high-throughput screening assays. A series of rhodol based Tl+ sensitive probes with the general structure of 127 manifest themselves as versatile reagents for Tl+ flux assays (Fig. 18).75 Probe 127 (R = Me) localizes in the cytoplasm in an analogous manner to thallium targeting probe Thallos,125 yet shows better pH tolerance and somewhat red-shifted absorption and emission spectra. The acetyloxy group is hydrolyzed in the intracellular volume thus opening the spiroform which activates the rhodol chromophore. Binding of the thallium ion gives rise to a more than fourfold increase in the fluorescence response (Fig. 21).

image file: c9cs00166b-f21.tif
Fig. 21 Cellular localization of Thallos (a), 127 (R = Me) (b), and rhodamine 123 (c). Confocal microscopy images were obtained in HEK-293 cells co-expressing GIRK1 and GIRK2 following incubation with Thallos (1 μM), 127 (R = Me) (5 μM), or rhodamine-123 (1 μM) for 1 h and counterstained with Hoechst 33342 (1 μg mL−1). Scale bar = 10 μm. Reproduced from ref. 75 with permission from The Royal Society of Chemistry.

Using the ESIPT phenomenon simultaneously with spiro/dye tautomerism in rhodol-based probes offers a large advantage as it allows for the switching of fluorescence regimes. The ESIPT exclusively takes place in the closed form as only in that structure there is a phenolic OH group capable of acting as the hydrogen bond donor. The rhodol-based probe 128 in the absence of Hg2+ or ClO ions exists in a hydrogen bond stabilized spiroform (Scheme 21).126 The mutual configuration of the benzothiazole moiety and the hydroxyl group leads to a blue emissive chromophore due to ESIPT. The presence of Hg2+ or ClO ions results in the activation of the rhodol chromophore and turns on fluorescence at 590 nm or 595 nm in 129 and 130, respectively.126

image file: c9cs00166b-s21.tif
Scheme 21 Sensing mechanism of 128 for Hg2+ and ClO.

Besides several other reports on mercury ion sensing,127–131 Chen et al. demonstrated further development of sensor 128. Compound 131 features the ability to selectively sense Hg2+ and Ag+ ions through activation of the rhodol chromophore from its spiro form or through aggregation-induced emission (AIE), respectively (Scheme 22).132 Sensor molecule 131 reacts with a mercury cation which promotes the formation of an oxadiazole moiety and the rhodol chromophore 132 with a fluorescence maximum at 595 nm. In contrast, silver cations form a complex 133 with the sensor molecule in such a manner that significant stacking occurs and AIE with a fluorescence maximum at 520 nm is observed.

image file: c9cs00166b-s22.tif
Scheme 22 Sensing mechanism of 131 for Hg2+ and Ag+.

Molecule 134 is another rhodol probe for sensing metal ions based on the reaction of the spiroform functionality (Scheme 23). This sensor, for the intracellular fluorescence imaging of copper(I) ions features a phosphonium functional unit which facilitates mitochondria targeting. In turn, the formation of the tripicolylamine copper complex cleaves the terminal C–O bond thus recovering the emissive rhodol chromophore from its spiroform.36

image file: c9cs00166b-s23.tif
Scheme 23 Sensing mechanism of 134 for Cu+.

Employing various rhodol structural features in one sensor molecule makes it possible to construct polyfunctional fluorescent probes. A combination of the ESIPT and the labile O-substituted chelating group, which governs the spiro/open form equilibrium, allows for the design of dual function fluorescence probes for separate sensing of glutathione and cysteine/homocysteine,83 the most abundant biological thiols. Rhodol probe 136 contains a benzothiazole moiety adjacent to the carbonyl group along with a nitrophenyl moiety (Scheme 24).133 In the free form probe 136 shows dimmed fluorescence as the nitrophenyl moiety causes fluorescence quenching. The addition of a Cys moiety to probe 136 in DMF/buffer solution increases the emission at 587 nm as transthioesterification removes the nitroaryl moiety thus terminating PET-induced fluorescence quenching. Next, the intermediate product experiences intramolecular S,N-acyl shift followed by cyclization to the spiroform 137. In the spiroform, the benzothiazole moiety in combination with the phenol group, enables the ESIPT with fluorescence at 454 nm. Since the cyclization is a reversible process, the fluorescence spectra after addition of Cys or Hcy, displays a strong band at 454 nm accompanied by weak emission at 587 nm. In the case of GSH the reaction stops at the transesterification step (138) as rearrangement is not possible, thus the addition of GSH leads to the growth of fluorescence at 587 nm.

image file: c9cs00166b-s24.tif
Scheme 24 Sensing mechanism of 136.

In probe 139, the spiroform of rhodol is secured by a propargyloxy group. In the presence of Au3+ ions intramolecular cyclization to 140 with a fused furanoxanthylium chromophore occurs showing strong absorption and fluorescence maxima at 493 and 526 nm, respectively (Scheme 25).134 Due to the dramatic increase in emissive strength, a very high sensitivity for Au3+ is achieved (7 ppb).

image file: c9cs00166b-s25.tif
Scheme 25 The cyclization of 139 induced by Au3+.

5.2. Biomolecule targeting

The design of probes where an enzymatic activity restores the rhodol chromophore from its spiroform has been found to be a promising approach for targeted imaging.9 In the following series of studies, a rhodol chromophore was chosen for the visualization of metastases originating from ovarian cancers which show an enhanced enzymatic activity of β-galactosidase in comparison with normal ovaries. The terminal oxygen atom of the rhodol spiroform was modified with a β-galactopyranoside group to image cells with an increased activity of β-galactosidase as here the rhodol would be converted to the active (open) form in the cytoplasma of certain cells (Scheme 26).37,135 An analogue of β-galactosidase targeting probe 141, Se-rhodol 142, contains all the functionality of probe 141, however, it exhibits a weak fluorescence response due to deactivation of the excited state via ISC to a greater extent.95 The main application of dye 142 is rather in the phototherapy of cancer cells, where the formation of a triplet state is key to its functionality. The advantage of photosensitizer 142 is that the Se-rhodol is activated only in cells with the targeted enzyme, suppressing undesired phototoxicity (Fig. 22).
image file: c9cs00166b-s26.tif
Scheme 26 Sensing mechanism of 141.

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Fig. 22 Se-Rhodol photosensitizer.

Probe 141 detects β-galactosidase activity in cultured cancer cells although it does not give adequate resolution due to high background fluorescence (Table 1).37 On the basis of DFT calculations Urano and co-workers optimized the structure of probe 141 to shift the equilibrium towards the non-fluorescent spiroform. Within a series of rhodol-based derivatives probe 145 demonstrates superior fluorescence reporting properties as at pH 7.4, more than 99% of the probe exists in the spiroform thus weakening background fluorescence.136 The detection limit of probe 145 to intracellular β-galactosidase activity in diverse ovarian cancer lines is very low and visualization of metastases as small as 1 mm in vivo can be achieved.

Table 1 Properties of β-galactosidase fluorescence probes136

image file: c9cs00166b-u1.tif

R1 R2 pKcycl Fluorescence enhancement by β-galactosidase (fold)
141 Et Et 6.9 76.137
143 Et H 6.3 313
144 (CH2)2CF3 H 5.6 582
145 CH2CF3 H 4.5 1420

While enzyme targeting probe 141 shows high selectivity of staining, the main drawback is that the activated fluorescence chromophore tends to leak out of cells during prolonged incubation. The next generation of β-galactosidase targeting probes were designed in a manner so that after activation, the chromophore would be immobilized inside a cell. In addition, cellular studies on probes synthesized with anchoring moieties such as 146 revealed that they show enhanced fluorescence activation (Scheme 27).137

image file: c9cs00166b-s27.tif
Scheme 27 Sensing mechanism of 146 and 147.

To extend the utility of β-galactosidase probes with single-cell resolution in the red region, a Si-rhodol based β-galactosidase targeting probe was designed. Probe 152 emits at 638 nm in the open form and demonstrates all the sensing properties of 146 (Fig. 23). It can label cells of interest in combination with GFP markers and imagine multiple cell types at single-cell resolution in living samples.103

image file: c9cs00166b-f23.tif
Fig. 23 The structure of 152.

Further development of specific cell targeting for analytical purposes requires the selective transport of therapeutic agents to a particular cell. The structure of the rhodol motif allows for the combination of cell targeting, drug release and activation of the chromophore to control the therapeutic effect. Rhodol based prodrug 153 consists of the biotin subunit as a moiety to provide tumor localization,138 and the topoisomerase I inhibitor SN-38139 connected via a linker with disulfide bonds. When prodrug 153 is localized in cancer cells it reacts with intracellular thiols to release SN-38 (155), one of the most efficient therapeutic agents used to treat various carcinomas, and activate the open form of rhodol probe 154 to monitor cytotoxicity (Scheme 28).140–142

image file: c9cs00166b-s28.tif
Scheme 28 Rhodol based prodrug 153.

Probe 156 combines a rhodol chromophore in the spiroform modified with an arginine–glycine–aspartate motif (cRGD), which targets αvβ3 integrin overexpressed cancer cells. In addition, the probe 156 consists of a photosensitizer motif, which due to AIE143 shows an emission maximum at 650 nm with a fluorescence quantum yield of 0.13 in a DMSO/water mixture and plays the role of an imaging agent to monitor endocytosis. When irradiated, probe 156 generates singlet oxygen and cleaves the aminoacrylate linker to release the green emissive rhodol 157 for in situ monitoring the singlet oxygen generation during PDT (Scheme 29).

image file: c9cs00166b-s29.tif
Scheme 29 Rhodol based targeting agent 156.

Probe 159 demonstrates a rare instance where the rhodol chromophore is the targeting motif itself (Fig. 24). The fluorinated hydrophobic rhodol 159, containing an electrophilic nitrofuran moiety, was revealed to accumulate in the endoplasmic reticulum (ER) due to the presence of the rhodol, and cause inhibition of the protein p97 of the ER.76 Thus the fluorinated rhodol motif was found to be suitable for the transport of small molecules to the ER.

image file: c9cs00166b-f24.tif
Fig. 24 Rhodol based agent 159.

5.3. Rhodol based redox probes

The design of fluorescence probes for the study redox processes in biological systems can employ the rhodol motif as it allows versatile modification of the structure combined with excellent optical properties.

The sensing effect of the probe 160 relates to the position of the indoloquinone LUMO depending on the redox state. In the conjugate 160 the LUMO of the indoloquinone possesses a lower energy compared to the rhodol chromophore (Fig. 25).

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Fig. 25 Redox probe 160.

As the result, probe 160 shows very weak fluorescence (λem = 550 nm, Φ = 0.0098 in water) as the emission is quenched by the electron transfer from the rhodol LUMO to the indoloquinone LUMO. In the cellular imaging of a human lung adenocarcinoma cells, A549, the reaction of probe 160 with NADPH:cytochrome P450 reductase under hypoxic conditions (0.02% oxygen) releases free rhodol chromophore which shows strong fluorescence. Under aerobic conditions (20% oxygen) probe 160 shows no fluorescence growth.144

Probe 161 features a ubiquinone moiety at the meso-position which makes this reporter sensitive to NAD(P)H (Scheme 30). It shows a strong fluorescence response for the quinone form (λem = 518 nm, Φ = 0.733 in PBS buffer), while in the reduced form fluorescence of dye 162 is quenched. The fluorescence microscopy of HeLa cells visualizes the reaction of quinone probe 161 with NAD(P)H in the presence of artificial promoter [(η5-C5Me5)Ir(phen)(H2O)]2+ which gives rise to an 8.6 fold decrease in the fluorescence efficiency over 10 min.12

image file: c9cs00166b-s30.tif
Scheme 30 Rhodol based redox probe 161.

Despite the large structural similarity of rhodols 44m, 44n68 and 163 these compounds show remarkably different fluorescence efficiency. Indeed, derivative 163 without N-aryl substituent is an efficient fluorophore, whereas both 44m and 44n show a fluorescence signal close to the background noise level (Fig. 26).

image file: c9cs00166b-f26.tif
Fig. 26 Peroxynitrite sensitive rhodol probes 44n,m and fluorophore 163 formed in the sensing process.

This difference in fluorescence response was used to design two families of rhodol-containing probes for the imaging of peroxynitrite (ONOO), a potent oxidant generated in cells from the reaction between nitric oxide (NO) and superoxide (O2˙) (Scheme 31). This species contributes to tissue injury in a number of human diseases. Probes 163 and 164 are sensitive to the peroxynitrite species which cleave the N-aryl bond to form highly fluorescence rhodol derivatives 114a and 165. These probes demonstrate a great tolerance to reactive oxygen species and reactive nitrogen species (ROS and RNS) such as H2O2, 1O2, ˙NO, O2˙, ROO˙ and very weak sensitivity toward HOCl and ˙OH. Still the fluorescence response over ONOO for probe 164 is more than 18 times higher than those for HOCl and ˙OH.70,72,105,145

image file: c9cs00166b-s31.tif
Scheme 31 The mechanism for peroxynitrite sensing of probes 163 and 164.

As a probe for nitroxyl (HNO), which influences both the physiological and pathological processes in mammalian organisms and mediates their immune system response, a rhodol chromophore was modified with a triarylphosphine moiety. The reaction of rhodol probes 16671 and 16759 with HNO selectively cleaves terminal ester bond converting them into fluorescent open forms (Scheme 32).

image file: c9cs00166b-s32.tif
Scheme 32 Nitroxyl sensing probes 166 and 167.

Fluorescence visualization of intracellular H2O2 employs the modified boronate-protected xanthene chromophore in the spiroform 168, which, in the presence of hydrogen peroxide cleaves the C–B bond to turn the chromophore into the fluorescent open form rhodol 8. This probe, within a range of diverse xanthene-based multicolor probes,28 shows a selective turn-on fluorescence response to H2O2 in live-cell imaging assays over a range of biologically relevant ROS (Scheme 33).45

image file: c9cs00166b-s33.tif
Scheme 33 H2O2 Sensing probe 168.

Endogenous hypochlorous acid is a biologically important oxidative species. Sensors for the hypochlorite anion have been designed that contain the Schiff base fragment derived from formylrhodol. The barely fluorescent probe 169 (Φ = 0.04) displays a high sensitivity and selectivity toward ClO to form formylrhodol 170 accompanied by an increase in fluorescence (Φ = 0.35). Probe 169 shows a high efficiency of in vivo imaging ClO in living mice (Scheme 34).146,147

image file: c9cs00166b-s34.tif
Scheme 34 Probe 169 for in vivo imaging of ClO.

As rhodol dyes demonstrate high photostability69 these molecules are suitable for super-resolution imaging techniques i.e. stimulated emission depletion (STED)93 and photoactivated localization microscopy (PALM). Rhodol 171 is sensitive to light irradiation as it experiences light-induced Wolff rearrangement.

The photoproduct 173 consists of a seven-membered ring and demonstrates a very weak fluorescence response. Nevertheless, the removal of the acetyl group by carboxylesterases leads to hydroxyl derivative 172 which upon the light irradiation (depending on the wavelength) gives rise to either photoproduct 174 or 175, both of which show bright emission (Scheme 35). Rhodol derivative 171 is an efficient reporter for probing esterase activity in live cells using PALM imaging technique as this molecule does not diffuse substantially from its activation site, allowing precise localization of the enzymatic event.148

image file: c9cs00166b-s35.tif
Scheme 35 The efficient reporter for probing enzymatic activity in live cells using PALM imaging probe 171.

Scientists are chiefly attracted to rhodols over related xanthene dyes due to the combined beneficial aspects of better stability, strong fluorescence, their dipolar nature, better pH tolerance, higher sensitivity of fluorescence intensity to structural changes and the fact that they are comparatively under-explored. Importantly, they retain the advantage of lactone-forming switchability i.e. the transformation of the colorless spiro tautomer into the fluorescent dye (in analogy to fluoresceins and rhodamines). The nitrogen and oxygen atoms of rhodol are often used as linking positions to introduce active functional groups, which can govern fluorescence signaling in biological media and introduce extra functionality. Moreover, these features enable probes based on rhodol and its heteroanalogues to be used in super-resolution fluorescence microscopy. Paradoxically the very fact which is responsible for their relatively complex synthesis is also responsible for the ability to introduce various functionalities required for sensing. This in turn affords the construction of truly complex probes. In addition to the modular and adjustable design of the rhodol chromophore, enhanced two-photon absorption compared to other dyes commonly used in fluorescence imaging (i.e. rhodamines, fluoresceines, coumarins and BODIPY) enable the design of multifunctional fluorescence probes for sensing a range of ions, imaging thick tissue, biomolecules, intracellular redox processes, sensing enzymatic activity, intracellular targeting and intracellular drug delivery.

6. Summary and outlook

In 1990, it would have been impossible to imagine that within 30 years the chemistry of rhodols would expand to create an independent field of study. It can be hypothesized that such developments will continue with the recent emergence of various core-modified and π-expanded rhodols.

The synthesis of rhodols, unlike rhodamines and fluoresceins, still utilizes the most classical methodology i.e. the reaction of anhydrides with aminophenols followed by the reaction of a second nucleophile. Plausibly, this is related to the fact that many probes rely on the ring-opening/ring-closure transformation of the carboxyl group intrinsic to this synthesis. The accumulated knowledge of rhodol dyes over the last 130 years has allowed thorough analysis of their structures and properties. Bathochromic and hypsochromic shifts can be readily achieved through judicious inclusion of functional groups or heteroatoms, and fluorescence efficiencies through amine substitution. As a result, a number of functional biological probes based upon the rhodol core have been prepared. Among rhodols and their analogues, a particularly promising and beneficial feature with respect to both rhodamines and fluoresceins is their dipolar nature, as this allows operating voltage sensitivity for in brain imaging without additional functional groups in the one- and two-photon excitation regimes. Interest in rhodols has shifted in the last few years from generic fluorescent dyes towards cutting-edge applications related to fluorescent imaging chiefly thanks to the work of Chang, Miller, Lavis, Rivera-Fuentes and Urano. Appreciation of rhodols’ physicochemical properties have made them attractive scaffolds for fluorescence imaging. The possibility of flexible modification of the rhodol chromophore allows the design of multifunctional fluorescence probes for sensing intracellular redox processes, enzymatic activity, intracellular targeting, intracellular drug delivery, sensing of diverse biomolecules etc. Enhanced 2P absorption along with the high photostability of rhodol-like chromophores prompts applications related to fluorescence sensing of the membrane potential in cells. In addition, it enables the usage of probes based on rhodols and their heteroanalogues for state-of-the-art imaging techniques such as super-resolution fluorescence microscopy.

Needless to say a great deal of an additional effort is required to fully explore the possibilities of these molecules. We hope that this Review, in addition to organizing knowledge on this topic, will serve as a catalyst to spark further studies. Possible targets may be more densely substituted rhodol chromophores, modified in such a way to use the many benefits of this fluorescence platform in new fields of application. For practical applications, it is important to seek a balance between high fluorescence efficiency, large Stokes' shift and photostability. We believe that many scientific problems can be resolved and many yet unknown compounds can be discovered with the help of this Review and that some of them will find their way towards practical applications. The analysis of research performed within the last twenty years suggests that the variety of future compounds will be only limited by our imagination.

Conflicts of interest

There are no conflicts to declare.


We thank for financial support from the Foundation for Polish Science (grants FNP TEAM POIR.04.04.00-00-4232/17-00 and FNP TEAM POIR.04.04.00-00-3CF4/16-00) and from Global Research Laboratory Program (2014K1A1A2064569) through the National Research Foundation (NRF) funded by Ministry of Science, ICT & Future Planning, Korea.

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Electronic supplementary information (ESI) available. See DOI: 10.1039/c9cs00166b

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