An open and shut case? Chemistry to control xanthene dyes

Abstract

Fluorescent dyes are an indispensable part of the scientific enterprise. Xanthene-based fluorophores, like fluorescein and rhodamine, have been in continual use across numerous fields since their invention in the late 19^th century. Modern methods to synthesize and expand the scope of xanthene dye chemistry have enabled new colors, enhanced stability, and improved brightness. Modifications to the 3-position of xanthene dyes have been, until recently, less well-explored. Here, we discuss how small changes to the identity of the substituent at the 3-position of fluoresceins and rhodamines can profoundly alter the properties of xanthene dyes, with the potential to unlock new applications at the interface of chemistry and biology.

Introduction

The xanthene dyes fluorescein and rhodamine (Figure 1a) are among the most widely used fluorophores in chemistry, biology, and biomedical research.¹ The syntheses of fluorescein and rhodamine were first reported in the late nineteenth century, and access to these small, highly fluorescent molecules and their derivatives have enabled applications in basic and applied research as well as in clinical settings.^[3-7] Derivatives of fluoresceins have been co-opted for use as responsive indicators or sensors, reporting the presence of ions, metabolites, or cellular viability. Modulating the brightness of fluorescein derivatives in response to changes in the local environment can be achieved in a number of ways; among the most common are alkylation of the phenolic oxygen at the 3' or 6' position, controlling lactonization, or by changing the rate of non-radiative decay via photoinduced electron transfer (PeT).

Figure 1.

Chemical modification of xanthene fluorophores. (a) general structures of 3-carboxyfluorescein and 3-carboxyrhodamine. (b) amine and 10’ substitution influence photophysical properties of xanthene fluorophores. (c) examples of 3-substitutions that maintain orthogonality between the pendant ring and xanthene core and have large implications on subsequent applications.

Other synthetic modifications to the identity of the 3' and 6' substituents dramatically change the properties of xanthene dyes. When the oxygen of fluorescein is replaced with nitrogen, the resulting rhodamines dyes display red-shifted excitation and emission profiles, remarkable photostability, and pH-independent fluorescence emission.² Systematic exploration of the substitution pattern of the 3' and 6' nitrogen of rhodamine dyes has revealed generalizable methods for improving the brightness and/or photostability of containing rhodamines through restriction of C-N bond rotation,³ electron-withdrawing groups,^4-5 or deuteration (Figure 1b).^{3, 6}

More recently, heteroatom substitution at the 10' position, generating carbon-,^7-9 silicon-,¹⁰ phosphorous-,^11-13 or sulfur-containing¹⁴ xanthene dyes have pushed the excitation and emission spectra of these dyes even farther into the red and near infrared range (Figure 1b). This avoids phototoxicity and autofluorescence associated with short wavelengths of light. The proliferation of long-wavelength xanthene dyes has been a boon to areas like fluorogenic labeling, voltage-sensing, in vivo imaging, and super-resolution microscopy.

Substitution at the 3 position of the pendant ring of xanthene dyes is essential for a high quantum yield. Although a carboxylic acid at the 3 position is the traditional substituent, ground-breaking work showed that an important role of the 3-substituent is to maintain orthogonality between the xanthene chromophore and the pendant benzene ring.¹⁵ In other words, as long as there is some substituent to restrict free rotation of the pendant ring, a high fluorescence quantum yield can be maintained. This was demonstrated beautifully in the case of TokyoGreen, which bears a methyl substituent at the 3-position and possesses a large fluorescence quantum yield.¹⁵ Since that time, a number of new chemical functionalities at the 3-position have been introduced (Figure 1c).¹⁶ These modifications have a substantial influence on the chemical properties and behavior of xanthene fluorophores, while the orthogonality of the ring systems leaves the fundamental photophysical properties of the fluorophore relatively unperturbed.

Here, we review the implications 3-subsitution can have on the properties of xanthene fluorophores and the subsequent biological applications for which they may be used. We first discuss carboxy-substituted xanthenes and the ways in which dynamic cyclization opens new opportunities. Then we discuss non-carboxy substitutions that display dynamic equilibria before moving to examples of fluorophores that remain in the open configuration.

Carboxy 3-substitution

A key feature of xanthene fluorophores brought about by the presence of the 3-carboxylate is the propensity to spirocyclize into a closed, colorless lactone (Figure 2a). This dynamic equilibrium is sensitive to several environmental factors, including pH, polarity and temperature. The fluorogenic nature of the closed-open equilibrium has been fundamental for developing fluorescent tools with unprecedented contrast.

Figure 2.

Applications of 3-carboxy substituted xanthene fluorophores. (a) Spirocyclization equilibrium of fluorescein. (b) Chemical structure of fluorescein diacetate. (c) Enzymatic, reactivity based and photoactivated fluorogenic uncaging of xanthene fluorophores. (d) General method for fine-tuning K_L-Z of rhodamines by xanthene or pendant ring substitution. (e) Fluorogenic turn on of rhodamines upon protein labelling. (f) Structures of rhodamines with K_L-Z constants altered via through fluorination. (g) Neighboring groups profoundly shape the K_L-Z equilibrium.

Fluoresceins predominantly adopt the open, fluorescent form under physiological conditions. Chemical trapping of the closed lactone is a powerful method for developing fluorogenic sensors.¹⁷ Acetylation of phenolic oxygens is perhaps the most common strategy for locking fluoresceins in the closed form. For example, fluorescein diacetate (Figure 2b) is a versatile cell viability reagent. It readily diffuses across biological membranes where cellular esterases uncage the fluorophore rendering it fluorescent (Figure 2c).¹⁸ Expansion of this strategy, including and beyond esterases has led to a plethora of reagents facilitating the measure of specific enzymatic activity in complex systems or site selective uncaging at subcellular precision.^{17, 19}

Rhodamines can also be locked in the closed form. Dipeptidyl rhodamines have served as important substrates for measuring protease activity. Beyond enzymes, reactivity based fluorogenic uncaging enables the fluorescent detection of diverse biological analytes, including metal ions, reactive oxygen species and endogenous nucleophiles (Figure 2c).²¹

The dynamic equilibrium between the fluorescent zwitterion (Z) and lipophilic lactone (L) forms of rhodamines (Figure 2d) has profound implications on their biological imaging applications. Those with high K_L-Z values, and thus a preference for the fluorescent zwitterion are environmentally insensitive making them useful for bioconjugation, but their lack of cell permeability often limits their intracellular use. The K_L-Z values of 3-carboxy siliconrhodamines, or rhodamines with Si – usually dimethyl – at the 10' position (SiRs), are sufficiently low that the closed lactone predominates in aqueous environments, facilitating passage through lipophilic cellular membranes. However, upon binding to biological targets, the local environment shifts the equilibrium to the open fluorescent zwitterion (Figure 2e).²² This fluorogenic turn on is highly attractive since background fluorescence is minimal. This leads to exceptional contrast in labelling experiments without the need to remove excess fluorophore. Combining SiRs with covalent targeting ligands such has HaloTag or SNAPTag has led to widespread use in the labelling of many intracellular structures and live-cell super resolution microscopy.²³ Additionally, this environmental sensitivity has led to the development of chemi-genetic probes of voltage and calcium dynamics in which changes in local protein structure in response to changing [Ca²⁺] or membrane potential modulate fluorescence output through the K_L-Z equilibrium.²⁴

More recent advances have sought to adjust the K_L-Z equilibrium of 3-carboxy rhodamines and expand the color palette of fluorogenic labelling probes.^{22, 25} The K_L-Z equilibrium position is determined by a balance between the electronics of the xanthene fluorophore and the nature of the pendant ring 3-substituent. Computational approaches provide a theoretical framework for assessing the magnitude of K_L-Z for a number of xanthene types.^26-27 Paired with the growing body of experimentally-determined open-closed positions for xanthene dyes, this can be a powerful approach for dialing in the desired K_L-Z parameter for a specific application.

The K_L-Z equilibrium inversely correlates with the electron-richness of the xanthene. Electron-rich rhodamines, with shorter wavelength excitation, usually exist as the open zwitterion. Incorporation of electron withdrawing groups on terminal azetidines, or fluorination of the xanthene core decrease K_L-Z, improving tissue permeability and fluorogenicity of shorter wave rhodamines (Figure 2d, f).^{22, 25} Another strategy is to use an amide at the 4 position to stabilize the closed lactone through a neighboring group effect (Figure 2g).²⁸ On the other hand, long wavelength, near infrared 3-carboxyrhodamines (with P or S at the 10' position) often exhibit prohibitively low K_L-Z values with insufficient tendency to open, rendering them essentially unusable for fluorescence imaging.²⁹ Fluorination of the pendant ring shifts K_L-Z to values within the threshold for super resolution imaging applications (Figure 2d, f).²⁹

Non-carboxy substitution: Nucleophiles

Substitution of the 3-carboxylate for nucleophiles such as hydroxymethyl, aminomethyl or mercaptomethyl can further shift the equilibrium to the closed colorless spirocyclic form (Figure 3a).^30-31 Interestingly, these fluorophores tend to spontaneously switch to the open fluorescent form in a transient manner. The thermal equilibrium of this spirocyclization and the lifetime of the open form are parameters that influence the blinking behavior and are finetuned by the nucleophilicity of the 3-subsituent and electrophilicity of the xanthene fluorophore.³⁰ 3-hydroxymethyl siliconrhodamines, HMSiRs (Figure 3b) have gained particular attention as spontaneously blinking fluorophores for single molecule localization microscopy (SMLM) applications.^{23, 30, 32-33} Under physiological conditions, HMSiRs exist predominantly (>99%) in the closed spiroether and the stochastic switching to the fluorescent form facilitates the construction of super resolution images (Figure 3c). This mitigates the need for any fluorophore activation with UV light or strongly reducing buffers required in some SMLM techniques.³⁴ A detailed understanding of blinking kinetics of HMRs has led to the expansion of the color pallet for multicolor super-resolution imaging.³⁵ Fluorogenic bioconjugation has further enhanced the applications of these fluorophores.³⁶

Figure 3.

(left) 3-nucleophile substitution for super resolution microscopy. (a) Equilibrium of spontaneously blinking fluorophores influenced by the pK_cyc and lifetime, τ spent in the “on” form. (b) Chemical structure of HMSiR and properties suitable for single-molecule localization microscopy. (c) Schematic of single-molecule localization microscopy with spontaneously blinking fluorophores. (right) Modular tuning of spirocyclization with 3-amide functionality. (d) Equilibrium between open fluorescent and colorless spirolactam of 3-amide substituted rhodamines. (e) Modular amide substitution influences spirolactam equilibrium for various applications.

3-hydroxymethyl rhodamines and rhodols have been incorporated into fluorogenic sensors by chemical trapping of the spiroether with substrates for enzymes such as glycosidases and peptidases. This has led to a range of indicators for visualizing enzymatic upregulation in a variety of cancers such as breast, pancreatic, and lung cancer, demonstrating the promise for optical discrimination of benign vs. malignant tumors.^37-38 Tuning of the spirocyclization equilibrium can be accomplished through substitution of the xanthene terminal amines and plays an important role in optimization of the fluorescent turn-on upon enzymatic uncaging.³⁹

Non-carboxy substitution: Amides

Modification of a 3-carboxylate to an amide dramatically alters the K_L-Z. 3-amide substitution shifts the equilibrium of rhodamines to the closed lactam (Figure 3d).^{2, 40} While carboxy-rhodamines are pH insensitive, 3-amidorhodamines spirocyclize in a pH dependent manner and exhibit spontaneous blinking behavior at physiological pH.⁴¹ Modification of the amide nitrogen facilitates a variety of ring-opening properties. For example extended conjugation finetunes the K_L-Z, photoswitching modules or phthalimides enable photoactivation,^42-43 while trifluoroethanamide substitution increases the probability of the open form of spontaneously blinking dyes (Figure 3e).⁴⁴

3-amides can be functionalized in a modular manner to alter the spirocyclization equilibrium (Figure 3e). Functionalization with electron withdrawing moieties such as sulfonamides enables precise adjustment of the spirolactam equilibrium for applications in fluorogenic labelling and super resolution imaging. ^44-45 In contrast to the finetuning approach using varying nucleophiles (discussed in previous section), the modular nature of amide substitution facilitates more precise control over K_L-Z across a larger range. The absorption and emission wavelengths remain relatively unperturbed in this approach, since the xanthene core is unmodified and is easily adaptable to other xanthene fluorophores thereby providing improved generalizability. Finally, amidorhodamines are readily synthesized from carboxyrhodamines, and the synthetic feasibility of this approach complements its modularity.

Fluorogenic spirolactam ring opening has been the basis of an abundance of fluorescent probes for specific detection of analytes and ions. Owing to the modularity of amide functionalization, often a chelator is appended and upon ion binding a shift in equilibrium to the open form results in a fluorescence turn on. Alternatively, reactivity-based probes undergo a chemical reaction or amide hydrolysis in the presence of a specific ion or analyte, resulting in a species with a higher propensity to adopt the open, fluorescent form. Several reviews have reported comprehensive summaries of fluorescent sensors that use this approach.^{2, 21, 40, 46}

Non-carboxy substitution: Sulfonate

The applications of 3-substititon we have highlighted have focused on systems with dynamic, or reversible interconversion between open and closed forms. However, in some cases, this useful property is a drawback: for example, antibody labeling or incorporation of dyes into the outer leaflet of the plasma membrane for voltage imagingA sulfonate at the 3-position typically prevents spirocyclization of xanthene fluorophores, on account of the exceedingly low pK_a of sulfonic acids. 3-sulfonate substitution imparts improved water solubility, an important property for use in biological applications. These properties make 3-sulfonates an important feature of voltage sensing fluorophores that localize to extracellular membranes. In these cases, the 3-sulfonate prevents cell permeability and ensures proper orientation within the membrane.⁴⁷ When 3-carboxy is used in this context, the dyes accumulate in internal membranes (presumably because the fluorophore can adopt a neutral, spirocyclized form and pass through the plasma membrane), making them poor membrane potential indicators.⁴⁸ Incorporation of a charged, anionic group that cannot spirocyclize restores localization to the plasma membrane.^48-49 A limitation of the use of sulfonates is that they do not spirocyclize, preventing fluorogenic targeting via previously discussed methods.

Non-carboxy substitution: Phosphonate

An under-explored substitution at the 3 position is the inclusion of phosphonates. Although carboxy and sulfonated xanthenes dyes were reported in the 19^th century, there were no reports of 3-phosphono xanthene dyes until recently. We recently reported the synthesis of fluoresceins with a 3-phosphonate substitution.⁵⁰ The use of 3-phosphonate substitution improves water solubility compared to 3-carboxyfluorescein.⁵⁰ 3-phosphonofluoresciens behave like their 3-sulfono counterparts, displaying no propensity to spirocyclize. Importantly, phosphonates are readily functionalizable, allowing modulation of their cell permeability, unlike sulfonates. Esterification of 3-phosphonofluorescein facilities cell permeability where we observe superior accumulation and retention of the fluorophore in living cells, with nearly 70-fold improvement in cellular brightness compared to the analogous carboxyfluorescein. One limitation of the phosphonofluorescein approach is the current low-yielding synthesis.⁵⁰

More recently we have expanded this phosphonate substitution pattern to rhodamine fluorophores.⁵¹ The increased electron richness of the aniline precursors to rhodamines compared to resorcinols enables a mild and high-yielding synthesis of a variety of rhodamines bearing 3-phosphono substitution. Yields are up to 97% and have been performed on the >1 mmol scale (Figure 4a).⁵¹ In addition, 3-phosphono-tetramethylrhodamine is 12- and 500-fold more water soluble than the corresponding carboxy- and sulfono-analogs. Esterification of the phosphonic acid oxygens dramatically alters cellular permeability (Figure 4b-e); cells stained with the bis-ester of 3-phosphono-tetramethyl rhodamine is ~2,000-fold brighter than the free acid or mono-ester (Figure 4f). Additionally, the nature of the phosphonate ester influences subcellular localization. The diethyl ester (Figure 4d) of phosphonoTMR shows different sub-cellular localization compared to the bis acetoxymethyl ester of phosphonoTMR (Figure 4e). 3-phosphonate substitution is also well suited for incorporation into voltage sensing scaffolds, where the free phosphonic acid functional group prevents voltage-sensitive fluorophores from crossing the cell membrane. Both fluorescein and rhodamine phosphonates have been incorporated into voltage sensing scaffolds.^50-51

Figure 4.

a) Synthesis of 3-phosphonotetramethyl rhodamine (phosTMR). b-e) Tunable localization of 3-phosphonoTMRs. Widefield fluorescence (b-e) images of HEK293T cells stained with 500 nM dyes in HBSS 16 (b), 23 (c), 24 (d), or 25 (e) for 20 min at 37 °C. Quantification (f) of cellular fluorescence. Values are mean fluorescence intensity ± S.E.M. for n = 21, 32, 45, and 19 regions of interest (ROI) for each dye. Each ROI contained between 2 and 20 individual cells. “n.s.” means that the cellular fluorescence values were not above background fluorescence. Coverslips were placed into fresh HBSS prior to imaging. Scale bar is 20 μm.

Reproduced from Turnbull, et al. Chem Sci 2023, 14, 11365 with permission from the Royal Society of Chemistry.

Conclusions and outlook

We have highlighted examples of how substitution beyond a 3-carboxylate over recent years has led to the evolution of novel xanthene fluorophores for new applications. This has largely focused on perturbing the spirocyclization equilibrium for super resolution imaging, fluorogenic labelling, or reaction-based analyte detection. Tuning this equilibrium by 3-substitution has advantages over approaches that rely on chemical modification of the xanthene scaffold. For example, changes to the xanthene electronics with withdrawing groups are accompanied by spectral shifts, often lower quantum yields and can leave the xanthene prone to nucleophilic attack.²⁹ Recent reports of altering spirocyclization propensity with 3-amide substitution are highly attractive due to the modularity of the approach facilitating easy incorporation of a wide number of amines.⁴⁴

On the other hand, there are instances where spirocyclization is undesirable and it is important for the fluorophore to remain in the open fluorescent form. This is useful in examples where fluorescence is being modulated through other pathways such as PeT, where water solubility is key, or where membrane permeability needs to be kept at a minimum, such as in antibody labeling, extracellular applications, or for improving retention once in cells.

Modification of the 10' position of xanthene dyes, in the context of 3-carboxy substitution, has provided xanthene dyes with excitation and emission profiles ranging from blue to far-red and near infrared. Our group has applied this 10' substitution to sulfonate-containing rhodamines;⁵² in the future, expansion of 10' substitution to 3-phosphono fluoresceins and rhodamines will enable new applications for functional dyes. For example, functionalization of phosphonate substitution with esters or other functional groups may enable large-scale alterations to the open/closed equilibrium constant. Ultimately, 3-functionality provides the unique advantage of having major influences on the characteristic properties of xanthene fluorophores, such as spirocyclization and water solubility, while leaving photophysical properties like absorption and emission wavelengths relatively unperturbed. As interest in the chemical exploration of this substituent has grown over recent years, several new biological imaging applications of xanthene fluorophores have emerged including in super-resolution microscopy – enabled by single molecule localization – and voltage sensing. Perhaps one of the greatest challenges accompanied with new 3-functionalities is synthetic tractability. Often, methodology advances that focus on synthetic generalizability do so in the context of modifications to the xanthene chromophore, thus methods that facilitate expansion of our repertoire of 3-substituents would be highly sought after (Outstanding Question). Novel modalities at the 3-position, and methods to make them, will enable the evolution of fluorophores with new properties. Computational approaches^26-27 to predict these properties like extinction coefficient, fluorescence quantum yield, or K_L-Z would be exceptionally valuable (Outstanding Question). The expansion of our fluorescent molecular toolkit (Outstanding Question) will help drive our ability to explore complex biological systems with increasing precision and clarity. Recently, spirocylization methodologies have been expanded to the cyanine class of fluorophores,⁵³ offering new opportunities to bring the power of the cyanine dyes to fluorogenic and superresolution imaging in new ways. Combinations of new synthetic methods and computational guidance should speed the development and application of fluorogenic cyanine dyes for cellular imaging (Outstanding Question).

Outstanding Questions.

-Can synthetic methods adapted to the generation of new xanthene core structures be readily modified to enable exploration of novel functionality at the 3 position? Specifically, can new synthetic routes to phosphonofluoresceins increase the availability of these useful dyes?

-Can 10' modifications to access far-red dyes be combined with 3-phosphono substitution to allow new applications?

-Can computational or machine learning approaches guide the synthesis of new xanthene dyes with defined properties?

-To what extent can lessons from xanthene fluorophore spirocyclization be applied to other classes of small molecular fluorophores, for example, cyanine dyes and related derivatives? -For superresolution imaging and related cell-based technologies, is it always true that “redder is better?” Does optimization of the 3 position and N-substitution patterns to improve photostability, brightness, and alignment with excitation sources and detector sensitivity represent a fruitful avenue of research?

Highlights.

-fluorescent dyes like fluorescein and rhodamine have been in use since the late 19^th century

-a proliferation of synthetic methods to access new derivatives of fluorescein and rhodamine has opened new areas of research at the chemistry-biology interface

-synthetic modifications to the 3 position on the pendant ring of fluorescein and rhodamine profoundly alter the properties of the dyes, including open-close equilibria, water solubility, cellular permeability, and fluorogenicity.

Glossary

fluorescein

a xanthene fluorophore featuring oxygen substitution at the 3' and 6' positions

rhodamine

a xanthene fluorophore featuring nitrogen substitution at the 3' and 6' positions

rhodol

a hybrid xanthene fluorophore featuring one nitrogen and one oxygen at the 3' and 6' position

xanthene fluorophore

an aromatic, three-ring structure that absorbs and emits light. Modifications at the 3', 6' and 10' positions determine the classification as fluorescein, rhodamine, or rhodol. Modifications at these positions also largely determine the wavelength of absorption and emission

fluorescence quantum yield

the efficiency of a fluorophore, conceptually defined as the fraction of photons emitted divided by the number of photons absorbed

fluorophore

a molecule which is fluorescent

fluorophore brightness

the brightness of a fluorophore is defined as the product of its fluorescence quantum yield and molar extinction coefficient

fluorogenic

a property describing a fluorophore that changes its brightness in response to changes in the local environment (pH, dielectric, ion concentration, membrane potential, receptor binding, etc.)

spirocycle

the non-fluorescent form of fluoresceins and rhodamines. For traditional 3-carboxy fluorescein and rhodamine, this spirocycle is a lactone. For other derivatives, the term spirocycle is used, since it is more inclusive and covers a range of chemical structures, including lactones, lactams, and other 5- and 6-membered rings

Super-resolution microscopy

any of the light microscopy techniques that can provide resolution below the diffraction limit of light. For visible light techniques, this is a resolution that is below ~300 nm