Heteroatom-Substituted Xanthene Fluorophores Enter the Shortwave-Infrared Region

Abstract

This article is a highlight of the paper by Ivanic and Schnermann et al. in this issue of Photochemistry and Photobiology (Daly et al. Photochem. Photobiol. 2022). The collaborative team utilized computational approaches to investigate the influence of electron-withdrawing groups at the 10′ position of tetramethylrhodamine (TMR). Leveraging this information, the team was able to extend the emission of the TMR scaffold into the shortwave-infrared region (SWIR, 1000–2500 nm) by incorporation of a ketone functional group at the 10′ position (Daly et al. Photochem. Photobiol. 2022). This work provides the first example of a TMR derivative with peak SWIR emission (λ_abs: 862 nm, λ_em: 1058 nm). The authors utilize the ketone rhodamine scaffold to generate fluorogenic, pH-responsive reporters. This work demonstrates the potential of the classic xanthene scaffold for use as a SWIR reporter, an important step in the ultimate expansion of the repertoire of small-molecule organic fluorophore scaffolds available for deep-tissue imaging applications.

COMMENTARY

Organic small-molecule dyes are excellent tools for optical imaging due to their high tunability, bioavailability and relatively low toxicity (1). Fluorescence imaging in the visible range of the electromagnetic spectrum (400–700 nm) has been instrumental in the investigation of cellular structures and biochemical processes (2–9). However, in vivo animal imaging applications in this spectral range are limited due to shallow light penetration caused by attenuation and scattering in tissues along with autofluorescence from endogenous chromophores, giving rise to high background signal (10). Thus, designing optical agents with absorption and emission in the near-infrared (NIR, 700–1000 nm) and SWIR (1000–2500 nm) range has gained traction as an approach to overcome the limitations of visible reporters in in vivo imaging (11). Unfortunately, there is currently a lack of fluorescent scaffolds in this spectral window, especially in the SWIR, underscoring the need for chemistry-focused efforts to further identify small-molecule organic dyes compatible with SWIR imaging (12). The demonstration that off-peak SWIR emission from indocyanine green can be used for in vivo imaging further highlights the need for dye development in the SWIR (13).

In the NIR and SWIR realms, polymethine dyes remain the most common scaffold. Nitrogen-containing cyanines are the classic example of polymethine dyes and are characterized by relatively large molar extinction coefficients (ε) (1). SWIR polymethines such as IR-26 and IR-1061 contain sulfur heterocycles and emit above 1000 nm. However, the quantum yield (Φ) of these dyes is often decreased due to the heavy atom effect of sulfur. Thus, brightness (ε × Φ) for these dyes often suffers relative to their NIR counterparts. Recently, promising efforts in this area have focused on the clever use of new heterocycles, such as dimethylamino flavylium, to obtain polymethine dyes with bright SWIR emission (14,15). Because of these important advances, polymethines continue to dominate SWIR imaging efforts with relatively little attention paid to other classic dye scaffolds.

Xanthenes, such as TMR, represent one such classic dye scaffold that has been widely used for visible imaging applications (1,16,17). Common approaches to red-shifting the absorption and emission of rhodamine dyes are accomplished by (1) modulating the push–pull system via the introduction of auxochromes at the 3′ and 6′ positions of the xanthene core or (2) heteroatom substitution at the 10′ position (Fig. 1). In the first approach, bathochromic shifts have been imparted on rhodamine structures bearing novel auxochromes such as para-substituted styryls and indolizine, yielding xanthene derivatives with peak emission in the SWIR (18,19). However, the alternative approach of heteroatom installation at the 10′ position had not produced a SWIR emissive compound prior to the work of Ivanic and Schnermann et al. in this issue (20). In this work, the collaborative team investigated the influence of a ketone at the 10′ position, obtaining a new class of ketone rhodamines with absorption at 862 nm and emission at 1058 nm (20). Compared with the previously described heteroatom TMR analogs (21–25), the ketone TMRs display a significant red-shift in absorption and emission (Fig. 2), producing the first heteroatom TMR analogs with peak emission in the SWIR. The authors also designed fluorogenic, pH-responsive ketone TMRs which are likely to prove useful for in vivo imaging applications. Although the ketone TMRs have reduced photon output relative to polymethines in this wavelength class, the availability of a new SWIR scaffold represents a significant advance, which is expected to diversify the design of reporters in this wavelength region.

Figure 1.

Xanthene core with numbering shown. Efforts focused on red-shifting absorption and emission are centered on auxochrome modification at the 3′ and 6′ positions or substitution of electron-withdrawing groups at the 10′ (X) position. Fluorogenicity is commonly introduced by ortho-substituted aryl groups at the 9′ (Y) position.

Figure 2.

Subset of the previously described heteroatom functional group substitutions at the 10’ position of the TMR scaffold ordered by maximal absorption wavelength (see Fig. 1 for base dye structure, R and R′ are dimethyl amino substituents). The ketone functional group induces a remarkable red-shift in both absorption and emission, producing the first TMR analog with peak emission in the SWIR.

COMPUTATIONAL STUDIES

Before embarking on a synthetic campaign, the team employed in silico approaches to determine the effects of placing electron-withdrawing groups (EWG) at the 10′ position within the TMR structure. Interestingly, these computational efforts indicated that 10′ modification with an EWG most strongly affects and stabilizes the LUMO, while HOMO energies remain virtually identical. These observations are rationalized by the observation of incremental extension of a bonding lobe in the LUMO to the 10′ position in TMR analogs containing more highly EWGs as well as a clear correlation between the bond order of the 10′ atom and the dye framework versus experimental absorbance. This is consistent with the Dewar–Knott color rule which for cationic diarylmethanes, structurally analogous to TMRs, predicts that placing more EWGs at the bridging position leads to bathochromic shifts (26). These careful studies provided the authors with a possible mechanism for red-shifting xanthene absorption and emission by substitution of EWGs at the 10′ position. Using their computational framework to investigate the influence of a ketone functionality at the 10′ position indicated a substantial red-shift in absorbance and emission compared with the previously described heteroatom modifications. The authors hypothesized that the ketone modification, compared with other functional groups previously installed at the 10’ position, provides the unique advantage of introducing two electrons into the conjugated system as well as an empty antibonding π-orbital that the LUMO can extend into. These observations prompted the authors to embark on the synthesis of ketone TMR analogs. Taken together, this improved physical understanding of heteroatom modifications in xanthenes will likely enhance the design of NIR- and SWIR-emitting TMRs.

SYNTHESIS

Xanthene dyes are often synthesized through expedient one-step condensation reactions (1). However, this approach is not viable for heteroatom xanthenes. Thus, numerous synthetic approaches have been utilized to construct heteroatom-substituted xanthenes (25,27–31). In their initial synthetic route, the research team employed Friedel–Crafts acylation to obtain KR-1 (Fig. 3) at a modest 8% yield. Building on the previous work demonstrating the utility of electronically matching nucleophilic and electrophilic reactants during heteroatom rhodamine synthesis (31,32), the authors employed a bis-nucleophilic addition route where a bis(arylmetal) intermediate (the nucleophile) was added to an ester or anhydride (the electrophile) to provide KR-1 as well as KR-2 and KR-3 (Fig. 3) with increased yields (~25%). Interestingly, the team also utilized a double Heck reaction to generate KR-4 (Fig. 3). This double Heck reaction provides a new approach to generate stimulus-responsive xanthenes and may allow for facile derivatization of other TMR analogs.

Figure 3.

Ketone xanthene dye series. Absorption and emission maxima for KR-1 (PBS), KR-2 (5% TFA in DCM), KR-3 (PBS) and KR-4 (0.05 M acetate at pH = 3.6) are shown.

PHOTOPHYSICAL PROPERTIES

Characterization of the optical properties of KR-1 clearly validated the authors’ computational studies and provided the first heteroatom TMR derivative with peak emission in the SWIR, displaying significantly red-shifted absorption and emission compared with the previous heteroatom xanthenes (Figs. 2 and 3). Comparison with the recently described rhodamine analogs bearing auxochrome modifications resulting in peak emission in the SWIR (Fig. 4) (18,19) illustrates a significant reduction in the molecular weight of KR-1. Together with their recently described low molecular weight, NIR-emitting fluorocoumarins, this team has established themselves as leaders in the development of low molecular weight red-shifted dyes (33).

Figure 4.

Structures and properties of xanthene dyes with peak emission in the SWIR. (a) A general structure for SWIR-emitting xanthenes. (b) Absorption, emission and molecular weights of xanthene dyes with peak emission in the SWIR. The ketone modification in KR-1 yields the lowest molecular weight xanthene dye with peak emission in the SWIR. ^aSpectroscopic properties were determined in PBS except for RhIndz ethyl ester, which was analyzed in DCM.

Xanthene fluorophores are well known for their fluorogenic properties, caused by an equilibrium between a spirolactone “ring-closed” form (fluorescence off) and a “ring-opened” zwitterion form (fluorescence on). This general spiro-ring open/closed equilibrium has been utilized to generate turn-on fluorescence probes for a variety of analytes including reactive oxygen/nitrogen species, metal ions, pH and thiols as well as protein-labeling events (34–37). In this work, the authors demonstrated that KR-2 (Fig. 3) displays pH-dependent fluorogenic behavior, providing the possibility for further design of turn-on SWIR-emitting detectors.

Lastly, the research team demonstrated that a previously described endo/exo-isomerization can be employed in KR-4 to create a pH-responsive probe (38). At neutral pH, KR-4 displayed an absorption peak at 455 nm (attributed to the exo form), while at acidic pH, absorption at 874 nm was restored (attributed to the endo form, Fig. 4). This transition occurred with a pK_a = 4.4 implying possible applications in biological systems.

CONCLUSIONS

Ivanic and Schnermann et al. provide the community with a mechanistic understanding of the influence of 10’ EWG substitution on the spectral properties of TMRs. Utilizing this insight, the team demonstrated that ketone installation at the 10’ position can dramatically red-shift peak emission into the SWIR, providing a low molecular weight scaffold for further functionalization. Future efforts aimed at increasing the photon output of ketone rhodamines are expected to yield valuable SWIR imaging agents. While application of KR dyes for in vivo imaging has yet to be demonstrated, this work provides valuable proof-of-principle evidence for the potential viability of the classic xanthene scaffold as a SWIR reporter while simultaneously pushing the limits of the capabilities of low molecular weight small-molecule organic fluorophores.