Color-change Photoswitching of an Alkynylpyrene Excimer Dye

Abstract

We describe a photoswitchable DNA-based dimeric dye that visibly changes fluorescence from green to blue upon UV irradiation. A novel bis-alkyne-dependent [2+2+2] cycloaddition is proposed as a mechanism for the color change in air. The photoinduced structural switching results in spatial separation of stacked pyrene units, thereby causing selective loss of the excimer emission. We demonstrate and suggest several applications for this novel photoswitch.

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DNA-based Photoswitch: We describe a DNA-based dimeric dye that visibly changes color from green to blue upon UV irradiation. A novel bis-alkyne-dependent [2+2+2] cycloaddition with oxygen is proposed as a mechanism for the color change in air. We demonstrate and suggest several applications for this novel photoswitch.

Switchable fluorescent dyes have uses in multiple fields from cell membrane research to high resolution microscopy.^[1] A variety of fluorescent dyes and proteins that undergo a photoinduced shift in spectral emission properties have been exploited for in vivo protein tracking.^[2] Fluorescent probes that exhibit switchable phenomena have played an integral role for applications in super-resolution microscopy.^[3] Additionallly, photobleachable dyes that convert from emissive to dark states have shown utility in FRAP (fluorescence recovery after photobleaching), which is widely applied in study of mobility of species in lipid membranes.^[4]

Notably, most emissive fluorophores used in such switching applications are photoconverted from an emissive state to another based on oxidative or other chemical alteration of conjugation.^[5] Here we describe a very different and unprecedented dye switching phenomenon, in which the dimeric dye switches from one emissive state to another via a photoinduced [2+2+2] cycloaddition reaction with oxygen.

We observed this color change recently while characterizing a rapid photobleaching that was documented earlier in an oligodeoxyfluoroside (ODF) dye (Figure 1).^[6] ODFs are DNA-like molecules that incorporate multiple fluorescent chromophores as monomers into a single water-soluble assembly so that they interact closely with one another. This close association of chromophores results in complex and useful sequence-dependent changes to fluorescence behavior, which have been exploited in a variety of applications ranging from analyte sensing to multicolor fluorescence imaging with single-wavelength excitation.^[7] The dyes can be assembled conveniently in thousands of sequences using a DNA synthesizer.

Figure 1.

Color change of the VV dimeric chromophore. a) Visible green excimer fluorescence of SSVV before bleaching. b) Blue fluorescence of SSVV after irradiation at 365 nm. c) Fluorescence spectra of the dye in water before (green) and after (blue) light exposure at 365 nm.

A recent report described the synthesis of the phenylethynylpyrene (PEP) deoxyribonucleoside (one-letter abbreviation V (Figure 2)), and its incorporation into a cyan/green-emitting dyad SSVV, containing two adjacent V dyes (S is a nonchromophoric spacer adding solubility).^[6] Following up on this observation, we carried out new studies of the SSVV dye, which revealed that, under irradiation at 365 nm (the PEP absorption maximum) it undergoes rapid photobleaching (t_1/2 = 134 s) in air-saturated water in a spectrometer (Figure 1). Strikingly, spectral measurements (as well as inspection by eye) revealed that rather than simply becoming dark, the dye switched emission maximum from 509 nm (green) to 393–413 nm (blue-violet). We had noted color changes previously in one study of other ODFs over a decade ago, but had not characterized the phenomenon further.^[8] The simplicity of the VV dimer system offered us that opportunity.

Figure 2.

Structures of SSVV and Cy3-SVV oligodeoxyfluoroside dyes comprising S spacer and V dye monomers linked by phosphodiester bonds. The SSYY control dye (see Figure. 3) includes pyrene monomer Y.

The emission of the SSVV dye occurs initially as a broad excimer band (Φ = 0.87, abs_max = 365 nm, ε = 91000 L•mol⁻¹ cm⁻¹, τ = 42 ns^[6]) at 509 nm, whereas the monomeric V dye displays pyrene-like bands at 393 and 413 nm. The excimer emission of the dimer is efficient, dominating the monomer band in intensity. However, exposure to 365 nm light results in rapid and almost complete loss of the excimer emission and a small increase in monomer emission in water (or a larger increase in buffer, Supporting Information (SI) Figure S3). Intriguingly, experiments carried out with prior argon sparging to remove oxygen result in similar bleaching upon light exposure, suggesting no requirement for oxygen (SI Figure S4). This was surprising: most photobleaching mechanisms rely on oxygen disrupting conjugation. Pyrene excimers (excited-state charge transfer dimers^[9]) are not a conjugated system in the conventional sense; moreover, the monomer pyrene chromophores that make up the VV excimer are not apparently destroyed in the process based on the retention (or even increase) of short-wavelength pyrene monomer emission, which is stable. How, then, is the VV excimer bleached?

To investigate the structural origins of this unusual color change effect, we synthesized a number of related control molecules, denoted SSSV, SVVV, and SSYY (Figure 2; SI Figure S1), and examined their photobleaching behavior. The first of these, SSSV, contains only the monomeric PEP dye, and lacks any excimer emission. Photoirradiation of SSSV over 15 min at 365 nm yields essentially no photobleaching in air-saturated solution (Figure 3b). Thus the monomeric PEP chromophore is strongly resistant to bleaching, whereas an adjacent dimer of the same dye changes color rapidly, establishing that proximity of two such dyes is required for bleaching. Indeed, the trimeric dye SVVV, containing three adjacent PEP chromophores, displays even more rapid initial bleaching than SSVV (Figure 3e) and more pronounced switching from excimer to monomer emission (Figure 3c).

Figure 3.

Rapid bleaching of VV-containing chromophores. a–d) Emission spectra of aqueous dyes over 15 min with exposure to 365 nm light. e) Bleaching time profiles of SSVV and SVVV under the same bleaching conditions in air. f) Emission spectral change of Cy3-SVV over 15 min under 365 nm UV exposure. Photograph shows the VV-mediated Cy3-SVV color change before (yellow) and after (purple) 15 min of 365 nm UV exposure.

The comparison molecule SSYY, containing adjacent pyrene chromophores but lacking the alkynyl groups, also shows the expected long-wavelength excimer emission^[9] (490 nm) similar to SSVV (Figure 3d). Importantly, however, it undergoes almost no photobleaching under the same conditions that yield rapid and virtually complete photobleaching of the SSVV molecule (Figure 3d). This striking difference establishes that the phenylethynyl groups, and most likely the reactive alkynes, of chromophore V are the site of photoreaction. Moreover, two adjacent alkynyl groups are necessary for this phenomenon to occur, suggesting a reaction mechanism involving two alkynes.

To gather more data, we examined the photoreaction of SSVV in further detail. MALDI-TOF mass analysis after UV exposure of argon-sparged solutions revealed no change in mass, implicating a photorearrangement. Interestingly, in air-saturated solution the main product is different: MS analysis revealed a +32 mass negative ion as a chief product, consistent with a product incorporating one equivalent of dioxygen (SI Scheme S1). This is present only in the UV-irradiated SSVV samples and absent in freshly synthesized samples. Thus the experiments suggest two main mechanisms of bleaching, which vary depending on oxygen availability.

For this initial study we focused on the oxygen-dependent bleaching of the VV dimer, since it is more practically relevant. Based on the data, we posited a possible mechanism for O₂-dependent bleaching involving photoexcitation of ambient dioxygen to its singlet state^[10], followed by formal [2+2+2] cycloaddition to two adjacent alkynes, generating an unstable 1,2-dioxin intermediate (Figure 4). Although 1,2-dioxins have not been isolated, they have been analyzed in computational studies,^[11] are implicated in the generation of benzodioxins,^[12] and they are postulated to rapidly isomerize to stable enediones.^[13] We suggest, in analogy to the work by Block et al., that after reaction with oxygen, our bis- pyrene 1,2-dioxin cyclic intermediate rapidly isomerizes to the corresponding stable enedione product (Figure 4). We confirmed the existence of an enedione product by derivatizing it with 2,4-dinitrophenylhydrazine (DNPH, Figure 4 and SI). This yielded a product whose mass matches those of a pyridazinium adduct as well as structurally related variants of this adduct, which are known to form from reactions of enediones with arylhydrazines.^[14] Significantly, models show (SI Figure S5) that both the proposed dioxin intermediate and the final alkene-bridged structure project the terminal arenes away from one another. This explains the observed color change: the product’s geometry prevents pyrene-pyrene stacking in the excited state, leaving only pyrene monomer emission at the expected blue-violet wavelength.

Figure 4.

Proposed light-induced oxygen reaction with adjacent alkynes (green fluorescence) during bleaching of SSVV in air-saturated water, forming a 1,2-dioxin intermediate, which rearranges to the enedione (blue fluorescence). Enedione product was derivatized by 2,4-DNPH and masses of the adducts were confirmed by MALDI-TOF (see Figures S8,9).

To look for evidence of the ability of VV to photosensitize the production of singlet oxygen, we carried out the UV exposure in the presence of diaminobenzidine,^[15] which polymerizes in the presence of this reactive oxygen species. The experiments showed clear visual and spectral evidence of polymer formation in the cuvette (SI Figure S7). The control lacking this dye showed no such behavior. Taken together, these data are consistent with (1) the generation of singlet oxygen sensitized by the dye, and (2) the addition of singlet dioxygen to the alkynyl dimeric dye, ultimately forming (3) an enedione product that prevents excimer formation by the pyrene groups.

As noted above, bleaching of SSVV also occurs when oxygen is excluded, and mass spectrometry measurements implicate a different molecular rearrangement preserving the original mass. A mechanism to explain this must also involve two alkynes and leave pyrene chromophores intact, also leading to a similar color change. Although multiple mechanisms may be possible, we propose as one plausible mechanism the photoinduced cyclization of two alkynes and a phenyl ring, yielding a naphthalene-bridged product (SI Scheme S2) that separates the pyrene chromophores rigidly. This is proposed in analogy to previous studies of bis(phenylethynyl)cyclophanes, which undergo this same rearrangement.^[16] More work will be needed to elucidate the mechanism in the present case, but it is likely not relevant to the oxygen-dependent photobleaching of SSVV in air.

Having documented this unusual color change phenomenon, we then explored possible applications of the dimeric dye. Taking advantage of the photobleachable excimer band of SSVV, we carried out periodic FRAP experiments in a cuvette (Figure 5). In this experiment, bleaching occurs within the localized beam of the UV light in a cuvette; when the light is blocked, unbleached SSVV diffuses into this region, recovering the signal. Seven cycles of bleaching and recovery were demonstrated. The results suggest possible utility of VV and VVV dyes in membrane diffusion studies in the future. While traditional FRAP dyes simply become dark, the current dyes remain emissive at a different wavelength, allowing their further tracking and analysis.

Figure 5.

a) Illustration of the fluorescence bleaching and recovery setup. The center of a quartz cuvette is irradiated with UV, causing bleaching of the SSVV excimer band. When the light is turned off, diffusion of unbleached SSVV can occur back into the beam path, recovering the excimer emission upon excitation. b) Multiple bleach and recovery cycles gives a periodic fluorescence signal that diminishes with the reservoir of unbleached SSVV.

In principle, conjugation of a red-shifted dye to VV as a FRET acceptor might allow one to engender color-change photoswitching at a longer wavelength. To test this, the dye Cy3 was conjugated to VV, forming the assembly Cy3-SVV (see structure in Figure 1 and data in Figure 3f). Cy3-SVV shows very large Stokes shifts (213 nm) due to apparent FRET energy transfer from the VV excimer band to the overlapping Cy3 excitation band at 550 nm (Figure 3f). When Cy3-SVV is exposed to UV irradiation, the Cy3 emission band undergoes rapid bleaching, while the pyrene monomer band intensity increases, yielding blue fluorescence and a marked color change from orange to blue (Figure 3f). Absorption measurements before and after reaction confirm that the Cy3 chromophore remains nearly unchanged (SI Figure S10). Remarkably, this dye assembly is bleached without destruction of any of the main chromophore components. Indeed, one can still measure Cy3 emission after bleaching, using its own excitation wavelength (not shown). Thus we conclude that it is possible to use dye conjugation to tune the emission wavelength of such a color-changing dye assembly, employing VV as the switch.

Finally, in a test of the light-dependent color change of VV in imaging, we carried out localized bleaching of stained yeast (Saccharomyces cerevisiae) and human (HEK293T) cells under an epifluorescence microscope (Figures 6 and S11). Using the SSVV dye, images of stained yeast show clear changes in hue with light exposure, with the final blue product remaining stable for extended times. This utilizes SSVV as a dual-purpose label in the fluorescence microscopy of yeast cells, allowing both the tracking of exposure to UV via the ratiometric photobleaching of the cyan/green excimer band, as well as the continual visualization of cells with the persistent monomeric blue emission peaks. Staining of human cells with Cy3-SVV shows an even more dramatic orange-to-blue color change upon localized UV exposure (Figure 6b,c).

Figure 6.

Eukaryotic cell labeling with color-change dyes, imaged by epifluorescence microscopy. a) Color-change photoswitching of SSVV-stained S. cerevisiae cells from cyan (t = 0 s) to blue (t = 15 s) after 365 nm UV exposure. b) and c) Cy3-SVV-stained human kidney (HEK293T) cells show pronounced VV-mediated photoswitching from orange to blue in upon 365 nm UV exposure (region of exposure is at left in each image).

It is worth noting that ODF dyes such as SSVV have broad applications beyond the nucleic acids context, having shown utility as protein and antibody conjugates and in living cells and animals.^[6,7] Although more studies are needed to better understand the photoswitching mechanisms under varied conditions, we envision future uses of the VV-induced color change in FRAP studies, for super-resolution imaging, and ratiometric measurements of light exposure. It may also have utility as a control mechanism in optogenetics.^[17] Moreover, the structural switch documented here may have utility in photocontrol of biological activities^[18] or as a mechanical actuator in a molecular machine^[19]. The unusual mechanism of bleaching suggests future generations of alkyne-containing dye designs that respond to photoswitching in unusual ways, and as quantitative chemosensors of reactive oxygen.^[20] Finally, the VV assembly may also enable photoactivatable bioorthogonal labeling, making use of ketone-mediated hydrazone/oxime ligations.^[21]