Resolving the Fluorescence Quenching Mechanism of an Oxazine

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A: Kinetics, Dynamics, Photochemistry, and Excited States

Resolving the Fluorescence Quenching Mechanism of an Oxazine Dye Using Ultrabroadband Two-Dimensional Electronic Spectroscopy William P. Carbery, Brismar Pinto-Pacheco, Daniela Buccella, and Daniel B. Turner J. Phys. Chem. A, Just Accepted Manuscript • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 22, 2019

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The Journal of Physical Chemistry

Resolving the Fluorescence Quenching Mechanism of an Oxazine Dye using Ultrabroadband Two-Dimensional Electronic Spectroscopy William P. Carbery, Brismar Pinto-Pacheco, Daniela Buccella∗ , and Daniel B. Turner∗ Department of Chemistry, New York University, 100 Washington Square East, New York NY 10003, USA E-mail: [email protected]

∗

To whom correspondence should be addressed

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Abstract The design and optimization of fluorescent labels and fluorogenic probes relies heavily on the ability to distinguish among multiple competing fluorescence quenching mechanisms. Cresyl violet, a member of the 1,4-oxazine family of dyes, has generally been regarded as an exemplary fluorescent probe; however recent ultrafast experiments revealed an excited-state decay kinetic of 1.2 ps, suggesting the presence of a transient photochemical state. Here we present ultrabroadband two-dimensional electronic spectroscopy (2D ES) measurements of cresyl violet in the presence of the fluorescence quenching agent 3,6-di(2-hydroxyethyl)1,2,4,5-tetrazine. The broad spectral bandwidth allows for the evaluation of multiple fluorescence quenching mechanisms such as exciton formation, photoinduced electron transfer, resonance energy transfer, and excited-state proton transfer. The 2D electronic spectra in the presence and absence of quencher suggest that excited-state proton transfer drives the system’s excited state dynamics and leads to a cresyl violet tautomer involved in fluorescence quenching. The invocation of the tautomeric form of cresyl violet neatly resolves longstanding inconsistencies in the photophysics of oxazine dyes more generally. Although still under development, the application of ultrabroadband 2D ES to a molecular system represents a compelling demonstration of the technique’s future role in the study of photochemical reaction mechanisms.

Introduction The study of biological systems relies heavily on fluorescence-based techniques that track biomolecules and probe interactions in a diverse set of environments over a wide range of timescales. The mechanistic understanding of energy transfer and fluorescence quenching underpins the design principles and rational optimization of the fluorescent labels and indicators used in these investigations. 1 Oxazine dyes such as Nile Red, Nile Blue, Cresyl Violet, Oxazine 1, Resorufin, and other 1,4-oxazine variants, have long been used as a subset of these fluorescent labels where the red-shifted emission helps to avoid spectral interference from cellular autofluorescence. 2–5 The accessible redox chem-

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istry and favorable spectroscopic properties of these oxazine dyes have even been incorporated into biological imaging experiments with resolution below the diffraction limit. 6,7 Despite continuous use, the fundamental mechanistic understanding of the photophysical and photochemical properties of oxazines and other century-old dyes remains limited, and this imperfect understanding highlights a key difficulty in designing new fluorescence-based sensors. Namely, in separating and optimizing the broad range of competing but equally useful photo-switching mechanisms that include charge transfer, proton transfer, isomerization, and aggregation. 8 Recent ultrafast spectroscopy experiments on the oxazine molecule cresyl violet perchlorate (hereafter cresyl violet, CV+ ) revealed an unexpected excited state species with a short-lived, 1.2 ps lifetime. 9 Further investigation using the method of coherent wavepacket evolution analysis (CWEA) indicated that this transient species precedes the ultimate fluorescent state. Additional features in the CWEA spectra shift abruptly to the ground state absorption maximum, and were assigned, following Wang and Shank, 10 to nonradiative decay through a conical intersection. This assignment is unusual given that cresyl violet exhibits none of the diagnostic properties usually associated with conical intersections, particularly high photoactivity and low fluorescence quantum yields. 11 Indeed, cresyl violet has been generally regarded as an excellent fluorescent molecule with seemingly unremarkable photophysics, to the point where it is commonly used as a redemitting laser dye. 2 Nevertheless, a pattern of seemingly minor abnormalities in the spectroscopic properties of oxazine dyes more generally support the results of the ultrafast experiments indicating more complicated photophysics for cresyl violet. For instance, oxazines typically have large kinetic isotope effects, with fluorescence quantum yields increasing in deuterated solvents as vibrational and rotational energy loss is reduced. These same molecules, however, have unexpectedly short fluorescence lifetimes in ethylene glycol and glycerol despite the viscous solvents similar suppression of rotational and vibrational energy. 12–15 Furthermore, the fluorescence quantum yield of cresyl violet in alcoholic solutions varies by up to 10% according to dye concentration, 16 and efforts to increase the efficiency of laser dyes by adding triplet-state quenchers failed to yield emission recoveries

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in cresyl violet, actually halting lasing completely in a quarter of cases despite widespread success in other dye systems. 17 Taken together, these observations combined with the ultrafast spectra suggest that the excited state dynamics of cresyl violet extend beyond thermal equilibration and fluorescence, potentially impairing the utility of the dye in fluorescence-based investigations of biological systems. The data also suggest that the current understanding of energy transfer mechanisms in oxazine dyes is incomplete, and may be improved with the use of additional ultrafast spectroscopy techniques. In this report, we use ultrabroadband two-dimensional electronic spectroscopy (2D ES) to discern between possible fluorescence quenching mechanisms in a bimolecular system of cresyl violet and 3,6-di(2-hydroxyethyl)-1,2,4,5-tetrazine (hereafter tetrazine, Tz). This system came to our attention as part of a study on fluorogenic bioconjugation reactions involving tetrazines 18 and subsequent studies aimed at establishing the effect of the dienophile on the quenching properties of the conjugation product. Therefore, the first objective of performing 2D ES on the cresyl violet and tetrazine system is to define the mechanism of fluorescence quenching and inform future design principles for other oxazine or tetrazine based systems. More broadly, the quenching effect of tetrazine provides an excellent opportunity to explain unanswered questions about the excitedstate dynamics of cresyl violet that arose from prior ultrafast spectroscopy experiments. In doing so we also seek to highlight the significant utility of 2D ES in deciphering energy transfer mechanisms and the technique’s application for designing molecular systems used in fluorescence-based technology.

Experimental Synthetic Procedures Cresyl violet perchlorate was purchased from Exciton and used without additional purification. Inspection of the linear absorption spectrum of cresyl violet in methanol revealed no contaminating hydrolysis products. The synthesis of 3,6-di(2-hydroxyethyl)-1,2,4,5-tetrazine (tetrazine) 4


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was adapted from the procedure reported by Devaraj and coworkers for the synthesis of 3-methyl6-hydroxyethyl-1,2,4,5-tetrazine. 19 Detailed synthetic protocol will be reported elsewhere. All spectra, including the 2D electronic spectra, were collected in a 2:1 mixture of HPLC grade acetonitrile and aqueous PBS buffer (pH 7.4). Stock solutions of 1 mM cresyl violet and 100 mM ˘ S80 tetrazine were prepared in spectroscopic grade dimethyl sulfoxide and stored in aliquots at âA ¸ ◦C

until needed.

Linear Spectroscopy Measurements Linear absorption spectra were obtained using a Cary 100 UV-VIS Spectrophotometer by Agilent Technologies. Fluorescence spectra were collected on a QuantaMaster 40 Photon Technology International spectrofluorometer equipped with Xenon lamp source, emission and excitation monochromators, excitation correction unit, and PMT detector. All measurements were conducted at 25.0 ± 0.1 ◦ C maintained by a Quantum Northwest cuvette temperature controller. Emission and excitation spectra were corrected for the wavelength-dependent response and wavelength dependent lamp intensity. Fluorescence emission spectra of 2 ÎijM solutions of cresyl violet perchlorate in 2:1 acetonitrile/PBS buffer were collected at 25 ◦ C in the presence of increasing concentrations of tetrazine (0–1.20 mM). Experiments were performed in duplicate. The steady-state fluorescence quenching results were analyzed using the Stern–Volmer equation:

F0 F

= 1 + KSV [Q]; where F0 and F are the

fluorescence intensities in the absence and presence of quencher concentration, [Q], respectively, and KSV is the Stern–Volmer quenching constant. 20 The KSV value obtained for this system was 92 ± 9 M−1 .

Electrochemical Studies Cyclic voltammetry studies were performed with a CH Instrument potentiostat model 400A, using a three-cell-electrode arrangement consisting of a Platinum working electrode, a thin Pt wire counter electrode and a non-aqueous Ag/AgNO3 reference electrode, all purchased from CH In5



struments. Measurements were performed in acetonitrile with 0.1 M tetra-n-butylammonium hexafluorophosphate (TBAF) as supporting electrolyte. Prior to each experiment, the working electrode was cleaned by polishing with 0.3 Îijm and 0.05 Îijm alumina, followed by sonication in water and ethanol. For reversible peaks, the half-wave potential was obtained by taking the mean of the anodic and cathodic peak potentials. For irreversible peaks, peak potentials were reported. Both positive and negative scans were collected for each compound at 150 mV/s and identical electrochemical responses were obtained in both directions. Ferrocene (1 mM) was added as an internal reference (0.40 V vs. SCE) and potentials were reported vs. Standard Calomel Electrode (SCE).

Ultrafast Spectroscopy Measurements Ultrafast spectroscopy measurements were taken using a non-collinear optical parametric amplifier (NOPA) and four-wave mixing apparatus described elsewhere. 21,22 The ultrabroadband laser pulses used here necessitated one additional improvement to the compression of the NOPA output. These laser pulses (500-760 nm at 200 nJ and with ∼0.5% shot-to-shot RSD) were compressed using a prism-based pulsed shaper to 6.6 fs as measured by transient-grating frequency resolved optical gating (Figure S1). The use of a prism (Brewster, SF10) rather than a grating increased the ability of the pulse shaper to modulate the phase of the high-frequency region of the spectrum, while achieving greater achromatic power throughput. The sample apparatus consists of a demountable liquid cell (Harrick DLC-M25) connected to a low-flow pump head and drive (Micropump T-07002-32 and T-07002-39). The tubing is 1/16-in. PEEK (Supelco Z227307). The compression fittings on the cell (Beswick MCB-1016316-C) and pump (Grainger 20YX75) are stainless steel. The o-rings in the cell (McMaster-Carr 9568K114) and fittings are Kalrez and Chemraz materials, respectively. A 0.25-mm thick PTFE spacer separates the front (0.5 mm) and back (0.5 mm) UV fused silica windows. For some of the cresyl violet and tetrazine experiments, limited volumes of tetrazine required the sample to be measured without flowing. Spectra obtained from flowing and non-flowing arrangements were 6


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indistinguishable from each other. Long transient absorption (TA) measurements to 5 ps were taken of cresyl violet and cresyl violet with tetrazine prior to the 2D ES measurements. Phased 2D electronic spectra were obtained by measuring TA spectra at 60 waiting time (τ2 ) points centered at the 2D slice of interest in 1 fs steps. The 2D ES measurements were conducted on a slice-to-slice basis, with τ1 scanned from 0–90 fs in 1 fs steps. For TA measurements the pump power and number of averages was 54 µW per pump pulse at 2000 kinetic cycle pairs (KCP). For the 2D ES measurements the power was reduced by a third to 18 µW per pump pulse at 250 kinetic cycle quads (KCQ). Magnitude 2D ˘ ˘ Z´ according to the projection slice theorem, 23 by summing electronic spectra were âAŸphasedâ A the 2D magnitude spectra along the excitation axis and comparing it to the TA spectrum at the same waiting time. All spectra were phased to below 0.5% error. Measurements of cresyl violet with and without tetrazine were performed immediately following one another using the same laser pulse and compression. The experiments were repeated three times, while the spectra reported here stem from the same day of measurements for consistency. All TA spectra are reported as percent change in transmittance for direct comparison to, and phasing of, the 2D electronic spectra.

Results and Discussion Just as 2D NMR techniques isolate correlated changes in proton magnetization, 2D ES experiments reveal the interaction of electronic states over time. 24–26 The appearance, dynamics, and evolution of off-diagonal peaks corresponding to coupled optical transitions are uniquely valuable spectroscopic features that distinguish between photochemical processes. 27 For the cresyl violet and tetrazine system, fluorescence quenching likely falls into one of four broad mechanisms: excitonic coupling via aggregation, photoinduced electron transfer (PET), resonance energy transfer (RET), and excited-state proton transfer (ESPT). 28,29 Each of these mechanisms leads to unique features in time-resolved sets of 2D electronic spectra, presented schematically in Figure 1. The various possible mechanisms differ based on the strength of their electronic coupling and the char-

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acter of the transition state that leads to fluorescence quenching. a) Cresyl Violet

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Figure 1: Schematic 2D electronic spectra of (a) cresyl violet, (b) excitonic coupling, (c) photoinduced electron transfer (PET), (d) resonance energy transfer (RET), and (e) excited state proton transfer (ESPT). Each panel contains a simplified 2D electronic spectrum, an associated potential energy surface, and an example of the type of interaction present at the excited transition state. Only bleach and SE signals are included in the mechanistic panels for simplicity. For exciton formation mechanisms, relatively strong electronic coupling produces an exciplex species that connects the optical transitions of the donor and acceptor. The resultant 2D electronic spectra contain diagnostic cross peaks that form instantaneously and evolve together. 30 In PET mechanisms, this exciplex species is transient, and yields off-diagonal peaks in the 2D electronic spectra at both donor and acceptor excitation frequencies that evolve as an electron moves from donor, to charge-transfer complex, to acceptor. 31 In contrast to exciton formation and PET, the weak electronic coupling underpinning RET mechanisms allows the donor and acceptor molecules to maintain their unique optical transitions. 32 As a result, characteristic off-diagonal peaks should appear in the region of the 2D spectrum where donor excitation and acceptor emission overlap. Specifically, the frequency range of donor absorption on the excitation axis and acceptor fluorescence on the probe axis combine to form a 8


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‘RET box’, the region of a 2D spectrum where off-diagonal RET peaks evolve in time according to the system’s interaction length. 33 Lastly, fluorescence quenching through ESPT involves negligible electronic coupling between the donor and acceptor and therefore leads to few unique off-diagonal peaks. The low-frequency normal mode of the proton transfer, however, heavily influences the transition state, and results in 2D electronic spectra where excitation-dependent changes in the vibronic peaks indicates the formation of a tautomer through excess vibrational energy in the excitation. 34 Figure 2 presents a 2D spectrum of cresyl violet with key frequencies from steady-state measurements represented by gray dashed lines. Additional annotations appear at expected peak locations for different potential fluorescence quenching mechanisms in the presence of tetrazine and to indicate spectroscopic signatures from prior transient absorption data. A version of the 2D electronic spectrum without the annotations can be found in Figure S4. The spectrum in Figure 2 can be separated into three regions describing optical transitions that involve the first excited state, S1 . Absorption from the ground to first excited state appears as red ground-state bleach (bleach) peaks (S0 → S1 ) in the middle of the spectrum, while stimulated emission (SE) signal (S1 → S0 ) appears as red peaks at lower probe frequencies. Blue excited-state absorption (ESA) peaks (S1 → Sn ) indicate transitions into higher-lying excited states, and appear at mostly high probe frequencies on the right side of the spectra. The linear absorption and fluorescence spectra of cresyl violet and tetrazine in a 2:1 mixture of acetonitrile and phosphate buffered saline are presented in Figure S2. Stern-Volmer analysis of the steady-state fluorescence spectra at increasing concentrations of tetrazine (Figure S3) show the quenching effect of the latter, with ∼10% quantum yield reduction at 45 mM tetrazine. To probe the mechanism of fluorescence quenching in cresyl violet, we acquired 2D electronic spectra of cresyl violet and tetrazine at this concentration, along with cresyl violet alone (0.075 mM), at a wide range of waiting times. Figure 3 presents the 2D spectra of both systems at 100 fs, 1 ps, and 10 ps. The low extinction coefficient of tetrazine (546 M−1 cm−1 ) compared to that of cresyl violet (80, 300 M−1 cm−1 ) results in no appreciable 2D electronic signal for tetrazine.

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Probe (THz) Figure 2: Annotated 2D electronic spectrum of cresyl violet at a waiting time of 100 ps. Each 2D electronic spectrum in this report is presented as change in transmittance, with ground state bleach (bleach) and stimulated emission (SE) peaks positive (red) and excited state absorption (ESA) peaks negative (blue). The spectra are normalized to each spectrum’s central bleach and SE peak. Each contour line represents a step of approximately 6% in signal amplitude, while each shaded contour represents a step of approximately 3%. An example of the RET box and approximate excitonic peak locations appropriate for the CV and Tz system overlay the spectrum.

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In general, the 2D spectra of cresyl violet closely match previous TA and linear spectroscopy data that show absorption/bleach at 509 THz (589 nm), fluorescence/SE at 481 THz (619 nm), and ESA at 570 THz (525 nm). 9,21,35 Prior 2D ES measurements of cresyl violet, however, have often served to demonstrate new ultrafast spectroscopy techniques rather than probe the photochemistry of the dye itself. 36–41 While those spectra bear some qualitative resemblance to the ones published here, to our knowledge, the 2D spectra in Figure 3 represent the first 2D ES measurements of cresyl violet with sufficient bandwidth and resolution to distinguish between competing mechanisms of fluorescence quenching. The addition of tetrazine to cresyl violet results in two major alterations to the features in the 2D electronic spectra. A new ESA feature appears in the top left corner of the spectrum, at excitation frequencies above 550 THz and probe frequencies below 480 THz. The peak appears as soon as 100 fs and persists for the duration of the experiment. On the right side of the spectrum, at probe frequencies above 525 THz, an increase in bleach signal at excitation frequencies above 550 THz coincides with a slight probe frequency shift in the main ESA feature from 570 THz to 585 THz and increased ESA signal extending into lower probe frequencies along the 528 THz excitation line. Overall, the changes in the spectra occur within three to four colored contours, which corresponds to an approximately 9–12% reduction of the excited state population of cresyl violet. This is consistent with the approximately 10% fluorescence quenching expected from steady-state measurements at the same tetrazine concentration employed in the 2D experiment. Comparing the experimental 2D electronic spectra in Figure 3 to the anticipated spectroscopic features illustrated in Figure 1 allows us to evaluate the plausibility of each fluorescence quenching mechanism in turn. Of the four mechanisms considered here, RET is the least plausible due to the minimal overlap between the cresyl violet fluorescence and tetrazine absorption spectra. 20 In the cresyl violet and tetrazine system the ‘RET box’ is located in the top left corner at excitation frequencies above 521 THz and probe frequencies below 481 THz. The scheme in Figure 1d portrays the RET peak as an acceptor-based SE signal, but since tetrazine has negligible fluorescence and unknown excited-state dynamics, the RET box can, in principle, include the low-frequency probe

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Figure 3: Two dimensional electronic spectra of cresyl violet alone (a, top) and with tetrazine (b, bottom) at waiting times of 100 fs, 1 ps, and 10 ps. The horizontal dotted line indicates the cresyl violet absorption maximum (509 THz) while the three vertical dotted lines represent, from left to right, the fluorescence maximum (481 THz), absorption maximum, and ESA maximum (570 THz).

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range near 425 THz where the new ESA peak appears. 42 The RET box does not include, however, the changes in the bleach region between probe frequencies of 500 THz and 550 THz, and their appearance by 100 fs alongside the ESA peak at 425 THz contradicts the slower, non-pertubative, incoherent energy transfer expected from a RET mechanism. Energy transfer on the order of 100 fs is far more consistent with excitonic coupling mechanisms. In well-characterized systems like CdSe nanocrystals, the diagnostic 2D ES feature attributed to excitonic coupling is the pair of cross peaks at the intersection of the coupled excitations. 43 A molecular system typically achieves this coupling through aggregation, 44 a process known to alter the spectroscopic properties of cresyl violet at particularly high concentrations in water. 45,46 Neither cross peak expected from excitonic coupling is found in the 2D electronic spectra of cresyl violet and tetrazine. While the new ESA peak appears at an excitation frequency reasonably close to the absorption of tetrazine, it is located at a probe frequency well to the red of the cresyl violet absorption. The second peak due to excitonic coupling, expected at 510 THz excitation and 600 THz probe frequencies, is not observed, with this region remaining virtually unchanged after the addition of tetrazine. This is consistent with the deconvolution of the cresyl violet and tetrazine absorption and fluorescence spectra, which show no significant perturbation of the electronic states due to excitonic coupling. Fluorescence quenching through PET shares some similarity with excitonic coupling, but differs in the location of the new peaks generated during the donor and acceptor interaction. The transient donor-acceptor complex that underpins all PET mechanisms produces at least two new peaks at unique probe frequencies along the main donor excitation line, one each for the oxidized or reduced version of the donor and acceptor. The lifetime of this donor-acceptor complex, and the strength of the electronic coupling between both molecules in the excited state, determines the subsequent dynamics of the transient features in the 2D spectrum. The coupling strength, and by extension the rate of PET, is understood through the reduction and oxidation potentials of the donor and acceptor. 47 For this reason we electrochemically characterized both cresyl violet and tetrazine. Cyclic voltammograms of each compound in acetonitrile

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are shown in Figures S5 and S6. With a measured reduction potential of E 1 (red) = −0.18 V vs. 2

SCE, cresyl violet (E00 = +2.0328 eV) is not strong enough of a photo-oxidant to oxidize tetrazine, Ep = +2.045 V vs. SCE, indicating that PET is not a favorable process. The 2D spectra of the mixture of cresyl violet and tetrazine confirm that fluorescence quenching through PET is an unlikely mechanism. The PET mechanism results in either diffusion of two new species, photoproducts that would appear as ESA features in the 2D spectra, or back electron transfer that recovers the original system. A number of pump-probe studies on oxazine 1 (Ox1) in the electron donating solvent N,N-dimethylaniline (DMA) showed an ultrafast, less than 100 fs depletion of the excited state followed by a recombination through back electron transfer on the order of tens of picoseconds. 48,49 Importantly, electrochemical studies of the Ox1/DMA system found PET to be a favorable process, with a ∆G◦PET of −0.68 eV. 50 The two ESA features in the 2D spectra of cresyl violet and tetrazine have an ultrafast rise time but persist on the order of hundreds of picoseconds, suggesting that, if the fluorescence quenching by tetrazine followed a PET mechanism, it would have formed long-lived photoproducts, particularly a reduced cresyl violet species. Linear absorption measurements of the neutral species, however, indicate a broad absorption centered around 475 nm (631 THz), and this region of the 2D spectra shows no change in the ESA feature from the main excited sate of cresyl violet at low excitation frequencies. While a PET mechanism may be appropriate for other members of the oxazine family, the 2D electronic spectra discussed here ultimately do not support PET as the mechanism for fluorescence quenching in cresyl violet. In the Ox1/DMA system, the observation of persistent oscillatory features in pump-probe measurements spurred debate on whether PET involved the coherent transfer of an electron, and to what extent this was mediated by molecular vibrations and solvent interactions. 51–53 While PET remains a poor fit for the cresyl violet and tetrazine system, the excitation dependence of the new ESA peak at 425 THz—located only at excitations above 550 THz —suggests that molecular vibrations and solvent mediation play an important role, pointing to ESPT as a possible mechanistic pathway for the fluorescence quenching. In the cresyl violet and tetrazine system, the two exo-

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cyclic amine/iminium groups of cresyl violet can act as weak photo-acids, while tetrazine can be considered a weak photo-base that mediates ESPT to the solvent. The transition state in ESPT mechanisms involves a barrier to tautomerization that is highly susceptible to excess vibrational energy, to the extent that sufficiently energetic excitations can alter whether proton transfer occurs through diffusion or tunneling. 54 Thus a key attribute of 2D electronic spectra involving ESPT is excitation-dependent changes in the SE or ESA regions, primarily focused on the vibronic peaks. The changes in the 2D spectra of quenched cresyl violet appear to be excitation dependent, occurring above 550 THz while otherwise differing little from the unquenched spectra. Overall these spectral changes fit well with the schematic representation of ESPT in Figure 1e. Despite adequate signal-to-noise in the 2D ES experiments, high tetrazine concentrations coupled with the compound’s limited stability under long term irradiation made detailed kinetic rate measurements such as the ones obtained from a full 3D dataset impossible to obtain. Nevertheless, some approximate decay rates obtained from summing across a region of the 2D spectrum for each measured waiting time allow for comparison between cresyl violet and cresyl violet with tetrazine. Figure 4 describes these regions of interest, which include the new ESA region (blue), the SE region (green), the main bleach and SE peak (orange, dotted), the region of the spectrum with a ‘delayed’ bleach recovery (magenta, dotted), and the main ESA feature (dotted, purple). The summed intensities for the new ESA region and the SE region, along with their fits to either a biexponential or triexponential function, feature alongside the 2D spectra, while the other regions are described in Figure S5. The time dependence observed for the new ESA region in both the cresyl violet and cresyl violet with tetrazine spectra yield similar decay rates when fitted with a triexponential function. An ultrafast (420 fs, 260 fs) growth in SE signal is followed by a longer lived decay (1.2 ps, 3.1 ps), that in turn is followed by the long lived, 3.2 ns relaxation of cresyl violet, which is undersampled in these measurements and thus appears as a decay rate in the hundreds of picoseconds. The similar time dependence in both systems confirms a dynamic observed by careful inspection of

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Figure 4: Selection of fitted intensity plots for cresyl violet and the mixture of cresyl violet and tetrazine for the new ESA region (blue) and SE region (green). The summed intensities within each region at each waiting point were fitted using either a biexponential or triexponential function (red, dotted), where the initial values were taken from decay parameters from prior transient absorption data. The inset includes data points out to 100 ps.

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the 2D electronic spectra in Figure 3, namely, that the SE signal is delayed and less intense at high excitation energies. The fluorescence quenching by tetrazine reveals this to be the result of a delayed SE peak overlapped with an initially present ESA peak that subsequently grows in over the course of 1 ps to 4 ps. While the time dependence of the SE region highlights the limits of kinetic analysis absent a full 3D dataset, the plots suggest that emission from cresyl violet is delayed relative to other optical transitions. This contrasts with what would be expected for RET and PET mechanisms, where a new feature would be expected to grow in on top of, but not in favor of, the existing SE peak. In fact, the decay rates obtained from the cresyl violet spectra without tetrazine match prior transient absorption data, where decay associated difference spectra isolated a 1.2 ps transient with optical transitions at low emission frequencies that match the ESA peak in the 2D spectra revealed by the addition of tetrazine. The acetonitrile and PBS solvent mixture, like the methanol used in prior TA measurements, is a protic solvent capable of mediating a proton transfer. The 1.2 ps decay observed in the 2D electronic spectra and in analysis of the TA data compares well with other intermolecular proton transfer rates, 55 and, having eliminated other mechanisms from consideration, supports the existence of two distinct tautomeric forms of cresyl violet in the excited state. These tautomers would be endemic to all systems of cresyl violet in protonated solvent and we propose that the spectroscopic observation of these tautomers here is enhanced by tetrazine. Taken together, the data leave ESPT as the most likely mechanism for the fluorescence quenching of cresyl violet and its anomalous ultrafast behavior more generally. We postulate a more detailed description based on the 2D spectra and prior ultrafast work in Figure 5. Each horizontal line in Figure 5 represents a crude adiabatic electronic state. 56 The ESPT process that suppresses fluorescence is only efficient at higher excitation frequencies, resulting in two pathways for excited-state relaxation; one in which low excess vibrational energy slowly forms a fluorescent excited state (Figure 5a), and another in which progressively faster ejections of the proton at higher excitation energy leads to a non-fluorescent excited state (Figure 5b). 57,58 In the first scenario, cresyl violet undergoes a slow charge recombination that eventually releases a

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a) Sn

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Figure 5: Excited state relaxation for cresyl violet involving proton transfer steps. Horizontal lines represent crude adiabatic electronic states. (a) Slow ejection of a proton post-excitation forms a neutral (CV–H∗ ) excited state (S1 ). Fluorescence yields a neutral ground state (S0 ) that scavenges a solvent proton to recover the original ground state, CV+ . Higher excitation energy (b) results in rapid ESPT leading to a tautomer with a buckled conformation (CVT) and nonradiative decay to both the charged and neutral ground states.

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proton. In the second scenario, high frequency excitations cause significant ESPT and lead to the formation of a cresyl violet tautomer (CVT), as a proton from one of the exocyclic iminium/amine groups is rapidly ejected and subsequent protonation occurs on the oxazine ring nitrogen. In this postulated mechanism, tetrazine acts as a weak photo-base, influencing the bulk solvent dynamics to ‘trap’ the cresyl violet in the CVT conformation where the central nitrogen atom is protonated, decreasing the available excited-state population for fluorescence. The tautomer is responsible for the ESA signal at 425 THz and the delayed bleach recovery between 580 THz and 500 THz in the 2D spectra. The proposed mechanism in Figure 5 has been adjusted to resolve key observations from prior TA measurements. 9 As observed in the CWEA spectra, the initial state is not the fluorescent one; rather, the emissive state forms as a proton from cresyl violet is transferred to the solvent. As the excited state relaxes to the ground state through radiative and nonradiative decay, the proton is scavenged from the solvent to recover the charged cresyl violet (CV+ ) species, building up spectroscopic signal at 500 THz from the neutral ground state (CV–H). In native cresyl violet this happens between 5–20% of the time according to the decay associated difference spectra. A sub-population involves the CVT conformation that leads to ESA signal at 425 THz and delayed bleach recovery. The CVT conformation bears qualitative resemblance to energetic excited states responsible for high nonadiabatic coupling, where the planar structure of cresyl violet is buckled upon protonation to the central oxazine ring and the ground and exited state can be brought close in energy by some reaction coordinate. 59 The abrupt shifts in the CWEA spectra likely stem from these regions of high nonadiabatic coupling that direct excited-state population to charged and neutral ground states as cresyl violet proceeds along the ESPT reaction coordinate. 60

Conclusions In summary, based on the features of the measured 2D spectra reported here, we rule out exciton generation, photo-induced electron transfer (PET), and resonance energy transfer (RET) as poten-

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tial mechanisms for the quenching of cresyl violet by tetrazine. 29 We propose that excited-state proton transfer (ESPT) is the most reasonable mechanistic explanation, and postulate that tetrazine enhances a pathway for ESPT that deactivates the excited state of cresyl violet in an ultrafast and nonradiative manner at the expense of the fluorescent pathway. Extended to the oxazine family more generally, ESPT should play a prominent role in the discussion of the basic photophysical properties of the dyes, and poses an important reaction coordinate to exploit in the design of fluorescent switches and sensors for biological applications. For the cresyl violet system, the 2D ES results also satisfactorily conclude prior ultrafast experiments 9 where the ultimate fluorescent state was shown to be preceded by an initial excited state and abruptly bifurcated into two systems after 1.2 ps. Through ESPT, we now consider this initial state to be the charged excited state (CV+ ), which can slowly form the neutral excited state species (CV–H) or, through excess vibrational energy in the excitation, rapidly tautomerize (CVT), leading to nonradiative decay to both a charged and neutral ground state. The selection of the tautomer as the cresyl violet species that involves nonradiative decay stems in part from the spectral observation of wavepacket motion from the excited state to ground state, where the tautomeric form qualitatively resembles the high energy conformations that give rise to regions of high nonadiabatic coupling. 59 More broadly, this work highlights the utility of 2D ES for mechanistic studies of photochemical reactions. The 2D spectra allow for the evaluation of competing mechanisms, and can be complemented with additional methods for further mechanistic interpretation. For example, the postexperimental analysis method of 2D decay associated spectra, useful for identifying kinetic intermediates, 61 can expand on the mechanistic investigation of molecular energy transfer by isolating spectral changes associated with specific kinetic rates. This method requires three-dimensional datasets that remain technically challenging, but are nevertheless becoming more commonplace as ultrafast experiments in general become more widespread. As the development of 2D ES instrumentation continues and gains more application across disciplines, the technique should become a powerful tool in enabling the study of systems rising to the level of biological complexity.

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Associated Content Supporting Information Available Experimental protocols, additional 2D electronic spectra including an annotated ‘map’ of cresyl violet signals, transient absorption spectra, linear spectra and electrochemical characterization of compounds. This material is available free of charge via the Internet at http://pubs.acs. org/.

Author Information The authors declare no competing financial interest.

Acknowledgement DBT acknowledges support from the Alfred P. Sloan Foundation and the National Science Foundation under CAREER Grant No. CHE–1552235. DB acknowledges support from NSF Grant No. CHE1555116, WPC acknowledges support from the Margaret Strauss Kramer Fellowship, and BPP thanks the NSF for a Graduate Research Fellowship under grant No. DGE1342536. The authors also acknowledge Charles Payne for designing the prism-based pulse shaper used to compress the laser pulses for the ultrafast measurements.

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Graphical TOC Entry

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Resolving the Fluorescence Quenching Mechanism of an Oxazine

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