Sensitized Two-Photon Activation of Coumarin Photocages - The

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Sensitized Two-Photon Activation of Coumarin Photocages Christopher A. Hammer, Konstantin Falahati, Andreas Jakob, Robin Klimek, Irene Burghardt, Alexander Heckel, and Josef Wachtveitl J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.7b03364 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 4, 2018

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The Journal of Physical Chemistry Letters

Sensitized Two-Photon Activation of Coumarin Photocages Christopher A. Hammer1,†, Konstantin Falahati1,†, Andreas Jakob2,†, Robin Klimek2, Irene Burghardt1, Alexander Heckel2,* and Josef Wachtveitl1,* 1

Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt am Main, Max-

von-Laue-Str. 7, 60438 Frankfurt am Main, Germany 2

Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt am Main,

Max-von-Laue-Str. 7, 60438, Frankfurt am Main, Germany †

Authors contributed equally

* To whom correspondence should be addressed: [email protected] [email protected]

ACS Paragon Plus Environment

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ABSTRACT Here we report the design of a new coumarin-based photolabile protecting group with enhanced two-photon absorption. Two-photon excited fluorescence (TPEF), color-tuned ultrafast transient absorption spectroscopy and infrared (IR) measurements are employed to photochemically characterize the newly designed ATTO 390-DEACM-cargo triad. Increased two-photon cross-section values of the novel cage in comparison to the widely used protecting group DEACM ([7-(diethylamino)coumarin-4-yl]methyl) are extracted from TPEF experiments. Femtosecond pump-probe experiments reveal a fast intramolecular charge transfer, a finding which is confirmed by quantum chemical calculations. Uncaging of glutamate is monitored in IR measurements by photodecarboxylation of the carbamate linker between the photolabile protecting group and the glutamate, showing the full functionality of the novel two-photon activatable photocage.

TOC GRAPHICS

KEYWORDS Two-Photon, Photolabile Protecting Groups, TPEF, Uncaging, Glutamate, Ultrafast Spectroscopy


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In many areas of material and life sciences, massive interest to control biological and chemical systems emerged in the past few years. Light-induced triggering in such systems is a promising approach to realize highly specific control strategies.[1-3] The ability to mask the biological activity of a compound by attaching a photolabile protecting group (“photocage” or “caging group”) and set it to its active or inactive state by irradiating the photocage with light has been exploited for biological applications since the end of the 1970’s.[4,5] Since then many interesting examples have emerged using photocages or photoswitches and have demonstrated, for example, light control of gene expression,[6,7] aptamer function,[8] protein function,[9] lipids[10,11] or signaling molecules.[12] In the small molecule domain the term “photopharmacology” was coined[13,1] and, for example, the light-control of the narcotic propofol and its application to living tadpoles was demonstrated in a spectacular study.[14] Among the known photocages the photolabile protecting group DEACM exhibits outstanding properties e.g. water solubility, absorption in the visible part of the spectrum and the possibility to be introduced into nucleic acids or DNA strands, which explains its widespread utilization.[1520]

Furthermore, the uncaging mechanism of (coumarin-4-yl)methyl derivatives is well-studied

and understood and therefore DEACM is the photocage of choice.[19,21-23] Nonetheless, controlled triggering with high spatial and temporal resolution represents a further obstacle. Techniques based on two-photon absorption are able to respond to this challenge, since they enable biological applications with high 3D resolution[18,24-27] where conventional one-photon techniques provide only two-dimensional control. Moreover, wavelength ranges in the “phototherapeutic window” (650-950 nm) are accessible, which are suitable for deeper penetration into blood-perfused tissue with lower phototoxicity.[28,29] By now, the field shows considerable promise, but only few potent two-photon activatable photolabile protecting groups


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are available to date, and the rational design or computational prediction of two-photon properties is still a formidable challenge. Hence, the present study addresses a novel molecular design strategy aimed at achieving an enhanced two-photon absorption of the DEACM cage. Our concept is based on the observation that fluorophores with large two-photon cross-section are readily available whereas the majority of the known photocages have not yet reached a comparable two-photon cross-section range.[30] Hence, we explore the idea to use suitable twophoton fluorophores as antennae to operate a photocage in an intramolecular dyad. As antenna fluorophore we chose ATTO 390 (labelled I in Figure 1A) due to its significant two-photon cross section of 14 GM. The fluorophore was connected to a DEACM core via an alkyne-containing linker (labelled II in Figure 1A). As model cargo to be released from this intramolecular dyad we chose glutamate (labelled III in Figure 1A) for its potential use as light-triggerable neurotransmitter.[31,32] For the synthesis of this compound we refer to the SI. In view of working towards rational design strategies adapted to supramolecular structures like I+II, the present detailed molecular-level study combines time-resolved spectroscopy with computational analysis. Besides examining the efficiency of the I+II system, we aim to gain a detailed understanding of the intermolecular interactions that determine the dyad’s performance. As will be discussed below, energy transfer, charge transfer, and electronic delocalization will be found to work together to create a robust and performant supramolecular assembly. Two-photon absorption cross sections are determined via the method of two-photon excited fluorescence (TPEF).[33,34] To validate our measurements, we used three reference compounds (Rhodamine B, Fluorescein and Coumarin 307), (referenced to each other) and detected broadband emission to diminish perturbations (see SI).


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“Figure 1. A) Chemical structure and labelling of the triad. B) Absorption of ATTO 390 (I), the dyad consisting of ATTO 390 and DEACM with a propargyl linker in 3 position (I+II) and the latter alone (II); the triangles indicate the excitation wavelengths used in the transient absorption measurements (see Figure 2) and two-photon absorption cross sections in values of GM for I (●), I+II (∆) and II (□) determined with the method of two-photon excited fluorescence and referenced to the values of Fluorescein. For more information see SI.”

Figure 1B shows the one-photon absorption and the two-photon absorption cross sections in units of GM for ATTO 390 I, the DEACM-derivative II and our newly designed two-photon sensitized photocage I+II for excitation wavelengths ranging from 740 – 900 nm. Spectra of the reference compounds and power-dependent measurements are provided in the SI. The absorption band of I (black line) exhibits a maximum at 390 nm, while II (green) has a maximum at around


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430 nm. The DEACM-derivative II alone shows a red-shifted absorbance compared to DEACM due to its propargylic substitution in the 3-position.

The absorption band of the dyad I+II (red) reveals a maximum at 400 nm and is most likely composed of the absorption bands of I and II. Nevertheless, the shift of bands of the isolated chromophores indicates electronic interaction within I+II. For two-photon cross sections, it is clearly recognizable that the values of II are the lowest of all three compounds. On the other hand I exhibits comparatively large GM values underlining that it is a good candidate for twophoton sensitization. I+II exhibits at all excitation wavelengths a higher two-photon absorption cross-section than II and even higher values than I in the range of 820-870 nm. At this point, it should be emphasized that TPEF is an indirect method of measuring the two-photon absorption cross-section, which is strongly coupled to the two-photon fluorescence quantum yield (φ2F). In a first approximation, the one-photon excited fluorescence is similar to two-photon excited fluorescence. The one-photon excited fluorescence quantum yields (φ1F) of I, II and I+II were determined to be 1, 0.85 and 0.7, respectively (see SI for further information). By taking the φ1F into account, we assume that the two-photon absorption of I+II is even higher, an important prerequisite for two-photon activation of the photocage.

Ultrafast UV/vis pump-probe transient absorption measurements have been performed to study the photophysics of I+II. We examined the ultrafast dynamics in a wavelength dependent fashion by photoexciting the sample at 365 nm, 388 nm and 475 nm. The control of the twophoton excitation pulse diameter in pump-probe experiments is quite challenging. Since the dynamics of I+II should be independent of the excitation mechanism, the photocage is excited with one photon. The resulting transient absorption spectra are depicted in Figure 2.


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“Figure 2. Transient absorption spectra of I+II excited at A) λexc = 365 nm, B) λexc = 388 nm and C) λexc = 475 nm. The gray bar indicates that data in this range were excluded from fitting, due to the photoexcitation at 475 nm. In the lower panel the corresponding decay associated spectra are shown.” On the basis of the absorption spectra in Figure 1B, the chosen excitation wavelength of 365 nm addresses predominantly I, whereas an exclusive photoexcitation of II at 475 nm is expected and therefore should only display the photodynamics of II. Photoexcitation at 388 nm presumably displays photodynamics of both. Figure 2C shows the transient absorption spectrum after photoexcitation at 475 nm, where a negative signal (shown in blue) centered at 450 nm is visible, which represents the ground state bleach of II. The second negative signal at around 500-600 nm is assigned to stimulated emission (SE). The negative signal of the SE and a positive signal (shown in red) related to the excited state absorption (ESA) of II (390-440 nm) decay with a time constant of 2.6 ps (Figure 2C, τ1). With this time constant a third negative signal centered at


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410 nm appears, which is assigned to the ground state bleach of I. Since an intramolecular energy transfer (IET) to higher energies and as well a direct excitation of I (Figure 1B) are, unlikely, we deduce that an intramolecular charge transfer (ICT) from II to I must occur. The decay of the SE with τ1, indicates that the molecule is no longer in the excited state, although the ground state is not recovered since the GSB at 450 nm is still present. The subsequent charge recombination leads to the decay of all transient absorption signals with a time constant of 130 ps (Figure 2C; τ2). The transient absorption spectrum recorded after photoexcitation at 388 nm depicted in Figure 2B also shows three negative signals. Similar to the excitation at 470 nm, the negative signal between 500 and 600 nm is assigned to the SE, which decays faster than the GSBs around 400-475 nm, indicating an ICT as described above. However, a long time constant τ4 is necessary to describe residual SE and long-wavelength GSB signals, while no contribution of the short-wavelength GSB is visible at late delay times. The transient absorption spectrum recorded after photoexcitation at 365 nm should represent mainly the photodynamics of I (Figure 2A). In the spectrum two major negative bands centered at 400 and 500 nm and a weak negative band at approximately 450 nm are present. We assign the first negative signal (400 nm) to the short-wavelength ground state bleach of I. The weak negative signal (450 nm) belongs to the long-wavelength GSB of II. Negative signals at 500 nm are assigned to the SE. Three positive signals (shown in red) in the very blue and red part of the spectrum and centered at 475 nm are assigned to ESAs. The long-wavelength GSB assigned to II is directly present, which is most probably due to direct photoexcitation of II. An ultrafast energy transfer is ruled out, since the short-wavelength GSB of I is not decreasing on this time scale. Between 0 and 100 ps an increase of the GSBs and a decrease of the SE is observed. Intriguingly, the ESA at 650 nm remains constant, implying complex dynamics in the excited state described by τ1 and τ2, a


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relaxation to the ground state is not observed. At delay times of few hundred ps to ns the longwavelength GSB persists to a certain extent, while the short-wavelength GSB completely decreases, indicating an IET from I to II (Figure 2A, compare DAS of τ3 and τ4). Photoexcitation at 475 nm indicates a CT from II to I (Figure 2B). However, the long-lived SE and the decay of the short-wavelength GSB is a compelling fact against an ICT from II to I after the IET following photoexcitation at 365 nm. This feature can be also explained by direct photoexcitation of II. However, Figure 2B displays that direct photoexcitation of II leads to a fast recovery of the ground state. Structural changes upon photoexcitation at 365 nm could explain our observations. As a result of conformational changes, a charge transfer from II to I after an IET from I to II is not possible anymore.

To summarize, transient absorption measurements disclose the charge transfer character of photoexcited I+II. Spectral signatures are tentatively assigned to charge transfer (II to I), and energy transfer (I to II). To validate our findings in the experimental section, we performed quantum chemical calculations, as now detailed.

Ground state geometry optimization of the model system at the ߱B97XD/SVP[35,36] DFT level of theory using the Gaussian09 program package[37] reveals a π-stacked aggregation of the hydrophobic moieties (see Figure 3). The relative energetics indicate selectively high stability for the stacked conformer both in gas phase as well as solvation model embedding (see SI for further thermochemical details). A linear and hence unfolded conformation was found to be roughly 25 kcal/mol higher in energy than the stacked conformer. Note that the stacked conformation further benefits energetically from a stabilizing intramolecular hydrogen bond as indicated in red in panel A.


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Subsequent excited state analysis was performed by means of TD-DFT calculations in gas phase and using a solvation model. The bright states (with excitation energies at 3.6 eV (343 nm) and 4.1 eV (306 nm), respectively) feature non-negligible ICT character at the Franck-Condon geometry as can be inferred from the Supporting Material, thus highlighting a close electronic entanglement of the ATTO 390 (I) and the DEACM (II) subunits. The S2 state at 3.9 eV (320 nm), however, features a dominant ICT character, which is reflected by an oscillator strength reduction of one order of magnitude. The mixed character of electronic excitation is indicated by the orbital transitions in the lower panel of Figure 3. The stacked structure of the photocage thus prohibits exclusive local excitation of either the I or the II moiety of the molecule effectively, a fact that matches our experimental findings displayed in Figure 2. The picture is somewhat altered in the case of the (energetically unfavorable) unfolded model system: TD-DFT calculations reveal rather local excitation patterns on either subunits of the molecule indicating that the degree of ICT contribution may be tuned via a potentially relevant mechanistic unstacking after initial excitation. However, since such a nuclear rearrangement upon unfolding corresponds to a considerable amount of correlated geometric motion the dynamics presumably does not occur on the ultrafast time scale. Nevertheless, as competing intramolecular IET and ICT processes might be inferred at least from the longer time traces of Figure 2 (100-ps regime) the interplay of π-stacked and unfolded conformations can be of importance regarding further elucidation of photochemical scenarios for this class of compounds.


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“Figure 3. A) Lewis structure of the calculated model system with indication of ATTO 390 (I) and DEACM moieties (II). Intramolecular hydrogen bond highlighted in red. B) Main orbital transitions resembling local (blue) and CT type (red) excitation character. Orbitals obtained in energetic order at the TD-߱B97XD/SVP level of theory.”

To demonstrate the functionality of the newly designed photocage we introduced a glutamate (III) into I+II linked via a carbamate to the coumarin moiety yielding compound I+II+III (see SI). We continuously irradiated the sample I+II+III with an LED with a central wavelength of 365 nm and performed UV/vis and FTIR measurements (see Figure 4).


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“Figure 4. Continuous illumination (λexc = 365 nm) of I+II+III monitored via A) UV/vis spectroscopy and E) FTIR spectroscopy (λexc = 365 nm). B) Absorption difference spectra of the UV/vis measurements. C) Transient at 421 nm with a linear fit to determine the uncaging quantum yield. The formation of dissolved CO2 is shown in D) and the corresponding decrease of the stretch mode of the C-O of the carbamate is depicted in F).”

Figure 4A shows that the absorption of I+II+III changes by continuous illumination. It can be clearly seen that the absorbance in the wavelength range between 390 and 420 nm increases while the shoulder in the long wavelength-range at around 475 nm of sample I+II+III merges into the blue region. Figure 4B shows the difference absorption spectra in the UV/vis with an isosbestic point underlining the formation of a photoproduct. To clarify the findings in the UV/vis region, measurements in the IR were additionally performed. FTIR-measurements reveal the formation of dissolved carbon dioxide (2337 cm-1) resulting from the decomposition of the carbamate (see Figure 4D). Moreover the band of the carbonyl stretch mode of the carbamate linker (1722 cm-1) decreases on the same time scale as the formation of CO2. The release of CO2 by uncaging a product linked via a carbamate has been shown in the past and monitors in this


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case the uncaging of glutamic acid.[19,38] Accordingly, we assign the absorption change observed in the stationary UV/vis measurements (Figure 4A) to uncaging of glutamate indicated by the isosbestic point and determined an uncaging quantum yield of 1.5 % (IR and UV/vis measurements on the reference compound II+III reveal an uncaging quantum yield which is one order of magnitude lower than of I+II+III, for further information see SI). In summary, we designed a photocage with a DEACM scaffold with enhanced two-photon absorption compared to regular DEACM. TPEF experiments reveal larger two-photon absorption cross sections of I+II than DEACM but not as large as ATTO 390. Transient absorption measurements disclose the charge transfer character of photoexcited I+II due to π-stacking confirmed by quantum chemical calculations. Charge transfer is detected from II to ATTO 390 (I), while most likely an energy transfer from I to II was observed. From stationary absorption measurements φunc = 1.5 % was determined. FTIR measurements reveal photodecarboxylation and hence uncaging of glutamate. We present a fully functional photocage with enhanced twophoton absorption. This work reports the functional intramolecular attachment of a two-photonsensitizing moiety, resulting in a molecular triad consisting of light harvesting unit, photocage and effector and should help to develop future design strategies for even better two-photon absorbing photocages.

EXPERIMENTAL METHODS The synthesis of I+II and I+II+III are described in detail in the SI. TPEF experiments were carried out using a tunable Ti-sapphire laser (Tsunami, Spectra-Physics) producing 150 fs laser pulses with a repetition rate of 80 MHz. The fluorescence after two-


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photon excitation was coupled into a spectrograph (SpectraPro 300i, Acton Research Corporation) with a CCD-camera (EEV 400_1340F, Roper Scientific). Transient UV/vis-pump-vis-probe experiments. Measurements were performed using a CLARKMXR CPA 2110 with a central wavelength of 775 nm (pulse length 150 fs, repetition rate 1 kHz). The excitation pulses with a central wavelength of 365 nm were generated by sum frequency of 690 nm with the fundamental laser beam. Excitation pulses with a central wavelength of 475 nm were generated by a home-built two stage non-collinear optical parametric amplifier (NOPA). Broadband probe pulses were produced by guiding the laser fundamental trough a sapphire crystal and were split for referenced measurements and subsequently recorded via photodiode arrays (Hamamatsu). For further information regarding the experimental setup see reference 39. Time-resolved data obtained from transient absorption measurements were analyzed with OPTIMUS, where a global lifetime analysis (GLA) was used to fit the data with a set of exponential decay functions.[40] Stationary absorption measurements UV/vis spectra were recorded with a Specord S600 (Analytik Jena), while FTIR measurements were carried out with a VERTEX 80 (Bruker, Ettlingen). For the irradiation of the samples a Thorlabs LED with a central wavelength of 365 nm was used.

ASSOCIATED CONTENT The Supporting Information contains: Chemical synthesis. One-photon absorption, two-photon excited fluorescence and wavelength-dependent TPEF spectra of Rhodamine B, Fluorescein and Coumarin 307. Power-dependent fluorescence spectra of Rhodamine B. Fit of transient absorption spectrum after photoexcitation at 475 nm. Calculations of uncaging and fluorescence


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quantum yield. Quantum chemical data concerning thermochemistry and excited state constitution.

AUTHOR INFORMATION Corresponding Authors *A.H.: [email protected] *J.W.: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Deutsche Forschungsgemeinschaft (DFG) by means of the research training group “CLiC” (GRK 1986, Complex scenarios of light-control). We thank Strahinja Lucic for help with the stationary spectroscopic characterization. REFERENCES (1) Lerch, M. M.; Hansen, M. J.; van Dam, G. M.; Szymanski, W.; Feringa, B. L. Emerging Targets in Photopharmacology. Angew. Chem. Int. Ed. 2016, 55, 10978–10999. (2) Klán, P.; Šolomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.; Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy. Chem. Rev. 2013, 113, 119−191. (3) Brieke, C.; Rohrbach, F.; Gottschalk, A.; Mayer, G.; Heckel, A. Light-Controlled Tools. Angew. Chem. Int. Ed. 2012, 51, 8446–8476.


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Table of contents 42x34mm (600 x 600 DPI)



Figure 1 77x101mm (300 x 300 DPI)


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Figure 2 125x95mm (300 x 300 DPI)



Figure 3 115x190mm (96 x 96 DPI)


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Figure 4 129x69mm (300 x 300 DPI)


Sensitized Two-Photon Activation of Coumarin Photocages - The

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