Letter

Cite This: J. Phys. Chem. Lett. 2018, 9, 1448−1453 pubs.acs.org/JPCL

Sensitized Two-Photon Activation of Coumarin Photocages

Christopher A. Hammer,†,§ Konstantin Falahati,†,§ Andreas Jakob,‡,§ Robin Klimek,‡ Irene Burghardt,†
Alexander Heckel,*,‡ and Josef Wachtveitl*,†
†
Institute of Physical and Theoretical Chemistry and ‡Institute of Organic Chemistry and Chemical Biology, Goethe University
Frankfurt am Main, Max-von-Laue-Straße 7, 60438 Frankfurt am Main, Germany
*
S Supporting Information

ABSTRACT: Here we report the design of a new coumarin-based

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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-
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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 experi-
ments. Femtosecond pump−probe experiments reveal a fast intramolecular
charge transfer, a finding that 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.

I n many areas of material and life sciences, massive interest to

control biological and chemical systems have 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
1970s.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 lipids10,11 or signaling molecules.12 In the
small molecule domain, the term “photopharmacology” was
coined13,1 and, for example, the light-control of the narcotic
propofol and its application to living tadpoles was demon-
strated 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.15−20 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 conven-
tional 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 photo-
labile protecting groups 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 a
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 two-photon fluorophores as
antennae to operate a photocage in an intramolecular dyad. As
the antenna fluorophore we chose ATTO 390 (labeled 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 (labeled II in Figure 1A). As model
cargo to be released from this intramolecular dyad we chose
glutamate (labeled 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 toward 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
Received: December 20, 2017
Accepted: March 2, 2018
Published: March 2, 2018

© 2018 American Chemical Society 1448 DOI: 10.1021/acs.jpclett.7b03364

J. Phys. Chem. Lett. 2018, 9, 1448−1453
The Journal of Physical Chemistry Letters
Figure 1. (A) Chemical structure and labeling 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 black 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 (Δ),
and II (□) determined with the method of two-photon excited
fluorescence and referenced to the values of Fluorescein. For more
information, see SI.
of the I+II system, we aim to gain a detailed understanding of
the intramolecular 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 supra-
molecular 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 com-
pounds (Rhodamine B, Fluorescein, and Coumarin 307),
(referenced to each other) and detected broadband emission to
diminish perturbations (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 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
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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 DEACM 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 two-
photon 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 measure-
ments 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 and 475
nm. The control of the two-photon 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.
On the basis of the absorption spectra in Figure 1B, the
chosen excitation wavelength of 365 nm addresses predom-
inantly I, whereas an exclusive photoexcitation of II at 475 nm
is expected and therefore should only display the photo-
dynamics 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 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 recombina-
tion 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 475 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. In the transient absorption spectrum recorded after
photoexcitation at 365 nm where mainly I should absorb
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The Journal of Physical Chemistry Letters
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.
(Figure 2A) 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 ≥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 residual 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 relaxation to the ground state is not
observed. At delay times of few hundred picoseconds to
nanoseconds, the long-wavelength 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 2C). 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 2C 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 observa-
tions. 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),
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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 ωB97XD/SVP35,36 DFT level of theory using the
Gaussian09 program package37 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.
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 1 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
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Figure 3. (A) Lewis structure of the calculated model system with
indication of ATTO 390 (I) and DEACM moieties (II). Intra-
molecular 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-ωB97XD/
SVP level of theory.
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 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.
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 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 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 1 order of magnitude lower
than that of I+II+III; for further information, see the 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-measure-
ments reveal photodecarboxylation and hence uncaging of
glutamate. We present a fully functional photocage with
enhanced two-photon absorption. This work reports the
successful intramolecular attachment of a two-photon-sensitiz-
ing 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 syntheses of I+II, II+III 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-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. Measure-
ments were performed using a CLARK-MXR 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 noncollinear 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
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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 panel D, and the corresponding decrease of the stretch mode of the C−O of the carbamate is depicted
in panel F.
regarding the experimental setup see ref 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
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jpclett.7b03364.
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 Rhod-
amine B; fit of transient absorption spectrum after
photoexcitation at 475 nm; calculations of uncaging and
fluorescence quantum yield; control experiments with II
+III; quantum chemical data concerning thermochemis-
try and excited state constitution (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: heckel@uni-frankfurt.de (A.H.).
*E-mail: wveitl@theochem.uni-frankfurt.de (J.W.).
ORCID
Irene Burghardt: 0000-0002-9727-9049
Alexander Heckel: 0000-0003-3541-4548
Josef Wachtveitl: 0000-0002-8496-8240
Author Contributions
§
Authors contributed equally
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Letter
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by the Deutsche Forschungsgemein-
schaft (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.
■
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