Sequential Uncaging with Green Light can be Achieved by Fine-Tuning the Structure of a Dicyanocoumarin Chromophore

Article abstract of DOI:10.1002/open.201700067

We report the synthesis and photochemical properties of a series of dicyanocoumarinylmethyl (DEAdcCM)- and dicyanocoumarinylethyl (DEAdcCE)-based photocages of carboxylic acids and amines with absorption maximum around 500 nm. Photolysis studies with green light have demonstrated that the structure of the coumarin chromophore as well as the nature of the leaving group and the type of bond to be photocleaved (ester or carbamate) have a strong influence on the rate and efficiency of the uncaging process. These experimental observations were also supported by DFT calculations. Such differences in deprotection kinetics have been exploited to sequentially photolyze two dicyanocoumarin-caged model compounds (e.g., benzoic acid and ethylamine), and open the way to increasing the number of functional levels that can be addressed with light in a single system, particularly when combining dicyanocoumarin caging groups with other photocleavable protecting groups, which remain intact under green light irradiation.

Full text of DOI:10.1002/open.201700067

DOI: 10.1002/open.201700067
Sequential Uncaging with Green Light can be Achieved by
Fine-Tuning the Structure of a Dicyanocoumarin
Chromophore
[
a]
[a]
[a, d]
[a]
[a, b, d]
Albert Gandioso, Marta Palau, Alba Nin-Hill,
Ivanna Melnyk, Carme Rovira,
[c]
[a, e]
[a]
[a]
Santi Nonell, Dolores Velasco,
Jaume Garcꢀa-Amorꢁs, and Vicente Marchꢂn*
We report the synthesis and photochemical properties of
a series of dicyanocoumarinylmethyl (DEAdcCM)- and dicyano-
coumarinylethyl (DEAdcCE)-based photocages of carboxylic
acids and amines with absorption maximum around 500 nm.
Photolysis studies with green light have demonstrated that the
structure of the coumarin chromophore as well as the nature
of the leaving group and the type of bond to be photocleaved
vations were also supported by DFT calculations. Such differen-
ces in deprotection kinetics have been exploited to sequential-
ly photolyze two dicyanocoumarin-caged model compounds
(e.g., benzoic acid and ethylamine), and open the way to in-
creasing the number of functional levels that can be addressed
with light in a single system, particularly when combining di-
cyanocoumarin caging groups with other photocleavable pro-
tecting groups, which remain intact under green light
irradiation.
(ester or carbamate) have a strong influence on the rate and
efficiency of the uncaging process. These experimental obser-
1
. Introduction
Light is an excellent non-invasive stimulus for controlling the
outcome of molecular processes with high spatiotemporal pre-
an exquisite control of complex biological processes has also
been achieved by using caged analogs of bioactive com-
pounds (for example, peptides, proteins, oligonucleotides, neu-
[1]
cision and without causing contamination of the sample.
[3]
Owing to these extraordinary properties and to the high versa-
tility and efficiency of light, the use of photocleavable protect-
ing groups (PPGs; also commonly referred to as caging
groups) have found widespread applications in several fields,
rotransmitters, antibiotics, agrochemicals), which can be pre-
pared by modifying an essential functionality with the appro-
priate PPG. Therefore, activation of the resulting caged com-
pound requires irradiation with light of the appropriate wave-
length and intensity to trigger the unmasking of such
functionality and, for instance, the recovery of the biological
activity of the parent compound.
[
2]
from organic synthesis to materials science. In recent years,
[a] A. Gandioso, M. Palau, A. Nin-Hill, I. Melnyk, Dr. C. Rovira, Dr. D. Velasco,
[2]
Dr. J. Garcꢀa-Amorꢁs, Dr. V. Marchꢂn
Among the large number of PPGs reported to date, cou-
marin derivatives fulfill some of the criteria for an ideal caging
group: they are relatively easy to synthesize from commercially
available precursors and amenable to structural modifications
both to facilitate the attachment of the compound to be
caged through different types of bonds (for example, ester,
amide, carbamate, or carbonate) and to improve their photo-
physical and photochemical properties. The latter are impor-
tant issues as most reported caging groups require UV irradia-
tion for photoactivation, which is toxic and has a poor capacity
of penetration in biological tissues. Among the coumarin-
based PPGs described to date, those possessing an N,N-dialkyl-
amino group at position 7 have larger molar extinction coeffi-
cients at longer wavelengths than their analogs with hydroxy
or methoxy substituents at that position and exhibit moderate
to high photochemical quantum yields. Moreover, the replace-
ment of such N,N-dialkyamino substituents at the 7-position
by azetidine, aziridine, or monoalkylated cyclic amines can be
used to improve the photophysical properties of coumarin-
Departament de Quꢀmica Inorgꢃnica i Orgꢃnica, Secciꢁ de
Quꢀmica Orgꢃnica, IBUB (AG, VM)
Universitat de Barcelona, 08028 Barcelona (Spain)
E-mail: vmarchan@ub.edu
[
b] Dr. C. Rovira
Instituciꢁ Catalana de Recerca i Estudis AvanÅats (ICREA)
0
8010 Barcelona (Spain)
[
c] Dr. S. Nonell
Institut Quꢀmic de Sarriꢃ
Universitat Ramon Llull, 08017 Barcelona (Spain)
[
d] A. Nin-Hill, Dr. C. Rovira
Institut de Quꢀmica Teꢄrica i Computacional (IQTCUB)
Universitat de Barcelona, 08028 Barcelona (Spain)
[
e] Dr. D. Velasco
Institut de Nanociꢅncia i Nanotecnologia (IN2UB)
Universitat de Barcelona, 08028 Barcelona (Spain)
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under https://doi.org/10.1002/
open.201700067.
ꢃ 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
[4]
based fluorophores. Recently, the modification of the carbon-
yl group of the lactone in the N,N-diethylamino(coumarin 4-yl)-
methyl platform has been proved to be a valuable way to fur-
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ther shift the wavelength of maximum absorption, which can
be exploited to trigger uncaging with blue, cyan, or green
no)coumarin-4-yl)ethyl (DEACE), generates a secondary cationic
intermediate upon the photo S 1 reaction of the correspond-
N
[
5]
light. Indeed, dicyanomethylenecoumarin derivatives, which
are obtained by conjugating two nitrile groups through posi-
tion 2 of the coumarin moiety, are particularly attractive as
they exhibit a maximum absorption around 500 nm in the 7-di-
ethylamino series (487 nm in a coumarin derivative containing
a benzyloxymethyl group at position 4 described by Jullien
ing DEACE-protected Asp derivative, no significant differences
were found with respect to the DEACM analog regarding the
photochemical properties, particularly the uncaging quantum
yield at 360 nm. By contrast, incorporation of this modification
in dicyanomethylene-coumarin-caged Asp monomers led to
a faster uncaging process with green light (505 nm), which
was attributed to the higher stability of the carbocation
[
5c]
and co-workers and 475 nm when this position was occu-
[6]
[5e]
pied by a methyl as described by Granberg and colleagues ).
Uncaging of dicyanocoumarin-caged cyclic morpholino oligo-
nucleotides has been exploited in the context of gene silenc-
intermediate.
Taking into account the potential of dicyanocoumarin deriv-
atives as PPGs, in this work we have synthesized a series of
N,N-diethylamino-dicyanocoumarinylmethyl (DEAdcCM)- and
dicyanocoumarinylethyl (DEAdcCE)-caged model compounds
(1–6, Scheme 1B) with the aim of studying their photophysical
and photochemical properties, particularly how uncaging is in-
fluenced both by the chemical structure of the coumarin chro-
mophore (R=H or Me) and by the nature of the leaving group
(that is, aliphatic or aromatic carboxylic acid or amine). Photol-
ysis studies with the corresponding caged model compounds
upon irradiation with green light (505 nm) as well as DFT com-
putational calculations have shown that both the structure of
the dicyanocoumarin caging group and of the leaving group
have a strong influence on the rate and efficiency of the pho-
tolysis process. Interestingly, by selecting the appropriate com-
bination of dicyanocoumarin-based caging groups, we have
demonstrated that carboxylic acid and amine functionalities
can be sequentially released from the corresponding caged
compounds by irradiation at 505 nm.
[
5a]
ing by Deiters’ and Chen’s group, and very recently we have
described the synthesis of a dicyanocoumarin-caged RGD pep-
[
5e]
tide for photocontrolled-targeted drug delivery.
Neverthe-
less, such modifications of the lactone in the N,N-diethylami-
no(coumarin 4-yl)methyl platform usually led to a significant
reduction of the uncaging quantum yield compared with the
[5c]
parent oxygenated coumarin or its thionated analog, which
could suppose a limitation for future applications of dicyano-
coumarins as PPGs.
The mechanism of photocleavage of coumarinylmethyl
esters has been well established for the classical 7-(N,N-diethy-
lamino)coumarin-4-yl)methyl (DEACM) caging group (R=H;
[
7]
see Scheme 1A). Upon electronic excitation, a solvent-assist-
2
. Results and Discussion
2.1. Synthesis of DEAdcCM- and DEAdcCE-Caged Model
Compounds
As shown in Scheme 2, the synthesis of the DEAdcCM-caged
model compounds (1, 2, and 5) was planned from 4-(acetoxy-
[5c]
methyl)-7-(N,N-diethylamino)-2-thiocoumarin (11), a key in-
termediate that can be prepared from the commercially avail-
Scheme 1. General mechanism of the photolysis of a DEACM- or DEACE-
caged carboxylic acid (A) and structure of the DEAdcCM- and DEAdcCE-
caged model compounds synthesized in this work (B).
[5c,e]
able precursor 7 in four steps.
Condensation of the thionat-
ed coumarin with malononitrile in the presence of triethyla-
mine and silver nitrate afforded 4-(acetoxymethyl)-2-(dicyano-
methylene)-7-(N,N-diethylamino)-coumarin (1) in 93% yield
after column chromatography. Hydrolysis of the acetyl group
with HCl followed by esterification with benzoic acid afforded
coumarin derivative 2. Reaction of the alcohol intermediate 12
with ethyl isocyanate in the presence of triethylamine afforded
compound 5 as a model for the protection of primary amines
with DEAdcCM through a carbamate linkage. All the com-
pounds were fully characterized by ESI mass spectrometry and
ed photoheterolytic bond cleavage produces the free carboxyl-
ic acid and the corresponding coumarinylmethyl alcohol,
which is generated by reaction of the cationic intermediate
with water followed by a fast deprotonation. Previous studies
have demonstrated that stabilization of this intermediate (by
incorporation of electron-donating substituents at the coumar-
in skeleton) and of the released carboxylate (by decreasing its
basicity) have a positive effect on the photocleavage of cou-
1
13
H and C NMR spectroscopy.
[
7]
marinylmethyl esters. Recently, del Campo and collaborators
have investigated the incorporation of a ethyl group at posi-
tion 4 of the DEACM moiety (R=Me, see Scheme 1A) for in-
creasing the stability of the corresponding caged aspartic acid
The synthesis of the DEAdcCE-caged model compounds (3,
4, and 6) is summarized in Scheme 3. First, reaction of the al-
dehyde 8 with CH MgCl in anhydrous THF at low temperature
3
[
5e]
(ꢀ788C) afforded coumarin alcohol 13 as a racemic mixture.
[8]
monomer during Fmoc-tBu solid-phase peptide synthesis.
Despite the fact that this new caging group, 7-(N,N-diethylami-
After esterification with benzoic acid followed by thionation
with Lawesson’s reagent and condensation with malononitrile,
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Scheme 2. Synthesis of dicyanocoumarinylmethyl-caged compounds (1, 2,
and 5).
Figure 1. Comparison of the absorption (solid lines) and fluorescence
dotted lines) spectra of DEAdcCM- (top) and DEAdcCE-caged (bottom)
(
model compounds with those of the corresponding dicyanocoumarin alco-
hols (12 and 16, respectively). Solvent: Tris buffer (20 mm, pH 7.5)/MeCN 1:1
(
v/v).
Table 1. Photophysical parameters for dicyanocoumarin-caged model
compounds (1–6) and for the corresponding photoreleased coumarin al-
[
a]
cohol derivatives (12 and 16).
[
a]
Compound
Absorption
Emission
[f]
l
max
e(lmax
[mM cm
)
l
Em
Dn
f
Em
[
b]
ꢀ1
ꢀ1 [c]
[d]
ꢀ1 [e]
[
nm]
]
[nm]
[cm ]
1
2
3
4
5
6
486
487
489
489
485
488
478
482
30.4
32.5
31.2
32.0
32.8
33.0
32.5
33.0
556
558
549
548
555
544
545
539
2633
2613
2193
2202
2600
2067
2572
2194
0.17
0.15
0.11
0.11
0.15
0.13
0.17
0.14
Scheme 3. Synthesis of dicyanocoumarinylethyl-caged compounds (3, 4, and
6).
1
1
2
6
the DEAdcCE-caged benzoic acid 4 was obtained. Hydrolysis of
followed by esterification with acetic acid or by reaction
[
a] Absorption and emission spectra were recorded in a 1:1 (v/v) mixture
4
of Tris buffer (20 mm, pH 7.5) and MeCN at 258C. [b] Wavelength of the
absorption maximum. [c] Molar absorption coefficient at lmax. [d] Wave-
length of the emission maximum upon excitation at 460 nm. [e] Stokes’
shift. [f] Fluorescence quantum yield.
with ethyl isocyanate, afforded the two remaining DEAdcCE-
caged model compounds (3 and 6, respectively).
2.2. Absorption and Emission Properties of Dicyano-
coumarin Derivatives
[5c]
The photophysical characterization of the dicyanocoumarin-
for 2. As shown in Table 1, esterification either with benzoic
acid or with acetic acid caused a slight redshift (about 5–
11 nm) with respect to the parent coumarin alcohol derivatives
(12 or 16), and in all cases the presence of the methyl group
on the coumarin skeleton caused an additional redshift com-
pared with non-methylated analogs (compare 1 and 3). A simi-
lar bathochromic effect was observed for the emission wave-
length of the caged compounds upon excitation at 460 nm
when compared with the corresponding coumarin alcohol de-
caged model compounds (1–6) was carried out in a degassed
1
:1 (v/v) mixture of Tris buffer (20 mm, pH 7.5) and MeCN. The
UV/Vis absorption and emission spectra of the compounds are
shown in Figure 1, and their photophysical properties are re-
ported in Table 1. The maximum absorption wavelength, the
shape of the absorption curve, and the molar extinction coeffi-
cients of the ester (1–4) and carbamate (5–6) derivatives were
very similar, and consistent with the values previously reported
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[
a]
Table 2. Photochemical properties of compounds 1–6.
Caged compound
R
Leaving compound Uncaging
2
[b]
%
2
Caged compound at irradiation time
min
k
u
e(505 nm)
[mm cm
10 ꢄfPh
e(505 nm)ꢄfPh
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
5 min
10 min
20 min
[min
]
]
[m cm ]
1
2
3
4
5
6
H
H
AcOH
PhCOOH
90
55
28
15
97
71
62
20
2
0
90
36
22
0
0
0
79
7
0
0
0
0
45
0
0.125
0.314
0.644
0.948
0.036
0.208
21.4
23.4
22.0
22.6
19.9
21.3
0.09ꢁ0.03 19
0.15ꢁ0.01 34
0.28ꢁ0.03 62
0.33ꢁ0.00 75
Me AcOH
Me PhCOOH
H
EtNH
2
2
0.03ꢁ0.01
5
Me EtNH
0.12ꢁ0.02 25
[a] Data presented: Percent of the starting caged compound at each irradiation time, uncaging first-order rate constant, molar extinction coefficient at the
irradiation wavelength, uncaging quantum yield, and uncaging efficiency at 505 nm. [b] Results are the mean ꢁSDs from two independent experiments.
rivatives. However, the emission maximum was blueshifted in
the DEAdcCE compounds with respect to the DEAdcCM coun-
terparts (compare lem for 1 and 3). The relative fluorescence
quantum yields were calculated by using fluorescein as the
standard (fEm =0.92 in 0.1m NaOH in water). All the com-
pounds exhibited good quantum yields (0.11<f <0.17) and
F
ꢀ
1
Stokes’ shifts around 2600 cm for DEAdcCM derivatives and
ꢀ
1
of 2000–2200 cm for the DEAdcCM analogs.
2.3. Photolysis Studies of Dicyanocoumarin-Caged
Compounds
From the comparison of the molar extinction coefficients of
DEAdcCM- and DEAdcCE-caged model compounds at their re-
spective lmax and at 505 nm (for example, for 1: e=
ꢀ1
ꢀ1
ꢀ1
ꢀ1
3
0.4 mm cm and 21.4 mm cm , respectively; see Table 1
and Table 2), we decided to explore the use of green light to
carry out the photoactivation studies. Green light is more suit-
able for biological applications than UV or blue light owing to
[
9]
its reduced photocytotoxicity and deeper optical penetration
[
10]
in tissues. A 505 nm LED source was used for irradiation of
solutions of compounds 1–6 in a 1:1 (v/v) mixture of Tris
buffer (20 mm, pH 7.5) and MeCN at 378C, and the course of
the uncaging process was monitored by reversed-phase HPLC-
ESI MS (see the Experimental Section for further details). As
shown in Figure 2, the concentration of all the compounds de-
creased gradually with time upon irradiation with green light.
In the case of DEAdcCM-caged compounds, coumarin alcohol
1
2 was released as the main photolytic product (Scheme 4).
Figure 2. Plot of the temporal evolution of the amount of caged compounds
–4 (top) and 5–6 (bottom) in a 1:1 (v/v) mixture of Tris buffer (20 mm,
pH 7.5) and MeCN at 378C. Irradiation was performed at 505 nm with con-
Conversely, photoactivation of DEAdcCE-caged compounds
gave coumarin alcohol 16 together with other minor photolyt-
ic byproducts, particularly vinyl coumarin 17 (Scheme 4). The
formation of this alkene can be explained by a b-elimination
reaction from the secondary carbocation intermediate generat-
ed upon photoheterolysis of the ester bond (see Scheme 1).
Overall, the results from the photolysis experiments revealed
the influence of 1) the structure of the dicyanocoumarin
caging group, 2) the nature of the leaving group, and 3) the
type of bond photocleaved (ester or carbamate), on the time
necessary to release the carboxylic acid or the amine from the
corresponding caged compounds. First, it is worth noting that
photolysis of DEAdcCE-caged compounds was much faster
than that of DEAdcCM (see Figure 2 and Table 2); this result
1
tinuous stirring.
can be attributed to the different stability of the carbocation
intermediates (primary or secondary) generated during photo-
heterolysis of the ester or carbamate bonds in the dicyanocou-
marin-caged model compounds. For example, the release of
acetic acid from 3 was almost complete after 5 min of irradia-
tion whereas it required 20 min to completely uncage com-
ꢀ
1
ꢀ1
pound 1 (k =0.125 min for 1 vs. k =0.644 min for 3). Sim-
u
u
ilar trends were found when comparing 2 and 4, and 5 and 6
(Table 2), in agreement with previous observations with (cou-
&
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2.4. Computational Studies
Having stablished how the photolysis rate of the dicyanocou-
marin-caged model compounds is influenced by both the in-
troduction of a methyl group in the coumarin moiety next to
the bond to be photocleaved and the nature of the leaving
group, we decided to get insight into these experimental ob-
servations from a theoretical point of view. DFT calculations
were carried out with the Gaussian 09 (G09) software package
Scheme 4. Photolysis of DEAdcCM- and DEAdcCE-caged model compounds.
(
see the Experimental Section for further details). As previously
stated, photocleavage of coumarin-caged compounds pro-
ceeds through a photo S 1 mechanism and the stability of the
N
[
7]
marin-4-yl)methyl esters (DEACM). For these compounds, the
rate of the overall uncaging process was found to be influ-
enced by the rate constant of the initial heterolytic bond cleav-
age (see Scheme 1A), which was higher when the primary cou-
marinyl carbocation intermediate was stabilized though the in-
corporation of substituents with strong electron-donating abili-
two components of the resulting ion pair (coumarinyl carbo-
cation and carboxylate) has a strong influence on the rate of
the first photocleavage step and, consequently, on the overall
[7]
rate of the uncaging process. For this reason, we calculated
the energy differences between the caged compounds (1–4)
and their corresponding intermediate species. Consistent with
the experimental data, DFT calculations predicted that photoly-
sis of DEAdcCE-caged compounds (3) gives a small stabiliza-
[
7]
ty (alkoxy or dialkylamino). In our case, the same tendency
was observed by shifting the nature of the carbocation inter-
mediate from primary to secondary through the incorporation
of a methyl group in the coumarin moiety in a position adja-
cent to the bond to be photocleaved.
ꢀ
1
tion of the ion pair intermediate (DE=1.2 kcalmol ) com-
pared with the DEAdcCM (1) analogs with the same leaving
group (Figure 3, top panel). A similar trend was obtained when
comparing 2 and 4 (see Figure S11 in the Supporting Informa-
tion). On the other hand, the calculations also supported the
experimental observations regarding the nature of the leaving
group: stabilization of the ion pair generated upon cleavage of
Second, the photocleavage process was also faster with
good leaving groups on the molecule to be caged (compare
benzoate vs. acetate in Table 2). Again, this can be attributed
to the stabilization of the second component of the carbocat-
ion–carboxylate ion pair and, consequently, to an increase of
[
7]
the rate constant of the first bond cleavage. As expected, the
ꢀ
1
best combination in terms of uncaging rate (k =0.948 min )
u
was obtained for compound 4 as the release of benzoate is ac-
companied by the formation of a secondary carbocation inter-
mediate. Third, the photolysis of carbamate-coumarin caged
amines (5 and 6) was much slower than that of the ester ana-
logs (1 and 2, respectively), which can be attributed to the
lower leaving-group ability of the carbamic acid anion com-
pared with carboxylate. This tendency has been found in other
carbonate-, carbamate-, and thiocarbonate-coumarin caged
compounds in which the final uncaged product is released
[
11]
after thermal decarboxylation of the intermediate. Neverthe-
less, our results point out that the introduction of an additional
methyl group in the skeleton of other coumarin derivatives
could be used to increase the deprotection rate of carbamate-
caged amines as well as that of carbonate-caged alcohols and
phenols, where photolysis is usually much slower compared
with uncaging of ester-caged carboxylic acids.
As shown in Table 2, the uncaging quantum yield (f ) of
Ph
the caged model compounds was also determined upon irradi-
ation at 505 nm (see the Experimental Section for further de-
tails). The product of the extinction coefficient at the irradia-
tion wavelength and the quantum yield can be considered as
an efficiency parameter of the process (eꢄfPhot), for quantify-
ing the uncaging process rate as a function of the photolysis
[
2b,12]
time.
In good agreement with results from photolysis
Figure 3. Free energy differences for the transformation of dicyanocoumar-
in-caged compounds in their respective intermediates (primary or secondary
dicyanocoumarinyl carbocation + acetate or benzoate): 1 vs. 3 (top) and
1 vs. 2 (bottom).
studies, the quantum yields for DEAdcCE derivatives were
higher than those of the DEAdcCM analogs (compare 1 and 3,
and 2 and 4), resulting in a high product eꢄfPhot
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ꢀ
1
the ester bond in 2 was about 4 kcalmol higher compared
with compound 1 (Figure 3, bottom panel), which agrees with
the increased acidity of benzoic acid (pK =4.2) compared with
a
acetic acid (pK =4.76). A similar value was obtained when
a
comparing 3 and 4 (Figure S11 in the Supporting Information).
2.5. Sequential Uncaging Studies
The kinetics of a photodeprotection process depend both on
the spectral properties of the PPG (lmax and e) and on the pho-
tolysis quantum yield (f ). As found by us and others, the re-
Ph
action quantum yield can be fine-tuned through the modifica-
tion of several parameters, including the structure of the chro-
mophore, the nature of the connecting atom in the molecule
to be caged, the type of photocleavable bond (ester, carba-
mate, etc.), and the basicity of the leaving group (aromatic or
[
2b,3n,13]
aliphatic carboxylate).
With the aim of controlling with
light multiple functionalities in a single system, two main ap-
proaches have been explored to selectively address several un-
caging processes. The first approach relies on using a set of
different caging groups, which can be sequentially photolyzed
at different wavelengths of light; this method requires the use
of two or more PPGs with significantly different spectral prop-
erties (lmax, e, and f ). A second approach is well exemplified
Ph
[
3n]
by the work of Heckel and collaborators, who demonstrated
that selective uncaging can be achieved by irradiation at
a single wavelength of light by exploiting the differences in
quantum yield (and for instance of deprotection kinetics) of
the same PPG when attached to the molecule to be caged
through a different connecting atom.
Figure 4. Plot of the temporal evolution of the amount of caged compounds
4, 5, and 10 (top) and HPLC chromatograms at a different time (bottom)
after irradiation at 505 nm in a 1:1 (v/v) mixture of Tris buffer (20 mm,
pH 7.5) and MeCN at 378C. *Unidentified coumarin side products.
On the basis of the photolysis studies and of the differences
in the uncaging quantum yield between DEAdcCM- and DEAd-
cCE-caged compounds (Table 2), we wondered if sequential
uncaging of at least two dicyanocoumarin-protected function-
alities in the same reaction mixture could be achieved by irra-
diating with the same green light source. First, an equimolar
mixture of compound 1 (DEAdcCM-caged acetic acid) and 4
ture of 4, 5, and 10, dicyanocoumarin-caged compounds (4
and 5) were sequentially uncaged whereas DEACM-caged
acetic acid (10) remained intact, even after 1 h irradiating at
505 nm (Scheme 5). Hence, the combination of two structurally
related dicyanocoumarin caging groups (DEAdcCM and DEAd-
cCE) with other PPGs with distinct absorption properties (for
example, removable with UV or blue light) allows us to in-
crease the number of levels of selectivity that can be ad-
dressed with light.
(DEAdcCE-caged benzoic acid) as dicyanocoumarin-caged
models of aliphatic or aromatic carboxylic acids, respectively, in
the presence of uracil as an internal standard, was irradiated at
505 nm. Unfortunately, HPLC analysis revealed (Figure S12 in
the Supporting Information) that despite the fact that 4 was
completely uncaged in 4 min, about 40% of 1 was also depro-
tected. However, irradiation at 505 nm of a mixture of 4 (DEAd-
cCE-caged benzoic acid) and 5 (DEAdcCM-caged ethylamine)
demonstrated that a carboxylic acid and an amine can be se-
quentially uncaged upon irradiation with the same wavelength
of light by exploiting the different deprotection kinetics pro-
vided by the two dicyanocoumarin caging groups when con-
nected to the caged functionalities through ester or carbamate
linkages: 4 was completely uncaged in 3 min leaving >90% of
5
intact (Figure S13 in the Supporting Information). Such as
promising results, led us to include compound 10 (see
Scheme 2) as a model for the protection of a carboxylic acid
with the classical DEACM coumarin, whose absorption at
Scheme 5. Schematic representation of the sequential uncaging with green
light of two dicyanocoumarin-caged compounds (4 and 5) in the presence
of DEACM-caged acetic acid (10).
505 nm is negligible. As shown in Figure 4 and Figure S14
(Supporting Information), upon irradiation at 505 nm of a mix-
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rupole detector coupled to an HPLC, and high-resolution (HR) ESI-
MS on a LC/MS-TOF instrument.
3
. Conclusions
We have described the synthesis and photophysical properties
of novel DEAdcCM- and DEAdcCE-based photocleavable pro-
tecting groups and their suitability for caging carboxylic acids
and amines through ester and carbamate bonds, respectively.
Such chromophores have a maximum absorption around
Synthesis of DEAdcCM-Caged Model Compounds
Compound 1: Malononitrile (1.1 g, 16.7 mmol) was added to a solu-
tion of 11 (1.01 g, 3.32 mmol; see the Supporting Information) in
anhydrous MeCN (100 mL). After addition of NEt3 (9.1 mL,
65.5 mmol) under Ar atmosphere and protection from light, the re-
action mixture, which acquired an intense red color, was stirred for
500 nm, which facilitates uncaging with the biologically com-
patible green light, which is non-toxic and penetrates deeper
into tissues than UV or blue light. Photolysis studies of the cor-
responding dicyanocoumarin-caged model compounds at
2
0 min. Then, silver nitrate (1.13 g, 6.66 mmol) was added and
stirred at room temperature for 4 h under an Ar atmosphere and
protection from light. Finally, the solvent was removed under
vacuum and the crude was purified by column chromatography
505 nm and DFT calculations have demonstrated that the
structure of the coumarin chromophore as well as the nature
of the leaving group strongly influence the rate and efficiency
of the uncaging process, which has been exploited to sequen-
tially photolyze two dicyanocoumarin-caged model com-
pounds (benzoic acid and ethylamine) in the presence of
DEACM-caged acetic acid. Such significant differences in the
deprotection kinetics open the way to increasing the number
of functional levels that can be addressed with light in a single
system, particularly when combining dicyanocoumarin caging
groups with other PPGs that remain stable upon irradiation at
(
silica gel, 50–100% CH Cl in hexanes, then from CH Cl to 0.5%
2
2
2
2
MeOH in CH Cl ), to give 1.04 g (93% yield) of an orange solid.
2
2
1
Characterization: TLC: R (2.5% MeOH in CH Cl ): 0.60. H NMR
f
2
2
(400 MHz, CDCl ): d=7.33 (1H, d, H5, J=9.2 Hz), 6.75 (1H, br s,
3
H3), 6.66 (1H, dd, H6, J=9.2, 2.4 Hz), 6.60 (1H, d, H8, J=2.4 Hz),
5
.24 (2H, d, CH , J=1.0 Hz), 3.45 (4H, q, CH NEt , J=7.2 Hz), 2.21
2 2 2
1
3
(
(
3H, s, CH ), 1.24 ppm (6H, t, CH3 NEt , J=7.2 Hz); C NMR
3 2
101 MHz, CDCl ): d=171.8, 170.2, 155.0, 151.7, 146.1, 124.9, 114.6,
3
1
13.8, 110.6, 106.9, 105.9, 97.4, 61.1, 55.5, 45.0, 20.8, 12.4 ppm; HR
ESI-MS (positive mode): m/z=338.1491 (calcd mass for C H N O
3
1
9
20
3
505 nm. Finally, our findings on the impact of the stabilization
+
[M+H] : 338.1505).
of the carbocation intermediate generated during photoheter-
olysis of ester or carbamate bonds in dicyanocoumarin-based
photocages could be applied to other coumarin-based systems
to improve their photochemical properties. Overall, this would
increase the potential of known coumarin-based caging
groups for designing wavelength-selective systems in which
multiple functionalities can be independently addressed by the
different wavelengths of light. Work is in progress to extend
this approach to other coumarin-based chromophores with im-
proved redshifted properties.
Compound 12: A solution of HCl in 1,4-dioxane (4m, 1.5 mL,
6 mmol) was added to 1 (0.80 g, 2.37 mmol) in absolute ethanol
(60 mL). The reaction mixture was heated at 908C for 12 h. Once
the reaction was finished, the solvent was removed under reduced
pressure and purified by chromatography column (silica gel, 50–
1
0
00% of CH Cl in hexane, then 0–0.8% of MeOH in CH Cl ) to give
2 2 2 2
.52 g of a yellow-orange solid (74% yield). Characterization: TLC:
1
R (2.5% MeOH in CH Cl ): 0.62; H NMR (400 MHz, CDCl ): d=7.34
f
2
2
3
(1H, d, H5, J=9.2 Hz), 6.93 (1H, br t), 6.65 (1H, dd, H6, J=9.2,
2.4 Hz), 6.60 (1H, d, H8, J=2.4 Hz), 4.88 (2H, d, CH , J=5.8 Hz),
2
3
1
.45 (4H, q, CH2 NEt , J=7.2 Hz), 2.06 (1H, t, OH, J=5.8 Hz),
2
.23 ppm (6H, t, CH3 NEt , J=7.2 Hz); ESI-MS (positive mode):
2
+
m/z=295.75 (calcd mass for C H N O [M+H] : 296.14).
17
18
3
2
Experimental Section
Compound 2: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
EDC)·HCl (51 mg, 0.33 mmol), coumarin 12 (75 mg, 0.25 mmol), 4-
Materials and Methods
(
Common chemicals and solvents (HPLC grade or reagent grade
quality) were purchased from commercial sources and used with-
out further purification. Aluminium plates coated with a 0.2 mm
thick layer of silica gel 60 F254 were used for thin-layer chromatog-
raphy analyses (TLC), whereas flash column chromatography purifi-
cation was carried out by using silica gel 60 (230–400 mesh). Re-
versed-phase high-performance liquid chromatography (HPLC)
analyses were carried out with a Jupiter Proteo C column (250ꢄ
dimethylaminopyridine (DMAP; 40 mg, 0.33 mmol), and benzoic
acid (45 mg, 0.37 mmol) were dissolved in dry CH Cl (2.5 mL) at
2
2
08C under an Ar atmosphere. After 10 min at 08C, the reaction
mixture was stirred at room temperature for 4 h in the dark and
under Ar. After evaporation under reduced pressure and purifica-
tion by column chromatography (silica gel, CH Cl ), 78 mg of an
2
2
orange solid were obtained (77% yield). Characterization: TLC: Rf
1
(5% MeOH in CH Cl ): 0.86; H NMR (400 MHz, CDCl ): d=8.10 (2H,
18
2
2
3
ꢀ1
4
.6 mm, 90 ꢅ, 4 mm, flow rate: 1 mLmin ) by using linear gradi-
m, Ph), 7.62 (1H, m, Ph), 7.49 (2H, t, Ph, J=7.6 Hz), 7.43 (1H, d, H5,
J=9.2 Hz), 6.89 (1H, br s, H3), 6.69 (1H, dd, H6, J=9.2, 2.4 Hz), 6.62
(1H, d, H8, J=2.4 Hz), 5.47 (2H, d, CH , J=0.8 Hz), 3.46 (4H, q, CH
ents of 0.045% trifluoroacetic acid (TFA) in H O (A) and 0.036%
2
TFA in MeCN (B). NMR spectra were recorded at 258C with
2
2
1
3
a 400 MHz spectrometer by using deuterated solvents. Tetrame-
Et, J=7.2 Hz), 1.24 ppm (6H, t, CH3 Et, J=7.2 Hz); C NMR
1
thylsilane (TMS) was used as an internal reference (0 ppm) for H
(101 MHz, CDCl ): d=171.8, 165.8, 155.1, 151.7, 146.0, 133.8, 129.8,
3
spectra recorded in CDCl and the residual signal of the solvent
128.9, 128.8, 125.1, 114.5, 113.8, 110.7, 107.1, 106.4, 97.4, 61.7, 55.6,
3
1
3
(
77.16 ppm) was used for C spectra. Chemical shifts are reported
45.0, 12.4 ppm; HR ESI-MS (positive mode): m/z=400.1643 (calcd
+
in parts per million (ppm) in the d scale, coupling constants in Hz,
mass for C H N O [M+H] : 400.1661).
24
22
3
3
and multiplicity as follows: s (singlet), d (doublet), t (triplet), q
(
quadruplet), qt (quintuplet), m (multiplet), dd (doublet of dou-
Compound 5: NEt3 (95 mL, 0.70 mmol) and ethyl isocyanate
(107 mL, 1.35 mmol) were added to a solution of 12 (101 mg,
0.34 mmol) in a mixture of dry MeCN (3 mL) and CH Cl (1.8 mL)
blets), td (doublet of triplets), ddd (doublet of doublet of dou-
blets), br (broad signal). Electrospray ionization mass spectra (ESI-
MS) were recorded with an instrument equipped with single quad-
2
2
under an Ar atmosphere. Once the reaction was finished (typically
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2
–3 h according to TLC), the solvent was removed under reduced
pressure and purified by chromatography column (silica gel, 0–
.35% of MeOH in CH Cl ) to give 85 mg of an orange solid (68%
was added to 4 (0.24 g, 0.58 mmol) in MeOH (50 mL). The reaction
mixture was heated at 908C for 15 h. Once the reaction was finish-
ed, the solvent was removed under reduced pressure and purified
by chromatography column (silica gel, 0–3% of MeOH in CH Cl ) to
0
2
2
1
yield). Characterization: TLC: R (2% MeOH in CH Cl ): 0.50; H NMR
f
2
2
2
2
(
6
400 MHz, CDCl ): d=7.33 (1H, d, H5, J=9.2 Hz), 6.75 (1H, br s),
.65 (1H, dd, H6, J=9.2, 2.8 Hz), 6.59 (1H, d, H8, J=2.8 Hz), 5.26
give 0.13 g of an orange solid (71% yield). Characterization: TLC: Rf
3
1
(5% MeOH in CH Cl ): 0.40; H NMR (400 MHz, CDCl ): d=7.46 (1H,
2
2
3
(
2H, br s, CH ), 4.97 (1H, br s, NH), 3.45 (4H, q, CH2 NEt , J=
d, H5, J=9.2 Hz), 6.91 (1H, s, H3), 6.66 (1H, dd, H6, J=9.2, 2.6 Hz),
2
2
7
7
.2 Hz), 3.29 (2H, qt, CH Et, J=7.2 Hz), 1.23 (6H, t, CH NEt , J=
.2 Hz), 1.20 ppm (3H, t, CH3 Et, J=7.2 Hz); C NMR (101 MHz,
6.59 (1H, d, H8, J=2.6 Hz), 5.19 (1H, m, CH-CH ), 2.10 (1H, d, OH,
2
3
2
3
13
J=3.6 Hz), 3.45 (4H, q, CH NEt , J=7.2 Hz), 1.57 (3H, d, CH-CH ,
2
2
3
1
3
CDCl ): d=172.0, 155.1, 154.9, 151.6, 147.3, 124.7, 114.8, 113.9,
J=6.8 Hz), 1.24 ppm (6H, t, CH3 NEt2, J=7.2 Hz);
C NMR
3
1
10.6, 106.9, 105.1, 97.3, 61.3, 55.2, 44.9, 36.2, 15.1, 12.4 ppm; HR
ESI-MS (positive mode): m/z=367.1752 (calcd mass for C H N O
3
(101 MHz, CDCl ): d=172.4, 156.1, 155.2, 151.4, 125.3, 114.9, 114.2,
110.5, 107.0, 104.2, 97.4, 66.0, 54.7, 44.9, 24.0, 12.5 ppm; HR ESI-MS
3
20
23
4
+
[M+H] : 367.1770).
(positive mode): m/z=310.1542 (calcd mass for C H N O
2
1
8
20
3
+
[
M+H] : 310.1556).
Compound 3: Acetic anhydride (85 mL, 0.77 mmol) and DMAP
94 mg, 0.77 mmol) were added to a solution of 16 (47.6 mg,
0.15 mmol) in anhydrous CH Cl (4 mL) under an Ar atmosphere.
The reaction mixture was stirred in the dark overnight. After re-
moval of the solvent under reduced pressure, the product was pu-
rified by column chromatography (silica gel, 0–0.1% MeOH in
Synthesis of DEAdcCE-Caged Model Compounds (3, 4, 6)
Compound 15: EDC·HCl (1.6 g, 10.6 mmol), coumarin 13 (2.52 g,
(
2
2
[5e]
9
.6 mmol),
DMAP (1.3 g, 10.6 mmol), and benzoic acid (1.4 g,
1
1.6 mmol) were dissolved in dry CH Cl (40 mL) at 08C under an
2
2
Ar atmosphere. After 10 min at 08C, the reaction mixture was
stirred at room temperature for 17 h in the dark and under Ar. Sub-
CH
tion: TLC: R
d=7.43 (1H, d, H5, J=9.2 Hz), 6.72 (1H, s, H3), 6.67 (1H, dd, H6,
J=9.2, 2.4 Hz), 6.60 (1H, d, H8, J=2.4 Hz), 6.08 (1H, q, CH-CH , J=
6.8 Hz), 3.45 (4H, q, CH NEt , J=7.2 Hz), 2.16 (3H, s, CH CO), 1.58
, J=6.8 Hz), 1.24 ppm (6H, t, CH NEt , J=7.2 Hz);
): d=172.1, 169.9, 155.3, 152.3, 151.6,
2 2
Cl ) to give 21 mg (40% yield) of an orange solid. Characteriza-
1
sequently, the organic solution was diluted with CH Cl2 (60 mL)
(1% MeOH in CH Cl ): 0.66; H NMR (400 MHz, CDCl ):
f 2 2 3
2
and washed with saturated NH Cl (25 mL), 5% aqueous NaHCO3
4
(
2ꢄ25 mL), water (25 mL), and dried over anhydrous Na SO . After
3
2
4
filtration and evaporation under vacuum, the desired product (14)
was obtained and used without further purification in the next
step. Lawesson’s reagent (2.5 g, 6.8 mmol) was added to a solution
of 14 in toluene (50 mL). The mixture was stirred at 1058C over-
night under an Ar atmosphere and protected from light. Then, the
solvent was evaporated under vacuum. After purification by
2
2
3
(3H, d, CH-CH
3
3
2
13
C NMR (101 MHz, CDCl
3
125.2, 114.8, 113.9, 110.7, 106.7, 104.3, 97.6, 67.2, 55.2, 45.0, 21.1,
12.5 ppm; HR ESI-MS (positive mode): m/z=352.1648 (calcd mass
+
for C20
H
N
22
O
[M+H] : 352.1661).
3
3
column chromatography (silica gel, 50–65% CH Cl2 in hexane),
2
2
.53 g of an orange solid (69% yield from 13) was obtained. Char-
Compound 6: NEt
4.8 mmol) were added to a solution of 16 (62 mg, 0.20 mmol) in
a mixture of dry MeCN (2.5 mL) and CH Cl (1.5 mL) under an Ar at-
3
(600 mL, 4 mmol) and ethyl isocyanate (380 mL,
1
acterization: TLC: R (5% MeOH in CH Cl ): 0.28; H NMR (400 MHz,
CDCl ): d=8.10 (2H, m, Ph), 7.62 (1H, m, Ph), 7.55 (1H, d, H5, J=
f
2
2
3
2
2
1
0 Hz), 7.48 (2H, t, Ph, J=7.6 Hz), 7.16 (1H, s, H3), 6.68 (2H, m,
mosphere. Once the reaction was finished (24 h according to TLC),
the solvent was removed under reduced pressure and purified by
H6+H8), 6.32 (1H, q, CH-CH , J=6.8 Hz), 3.44 (4H, q, CH NEt , J=
3
2
2
7
.2 Hz), 1.71 (3H, d, CH-CH , J=6.8 Hz), 1.23 ppm (6H, t, CH NEt ,
chromatography column (silica gel, 0–0.75% of MeOH in CH
give 42 mg of an orange solid (63% yield). Characterization: TLC:
Cl ) to
2 2
3
3
2
1
3
J=7.2 Hz); C NMR (101 MHz, CDCl ): d=197.4, 165.5, 159.5, 150.9,
3
1
1
6
47.9, 133.5, 129.8, 129.5, 128.6, 124.9, 119.2, 110.4, 108.0, 97.6,
7.6, 45.0, 21.0, 12.4 ppm. ESI-MS (positive mode): m/z=382.66
R
f
(2% MeOH in CH
(1H, d, H5, J=9.2 Hz), 6.73 (1H, s, H3), 6.67 (1H, dd, H6, J=9.2,
2.4 Hz), 6.59 (1H, d, H8, J=2.4 Hz), 6.03 (1H, q, CH-CH , J=6.8 Hz),
.90 (1H, br s, NH), 3.45 (4H, q, CH NEt , J=7.2 Hz), 3.24 (2H, qt,
2
Cl ): 0.46; H NMR (400 MHz, CDCl ): d=7.44
2
3
+
(
calcd mass for C H NO S [M+H] : 382.15).
3
2
2
24
3
4
2
2
Compound 4: A solution of malononitrile (52 mg, 0.79 mmol) and
NH-CH
2
-CH
, J=7.2 Hz), 1.17 ppm (3H, t, NH-CH
C NMR (101 MHz, CDCl ): d=172.3, 155.3, 154.8, 153.5, 151.6,
3
, J=7.2 Hz), 1.55 (3H, d, CH-CH
3
, J=6.8 Hz), 1.23 (6H, t,
NEt (0.2 mL, 1.54 mmol) in anhydrous MeCN (2 mL) was added to
CH
3
NEt
2
2
-CH , J=7.2 Hz);
3
3
1
3
a solution of 15 (202 mg, 0.53 mmol) in MeCN (8 mL) under an Ar
atmosphere. The reaction mixture was stirred in the dark for 2 h.
Then, AgNO3 (196 mg, 1.15 mmol) was added and stirring was
maintained for 2 h. After filtration and removal of the solvent
under reduced pressure, the product was purified by column chro-
matography (silica gel, 0–100% CH Cl in hexane) to give 158 mg
3
125.2, 115.0, 114.1, 110.6, 106.8, 104.0, 97.5, 67.2, 54.7, 44.9, 36.1,
15.1, 12.5 ppm; HR ESI-MS (positive mode): m/z=381.1922 (calcd
mass for C21
+
H
N
4
O
3
[M+H] : 381.1927).
25
2
2
(
72% yield) of an orange solid. Characterization: TLC: R (5% MeOH
f
Photophysical Properties
1
in CH Cl ): 0.63; H NMR (400 MHz, CDCl ): d=8.09 (2H, m, Ph),
2
2
3
7
7
.61 (1H, m, Ph), 7.54 (1H, d, H5, J=9.2 Hz), 7.49 (2H, t, Ph, J=
.6 Hz), 6.85 (1H, s, H3), 6.69 (1H, dd, H6, J=9.2, 2.6 Hz), 6.61 (1H,
Absorption spectra were recorded with a Varian Cary 500 UV/Vis/
NIR spectrophotometer at 258C. Emission spectra were registered
in a Photon Technology International (PTI) fluorimeter. Solutions
for emission measurements were adjusted to concentrations such
that the absorption was around 0.04 at the excitation wavelength
(l =460 nm). Fluorescence quantum yields, F , were calculated
d, H8, J=2.6 Hz), 6.33 (1H, q, CH-CH , J=6.8 Hz), 3.45 (4H, q, CH2
NEt , J=7.2 Hz), 1.73 (3H, d, CH-CH , J=6.8 Hz), 1.24 ppm (6H, t,
CH3 NEt , J=7.2 Hz); C NMR (101 MHz, CDCl ): d=172.1, 165.5,
3
2
3
13
2
3
1
1
55.4, 152.2, 151.6, 133.7, 129.7, 129.2, 128.7, 125.3, 114.7, 113.9,
10.7, 106.8, 104.6, 97.6, 67.9, 55.3, 45.0, 21.1, 12.5 ppm. HR ESI-MS
positive mode): m/z=414.1805 (calcd mass for C H N O
Ex
F
from Equation (1):
(
[
25
24
3
3
+
M+H] : 414.1818).
s
2
Abs A n
s
F
F ¼ F
ð1Þ
F
s
Abs A n2s
Compound 16: A solution of aqueous HCl (37%, 50 mL, 0.58 mol)
&
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ÝÝ These are not the final page numbers!

in which the superscript “s” stands for standard samples, Abs is the
mined by calculating the ratio between the initial slope and the
number of absorbed photons by the sample as determined by acti-
nometry.
absorbance at the excitation wavelength (l ), A is the integrated
Ex
[18]
area of the corresponding emission spectrum, and n is the refrac-
[14]
tive index of the solvent used. The uncertainty in the experimen-
tal value of F has been estimated to be approximately 10%. The
F
Computational Calculations
standard fluorophore used for the determination of the fluores-
cence quantum yield was fluorescein dissolved in aqueous sodium
hydroxide (0.1m) with a reported absolute fluorescence quantum
According to the Bell–Evans–Polanyi principle, it can be considered
that the changes in the exothermicity of the reaction for two simi-
lar dicyanocoumarin-caged compounds is proportional to the dif-
ferences in reaction activation energies. Thus, we computed the
free energy changes associated with the transformation of each
compound in its corresponding ion pair intermediate species and
compared the results for each pair of similar compounds (that is,
differing either in the type of carbocation or anion released in the
reaction). All calculations were carried out with the Gaussian 09
[15]
yield, F , of 0.92. All samples were measured in 1ꢄ1 cm quartz
F
cuvettes (Hellma).
Irradiation Experiments
Photolysis studies were performed at 378C in a custom-built irradi-
ation setup from Microbeam including a cuvette, thermostated
cuvette holder, and a mounted high power LED of 505 nm
[19]
(
G09) software package. The electronic structures were comput-
[20]
ꢀ2
ed by Density Functional Theory (DFT) by using the B3LYP func-
(
100 mWcm ). In a typical experiment, the irradiation samples
[21]
[22]
tional and the 6-31G** basis set. The most stable enantiomers
of all chiral centers were chosen for structural optimizations, which
were performed in an implicit water solvent (SMD solvation
contained the dicyanocoumarin-caged model compound (20 mm)
and 12 mL of a 30 mm solution of uracil (internal standard) in a 1:1
(
v/v) mixture of Tris buffer pH 7.5 and MeCN. After irradiation, the
[
23]
model ).
samples were analyzed by reversed-phase HPLC-ESI MS with a Jupi-
ter Proteo C column (250ꢄ4.6 mm, 90 ꢅ, 4 mm, flow rate:
1
8
ꢀ1
1
mLmin ) by using linear gradients of 0.1% formic acid in H O
2
(
A) and 0.1% formic acid in MeCN (B).
Acknowledgments
This work was supported by funds from the Spanish Ministerio de
Uncaging Quantum Yield Determination
Economꢀa
y Competitividad (grants CTQ2014-52658-R and
CTQ2015-65770-P MINECO/FEDER) and the Generalitat de Catalu-
nya (2014SGR187 and XRB). The authors acknowledge the assis-
tance of Dr. Irene Fernꢂndez and Laura Ortiz (MS), and Dr. Fran-
cisco Cꢂrdenas (NMR) from CCiTUB. A.G. is a recipient fellow of
the University of Barcelona.
The uncaging quantum yield for coumarin derivatives 1–6 upon il-
lumination at 505 nm was estimated by comparing the initial rate
of photo-uncaging with the rate of photobleaching of (E)-2-[1-(2,5-
dimethyl-3-furyl-9-ethylidene]-3-isopropylidene succinic anhydride
[16,17]
(
Aberchrome 540), a well-known chemical actinometer.
Indeed,
the ring-opening back reaction of this particular organic chromo-
phore can be easily exploited for actinometry in the visible region
of the electromagnetic spectrum by measuring the decrease in ab- Conflict of Interest
sorbance at 494 nm of a known volume of the red toluene solu-
tion, which was previously obtained by irradiating the ring-opened
isomer of Aberchrome 540 with UV light (Philips high pressure
The authors declare no conflict of interest.
mercury lamp, 500 W, 320 nm<lIrrad <390 nm) for 5 min.
Keywords: caged compounds · coumarins · photolysis ·
protecting groups · UV/Vis spectroscopy
The photon flow onto the sample was calculated by using
Equation (2):
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DA ꢂ V
¼
ð2Þ
F ꢂ e ꢂ Dt
l
2
2
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ꢀ1
ꢀ1
chrome 540 at 494 nm (8200m cm ). The quantum yield for the
photobleaching reaction in toluene at 218C, was calculated by the
following expression [Eq. (3)]:
ˇ
ˇ
ˇ
F ¼ 0:178ꢀ2:4 ꢂ 10ꢀ4 l
ð3Þ
l
Ac
where lAc is the activation wavelength (in this instance, l =
Ac
5
1
05 nm). The photon flow thus determined was 1.05ꢄ
ꢀ7
ꢀ1
0
Einsteins .
The kinetic traces for the uncaging step were fitted to a monoexpo-
nential decreasing function and the initial slope was determined
by differentiation. The uncaging quantum yield, F , was deter-
Ph
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ꢃ 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
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Published online on && &&, 2017
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ÝÝ These are not the final page numbers!

FULL PAPERS
Lighting the way: Photolysis studies
with a series of dicyanocoumarin-based
photocages of carboxylic acids and
amines have shown that both the struc-
ture of the coumarin chromophore, the
nature of the leaving group, and the
type of bond to be photocleaved (ester
or carbamate) strongly influence the
rate and efficiency of the uncaging pro-
cess upon green-light irradiation.
A. Gandioso, M. Palau, A. Nin-Hill,
I. Melnyk, C. Rovira, S. Nonell, D. Velasco,
J. Garcꢀa-Amorꢁs, V. Marchꢂn*
&& – &&
Sequential Uncaging with Green Light
can be Achieved by Fine-Tuning the
Structure of a Dicyanocoumarin
Chromophore
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ꢃ 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ