Light triggering of 5-aminolevulinic acid from fused coumarin ester cages

Article abstract of DOI:10.1039/c6nj03787a

The evaluation of the photorelease of 5-aminolevulinic acid (5-ALA), a small molecule which has considerable interest in the area of medicine as a photosensitizer prodrug in PDT and cosmetic treatments, and in agriculture as a herbicide/insecticide, was carried out by using a series of fused coumarin derivatives with different substituents and ring fusions in the preparation of 5-ALA photosensitive ester cages, in order to tune the photophysical and photolytic properties of the resulting conjugates. This study was intended to assess the viability of the photorelease of 5-ALA from lipophilic conjugates since it has hydrophilic character, does not penetrate efficiently through the skin or cell membranes and appropriate derivatisation can enhance its lipophilicity and its application scope in biological environment. Photolytic studies were performed on the ester cages by irradiation in a photochemical reactor at 254, 300, 350 and 419 nm, using methanol/HEPES buffer 80 : 20 solutions as solvent. The data obtained confirmed the suitability of the tested photosensitive moieties for the release of 5-aminolevulinic acid at the various wavelengths of irradiation. As well as the photolysis, the photophysics of the compounds was characterised by both steady state and time-resolved methods which uncovered the presence of different fluorescing species. Additionally, an on-off experiment was carried out by using two excitation sources (cleave and probe) to enable fluorescence to follow the kinetics of the process and to ascertain optical control over the bond scission.

Full text of DOI:10.1039/c6nj03787a

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The role of atropisomers on the photo-reactivity and fatigue of
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Journal Name
RSCPublishing
ARTICLE
Light triggering of 5-aminolevulinic acid from fused
coumarin ester cages
Cite this: DOI: 10.1039/x0xx00000x
Ana M. S. Soares,a Graham Hungerford,b M. Sameiro T. Gonçalves a and Susana P. G.
Costa a,*
Received 00th January 2012,
Accepted 00th January 2012
The evaluation of the photorelease of 5ꢀaminolevulinic acid (5ꢀALA), a small molecule which
has considerable interest in the areas of medicine as a photosensitizer prodrug in PDT and
cosmetic treatments, and in agriculture as herbicide/insecticide, was carried out by using a
series of fused coumarin derivatives with different substituents and ring fusions in the
preparation of 5ꢀALA photosensitive ester cages, in order to tune the photophysical and
photolytic properties of the resulting conjugates. This study was intended to assess the viability
of photorelease of 5ꢀALA from lipophilic conjugates since it has hydrophilic character, does
not penetrate efficiently through the skin or cell membranes and proper derivatisation can
enhance its lipophilicity and its application scope in biological environment. Photolysis studies
were performed on the ester cages by irradiation in a photochemical reactor at 254, 300, 350
and 419 nm, using methanol/HEPES buffer 80:20 solutions as solvent. The data obtained
confirmed the suitability of the tested photosensitive moieties for the release of 5ꢀ
aminolevulinic acid at the various wavelengths of irradiation. As well as the photolysis, the
photophysics of the compounds were characterised by both steady state and timeꢀresolved
methods which uncovered the presence of different fluorescing species. Additionally, an onꢀoff
experiment was carried out by using two excitation sources (cleave and probe) to enable
fluorescence to follow the kinetics of the process and to ascertain optical control over the bond
scission.
DOI: 10.1039/x0xx00000x
www.rsc.org/
a carbon chain attached to 5ꢀALA results in increased lipophilicity
and in consequence higher membrane and skin permeability. The
advantage of 5ꢀALA derivatives over 5ꢀALA can be mainly ascribed
Introduction
5ꢀAminolevulinic acid (5ꢀALA) is
a key precursor in the
to: (i) the rate at which these compounds reach the target site, (ii) the
rate at which they reach the intracellular space and (iii) the rate of
their enzymatic conversion into photoactive compounds.21 For MAL,
it has been reported that it also offers better tumour selectivity and
less pain during PDT with less patient discomfort compared to
ALA.22
Also, the herbicidal properties of 5ꢀALA are well documented. It
belongs to the group of photodynamic herbicides/insecticides and its
mechanism of action depends on its conversion to tetrapyrroles
within plants/insects after application in the dark. This provides plant
growth regulating properties at low concentrations and may enhance
agricultural productivity by enhancing photosynthesis, suppressing
respiration and stimulating carbon dioxide uptake.23ꢀ25
In prodrug design, it is of the utmost importance to control the
scission of chemical bonds between the active drug and the
promoiety that masks the activity, in order to regulate in time and
space the availability of molecules with biological function, in the
most biocompatible manner possible. Among the various triggering
stimuli that can be used, i.e. enzymes, reducing or oxidising agents,
temperature or pH, the use of light represents a fastꢀdeveloping
methodology for application in controlled drug delivery.26ꢀ28
Photochemical cleavage is a very mild method that allows the
cleavage of specific bonds within a structure with the consequent
removal of protecting groups/promoieties and the on demand/on site
release of an active molecule. Thus, photoactivable prodrugs
biosynthesis of porphyrins such as chlorophyll and heme. It is
frequently used with PDT because it can be interconverted to a
potent photosensitizer, protoporphyrin IX (PpIX), via the heme
biosynthetic pathway in mitochondria. 5ꢀAminolevulinic acidꢀbased
photodynamic therapy (ALAꢀPDT) is gaining increasing acceptance
in medicine as an effective technique for the treatment of a variety of
neoplastic lesions and premalignant disorders.1
The first topical application of 5ꢀALA in the treatment of basal cell
carcinoma was reported in 1990 and, since then, the clinical use of
ALAꢀPDT has steadily grown and the methyl ester of 5ꢀALA has
been approved for the treatment of basal cell carcinoma.2ꢀ4 Other
applications of ALAꢀPDT include treatment of actinic keratosis,
squamous cell carcinoma, Bowen’s disease, acne, onychomycosis,
verrucae and photorejuvenation,5ꢀ11 and it can also be used as a
diagnostic tool for the visualization of precancerous changes in the
mucosae by fluorescence spectroscopy.12,13
The main disadvantage of 5ꢀALA is that is poorly absorbed by cells
due to its high hydrophilicity, by virtue of its zwitterion nature at
physiological pH, resulting in low bioavailability. However, this
solubility enables fast clearance of 5ꢀALA from the body. In order to
overcome the solubility issues, numerous efforts have been made to
design 5ꢀALA prodrugs with a more favourable solubility profile
such as esters,14ꢀ18 and peptide derivatives.19,20 The most successful
5ꢀALA derivatives are its methyl ester (methyl aminolevulinate,
MAL) and hexyl ester (hexyl aminolevulinate, HAL). Elongation of
This journal is © The Royal Society of Chemistry 2013
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VJieowuArrtnicalelONnlainme e
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DOI: 10.1039/C6NJ03787A
incorporate a photosensitive promoiety (usually of heterocyclic nature) linked covalently to the active drug molecule.
O
H
Cl
Cl
Cl
O
N
Boc
MeO
MeO
O
O
O
O
O
O
O
O
O
1a
3a (20%)
O
H
O
N
Boc
H2N
O
H2N
O
O
1b
3b (42%)
O
O
δ
H
N
β
O
O
Boc
Boc
HO
N
α
H
O
2
H2N
H2N
O
O
KF, DMF, rt, 24h
1c
1d
3c (55%)
O
H
N
O
Cl
Boc
O
N
O
N
O
O
O
O
O
3d (62%)
O
H
Cl
O
N
Boc
O
O
N
O
O
N
O
O
O
O
O
O
O
1e
3e (56%)
hν
MeOH/HEPES
(80:20)
2
+ photo by-products
Scheme 1. Synthesis of ester cages of 5ꢀaminolevulinic acid 3aꢀe and photorelease of the Nꢀprotected carboxylic acid 2.
Results and discussion
Attempts to improve and tune the photosensitivity of the
abovementioned heterocyclic promoieties to be adequate for
biological applications have been achieved through synthetic
Synthesis of 5-aminolevulinic acid conjugates 3a-e
tailoring in terms of substituents present in the structure and/or
structural adjustments such as ring fusion and expansion of the
aromatic system.
In the last few years, our research interests involve the release of
bioorganic molecules from conjugates/cages possessing heterocyclic
moieties specifically designed for using light as the triggering
stimulus, to act as photoremovable protecting groups and
phototriggers. Benzocoumarins and oxazole fused coumarins are
examples of oxygen heterocycles used previously in the caging of
amino acids, neurotransmitters, and also butyric acid.29ꢀ38
Considering the photolytic behaviour of such moieties and the
consequent improvement as photolabile groups, the present work
aims to evaluate their behaviour in the photorelease of 5ꢀ
aminolevulinic acid, in an attempt to enhance its lipophilicity and its
application scope assisted by light in biological environment.
The photosensitive fused coumarins 1aꢀe were prepared as
previously reported by us in good to excellent yields, through
Pechmann
condensation
and
intramolecular
cyclisation
reactions.29,36ꢀ38 5ꢀ(NꢀtertꢀButoxycarbonyl)aminolevulinic acid 2 was
prepared from 5ꢀaminolevulinic acid (5ꢀALA) by a standard amino
group protection procedure with tertꢀbutylpyrocarbonate.17
The chloromethyl precursors 1aꢀe were reacted with NꢀBocꢀ5ꢀALA
2 in the presence of potassium fluoride in DMF,39 to afford the
corresponding ester conjugates 3aꢀe in fair to moderate yields
(Scheme 1). As a representative example, conjugate 3c was
subjected to acidic treatment with TFA in dichloromethane to
remove the Nꢀprotecting group yielding the corresponding conjugate
4c with the free amino group (Scheme 2).
2 | J. Name., 2012, 00, 1-3
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New Journal of Chemistry
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ARTICLE
DOI: 10.1039/C6NJ03787A
1a29 383 3.70 471
88
11
0.01
0.02
0.08
0.06
0.04
0.28
0.23
0.32
0.06
0.39
0.12
O
O
NH2
1b36 291 5.32 302
1c37 291 3.92 318
1d38 341 3.98 397
1e38 306 3.85 444
O
O
27
TFA
H2N
3c
O
56
DCM, rt, 1h
138
103
29
4c (68%)
376 5.81 479
291 3.66 320
291 3.71 340
340 3.89 417
347 3.86 453
291 3.70 339
3a
3b
3c
3d
3e
4c
h
ν
MeOH/HEPES
(80:20)
49
O
77
+
photo by-products
HO
NH2
106
48
O
5
Scheme 2. Synthesis and photolysis of deprotected 5ꢀALA conjugate
4c.
The fused coumarin oxazole conjugates 3d,e also showed a
bathochromic shift compared to the latter but not as pronounced (≈
Compounds 3aꢀe and 4c were fully characterised by high resolution 50 nm). The same trend was seen in the fluorescence wavelength
mass spectrometry, IR, 1H and 13C NMR spectroscopy. The IR maxima. Considering conjugates 3b and 3c which differ in the
spectra of compounds 3aꢀe and 4c displayed stretching vibration relative position of the electron donor amino group, the λem was
bands of the ester carbonyl group from 1699 to 1735 cmꢀ1. 1H NMR longer for substitution at position 7 (compound 3c), with a 20 nm
spectra showed signals of 5ꢀaminolevulinic acid at δ 2.78ꢀ2.89 ppm bathochromic shift when compared with substitution at position 6
(compounds 3b).
for αꢀ and βꢀCH2 and δ 3.98ꢀ4.09 ppm for δꢀCH2. The heterocycle
methylene group, adjacent to the ester link, was visible for all
compounds at δ 5.23ꢀ5.49 ppm. The newly formed ester linkages
were confirmed by 13C NMR spectra signals of the carbonyl group,
at about δ 171.47ꢀ171.80 ppm.
400
400
350
300
250
3e
3b
350
300
Rayleigh
Second order
Evaluation of the photophysical properties of 5-aminolevulinic
acid conjugates 3a-e and 4c
250
300
400
500
300
400
500
400
350
300
250
Fundamental UV/visible absorption and emission characterisation
was performed for conjugates 3aꢀe and 4c considering that their
photolysis would be monitored by HPLC with UV detection. The
absorption and emission spectra of degassed 10ꢀ5 M solutions in
methanol/HEPES buffer (80:20) solutions of ester conjugates 3aꢀe
and 4c were measured and the absorption and emission maxima,
molar absorption coefficients and relative fluorescence quantum
yields are reported in Table 1. Relative fluorescence quantum yields
were calculated using 9,10ꢀdiphenylanthracene in ethanol (ΦF
0.95)40 as a standard. For the ΦF determination, the fluorescence
standard was excited at the maximum absorption wavelength found
for each compound. In all fluorimetric measurements the absorbance
of the solution did not exceed 0.1. For easier comparison, the data
already published by us for precursors 1aꢀe was also included in
Table 1.
400
350
300
250
4c
3c
300
400
500
300
400
500
400
3d
350
300
250
Emission wavelength (nm)
300
400
500
From the data in Table 1 it was confirmed that the absorption and
emission of the conjugates were directly related with the nature of
the heterocyclic tag. As for the influence of the tag structure, by
comparison of conjugates 3aꢀc with a benzocoumarin with the same
ring fusion but different substituents at different positions (methoxy
or amino groups), it could be seen that the methoxy derivative 3a
exhibited longer wavelength of absorption (> 80 nm) than that of
amino derivatives 3bꢀc.
Figure 1. Excitation – emission matrices for selected compounds.
The influence of scattered excitation light (first and second order) is
also indicated.
Table 1. UV/Vis absorption and emission data for precursors 1aꢀe
and conjugates 3aꢀe and 4c, in methanol/HEPES buffer (80:20)
solutions.
Absorption
Emission
Cpd.
λmax
log ε
(nm)
λem Stokes’ shift
ΦF
(nm)
(nm)
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Photolysis studies of 5-aminolevulinic acid conjugates 3a-e and
4c
10.0
7.5
5.0
2.5
0.0
lifetime
434ps
772ps
2.85ns
lifetime
398ps
1.43ns
0.4
0.3
0.2
0.1
3e
3b
Conjugates 3aꢀe and 4c were irradiated at 254, 300, 350 and 419 nm
in mixtures of methanol with aqueous HEPES buffer in 80:20 ratio,
in a Rayonet RPRꢀ100 reactor, and kinetic data were collected. The
course of the photolytic reaction was followed by reverse phase
HPLC with UV detection. The plots of peak area (A) of the starting
material versus irradiation time were obtained for each compound, at
the considered wavelengths. Peak areas were determined by HPLC,
which revealed a gradual decrease with time, and were the average
of three runs. The irradiation time given represents the time
necessary for the consumption of the starting materials until less than
5% of the initial area was detected (Table 2). For each compound,
and based on HPLC data, the plot of ln A versus irradiation time
showed a linear correlation for the disappearance of the starting
material. This is indicative of a first order reaction, obtained by the
5.29ns
0.0
400
450
500
550
600
400
450
500
550
600
wavelength / nm
wavelength / nm
lifetime
185ps
1.36ns
3.3ns
0.30
0.25
0.20
0.15
0.10
0.05
0.00
206ps
0.4
0.3
0.2
0.1
3c
4c lifetime
1.44ns
3.59ns
0.0
400
450
500
550
600
400
450
500
550
600
wavelength / nm
wavelength / nm
linear least squares methodology for
photochemical quantum yields (Φphot) were calculated based on halfꢀ
lives (t1/2), molar extinction coefficients ( ) and the incident photon
a straight line. The
lifetime
10000
1000
100
10
“sum” decays compound
5
4
3
2
1
293ps
1.26ns
3.27ns
3b
3c
3d
3e
3d
ε
4c
IRF
flux (I0), which was determined by potassium ferrioxalate
actinometry.41
The results in Table 2 for the various wavelengths of irradiation
revealed the significant influence of the photoactive unit structure in
the irradiation time (tirr) necessary to release 5ꢀaminolevulinic acid.
Although a study at the shorter wavelengths of irradiation (254 and
300 nm) was carried out for comprehensiveness and the results are
included in Table 2, the main focus of this work was the
performance at 350 and 419 nm, as these wavelengths are more
adequate for biological applications. At these wavelengths, it was
found that the release occurred from the different cages with shorter
tirr for the irradiation at 350 nm, with the exception of cage 3b which
displayed similar irradiation time. Altogether, the best result at 350
nm was obtained with compound 3e, bearing the fused oxazole
coumarin with a chromone substituent, with 95% of the caged 5ꢀ
aminolevulinic acid being released in just 10 min.
0
1
400
450
500
550
600
0
20
40
60
wavelength / nm
time / ns
Figure 2. Decay associated spectra obtained from a 3ꢀexponential
global analysis for selected compounds. The (scaled) sum decays
made by the addition of all the decays in the timeꢀresolved emission
measurements (380 to 625 nm at 5 nm increments) are also shown.
Overall, the emission spectra appeared structureless, which may be
indicative of an emission that has a charge transfer nature. The fact
that the fluorescence originates from the tag is further reinforced by
the measurement of the excitation – emission matrix (EEM), which
produces a contour plot of fluorescence intensity in relation to
different excitation and emission wavelengths (Figure 1). The EEMs
show that even excitation towards shorter wavelengths causes the
longer wavelength emission and is thus indicative of an energy
transfer type of process. Although the final emission is apparently
from the tag, to further explore the mechanism timeꢀresolved
fluorescence measurements were performed in order to elucidate if
any other fluorescing species were present. In the case of this kind of
photocleaveable compounds it can be expected that, as well as any
locally excited state, there is the possibility of the formation of an
ion pair that can either recombine or cleave. A study was performed
using an excitation wavelength of 349 nm, which (considering the
photolysis data) is the longest which can produce photocleavage in
all of the compounds and opens this area to the use of twoꢀphoton
excitation.
To establish whether optical control over the bond scission could be
exercised, an experiment was attempted making use of fluorescence
to monitor the kinetics of this process. Cleavage of the conjugate
should remove a nonꢀradiative pathway, thus increasing the quantum
yield of the fluorophore. However, to exert control and check for an
“on – off” behaviour is not trivial as it involves modulating the light
source, and in the “off” condition this would also remove the source
of the fluorescence excitation. In an initial attempt to overcome this,
an approach was made using two excitation sources; one better
tuned, in terms of wavelength, to the photocleavage process and
another longer wavelength source to monitor the fluorescence.
Compound 3e was chosen as model, as the irradiation time for the
cleavage process at shorter wavelengths was considerably shorter
than that at longer wavelengths (see Table 2). To this end, an
excitation source at 295 nm was used to modulate the photocleavage
process and one at 392 nm was employed to monitor the
fluorescence. Although in principle both wavelengths can contribute
to both the fluorescence and the bond scission, as the data in Table 2
shows, the influence on photocleavage of the longer wavelength
source should be significantly smaller. The contribution of the
shorter wavelength source to the fluorescence intensity can also be
taken into account as a background level and corrected. The set up
made use of the timeꢀresolved fluorimeter with an extra excitation
channel added to the sample chamber opposite to the standard one.
Timeꢀresolved emission spectra were recorded (fluorescence decays
measured for the same time at different wavelengths ꢀ 5 nm
increments) and analysed globally to obtain decay associated
spectra. Overall, these data (Figure 2) show the need to fit to a threeꢀ
exponential decay model (indicative of three excited states) and
these uncorrected spectra are obtained from the preꢀexponential
factor weighted by the lifetime.
The fact that the shortest lifetime is also at the shortest wavelength,
with an increase in both lifetime and wavelength for the component
spectra, can indicate a common origin. It can also be noted that the
removal of the Nꢀtertꢀbutoxycarbonyl (Boc) group (3c to 4c) did not
have any significant effect.
4 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6NJA03R7T8I7CALE
Table 2. Irradiation times (tirr, in min) and photochemical quantum Comparison of the data for oxazole fused coumarins 3d and 3e,
yields (ΦPhot, ×10ꢀ3) for the photolysis of conjugates 3aꢀe and 4c at bearing a simple phenyl or a chromone ring linked to the oxazole,
different wavelengths in methanol/HEPES buffer (80:20) solution.
respectively, it was found that the extra heterocycle in 3e had a
beneficial effect in the photolytic reaction, which occurred faster for
all the wavelengths of irradiation. It should be noted that in the
decay associated spectra (Figure 2) these compounds exhibit
dominant spectra associated with a short lifetime, when excited at
349 nm. Longer irradiation times were required for compounds 3c
and 4c which exhibited dominant longerꢀlived spectra. As already
mentioned, photolysis at 350 nm and longer wavelengths is
preferable for biological applications, and the data obtained by
254 nm
tirr
89 0.0024 145 < 0.001 87 < 0.001 280 < 0.001
300 nm
350 nm
419 nm
Cpd.
tirr
tirr
tirr
ΦPhot
ΦPhot
ΦPhot
ΦPhot
3a
3b
3c
3d
3e
4c
25
38
0.94
38
0.33
47
0.19
43
232
0.25
0.50 111 0.085 162 0.060
0.039
irradiation at 419 nm revealed
a
promising result for
a)
31 0.070 45
21 0.082 18
0.13
51
0.12
>1200
aminobenzocoumarinꢀcaged 5ꢀALA 3b, with a practical irradiation
time of 43 min. Photolysis of the deprotected aminolevulinic acid
conjugate 4c was also carried out by irradiation at 350 nm and
revealed a dramatic difference in the irradiation time necessary for
the release of the active molecule in its free form 5. This behaviour
can be related to the higher propensity of the free amino group in the
conjugate form to engage in hydrogen bonding to the solvent which
in turn can lead to an increase in the rate constants of excited state
deactivation through processes other than bond scission.
0.41
10 0.086
360 0.037
798
0.010
a)
a)
a)
a)
a)
a)
a) not determined.
An experiment was conducted, firstly with the 295 nm source in
continuous irradiation, and then repeated with this source first turned
on, then off and finally on again. The outcome is shown in Figure 3
and shows an initial similar trend in the increase in fluorescence
intensity between the two runs, but when the shorter wavelength
source is turned off there is a “plateau” with no significant increase
until it is turned on again. This is indicative of the fact that light
modulates the photocleavage process and that this approach is
worthy of further study to obtain kinetic information.
In addition to monitoring the photolysis process by HPLC, the
release of 5ꢀ(NꢀBoc)ꢀaminolevulinic acid 2 was also followed by 1H
NMR in methanolꢀd4/D2O (80:20) solution. As a representative
example, in the photolysis of conjugate 3b at 300 nm the signal of
the benzylicꢀtype CH2 at position 4 of the benzocoumarin, visible at
about δ 5.4 ppm, gradually decreased with time. The same
observation occurred with the signals related to Bocꢀ5ꢀALA in the
conjugated form, at about δ 3.95, 2.90 and 2.80 ppm, giving rise to a
close set of signals corresponding to Bocꢀ5ꢀALA in its free form at
about δ 3.90, 2.75 and 2.60 ppm, respectively (Figure 4). NMR
monitoring was carried out with a 4.3×10ꢀ4 M solution, which led to
an expected increase in the photolysis time for the complete release
of the molecule, when compared to the irradiation times in Table 2
obtained with dilute solutions and can be related to the relatively
higher optical density.
Regarding the structure of the heterocyclic photosensitive moiety,
comparison of the behaviour methoxybenzocoumarin 3a with
aminobenzocoumarin 3b, bearing the substituents at the same ring
position (position 6), showed that the amino substituent improved
the photosensitivity in the release of the active molecule as the tirr
were consistently shorter with compound 3b at all the wavelengths
of irradiation tested. The amino substituent at position 6 was crucial
for the photolytic behaviour since the amino group at position 7
(compound 3c) lead to a significant increase in the irradiation times
when compared to 3b, at all wavelengths tested.
1.25
(a)
(b)
1.20
1.15
1.10
1.05
1.00
295nmOFF
295nm ON
295nm ON
0
500 1000 1500 2000 2500 3000 3500 4000
Figure 4. 1H NMR spectra in methanolꢀd4/D2O (80:20) of the
photolysis of conjugate 3b (C = 4.3×10ꢀ4 M) at 300 nm: (a) before
irradiation; (b) after irradiation for 45 min; (c) after irradiation for
135 min; (d) sample of free Bocꢀ5ꢀALA 2.
time / s
Figure 3. Change in fluorescence intensity (monitored at 465 nm)
for (a) continuous irradiation at 295 nm and (b) modulated
irradiation 295 nm. Fluorescence was “probed” at 392 nm and traces
have been corrected for both excitation source contributions.
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New Journal of Chemistry
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VJieowuArrtnicalelONnlainme e
ARTICLE
DOI: 10.1039/C6NJ03787A
the result at 419 nm. As for the oxazole fused coumarins, the best
results in the photorelease of aminolevulinic acid were obtained for
the chromoneꢀbearing derivative 3e, especially at 350 nm.
Bocꢀ5ꢀALAꢀCOꢀOCH2ꢀHet
hυ
Experimental
homolysis
CH2ꢀHet
Bocꢀ5ꢀALAꢀCOꢀO
electron transfer
+
+
Materials
All melting points were measured on a Stuart SMP3 melting
point apparatus. TLC analyses were carried out on 0.25 mm
thick precoated silica plates (Merck Fertigplatten Kieselgel
60F254
)
and spots were visualised under UV light.
heterolysis
CH2ꢀHet
Bocꢀ5ꢀALAꢀCOꢀO
Chromatography on silica gel was carried out on Merck
Kieselgel (230ꢀ240 mesh). IR spectra were determined on a
BOMEM MB 104 spectrophotometer in KBr discs (1%).
Absorption spectra (200ꢀ700 nm) were obtained using a
Shimadzu UV/2501PC spectrophotometer. NMR spectra were
obtained on a Bruker Avance III 400 at an operating frequency
recombination
MeOH
H2O
HOꢀCH2ꢀHet MeOꢀCH2ꢀHet Bocꢀ5ꢀALAꢀCOOH
2
Het = coumarinyl derivative
1
of 400 MHz for H and 100.6 MHz for 13C using the solvent
peak as internal reference at 25 oC. All chemical shifts are
Scheme 3. Proposed mechanism for the photolysis of 5ꢀALA
coumarinyl ester conjugates.
given in ppm using δH Me4Si = 0 ppm as reference and
J values
are given in Hz. Assignments were supported by bidimensional
heteronuclear correlation techniques. Mass spectrometry of the
photolysis mixtures were obtained in a Finnigan LXQ mass
spectrometer by ESI in positive ionization mode. Highꢀ
resolution MS spectra were performed at the “C.A.C.T.I. ꢀ
Unidad de Espectrometria de Masas”, at University of Vigo,
Spain. Fluorescence spectra were collected either using a
FluoroMaxꢀ4 spectrofluorometer (HORIBA Scientific) or a
FluoroLog 3 for the EEMs. Timeꢀresolved fluorescence
measurements were performed using a HORIBA Scientific
DeltaFlex equipped with a DeltaDiode DDꢀ350 excitation
source.
To obtain further proof of release of the target molecule,
through
a mechanism previously suggested by us and
others,38,42 involving ionic intermediates, MS spectra were
obtained for the mixture resulting from the photolysis of
conjugate 3e in MeOH/HEPES (80:20) buffer after irradiation
at 350 nm for 60 min (C = 3.7 ×10ꢀ4 M). In the obtained
spectrum, the most relevant peaks were assigned to the released
Bocꢀ5ꢀALA
in accordance with the result obtained by H NMR monitoring,
and to coumarinyl alcohol byproduct resulting from
2 as base peak with m/z = 254 (includes sodium),
1
a
nucleophilic attack of water to the intermediate carbocation
with m/z = 384 (includes sodium). Coumarinyl esters are
thought to photocleave through both homolytical or
heterolytical fission of the OꢀCH2 bond (the latter being
energetically favourable). The homolysis of the OꢀC bond,
followed by electron transfer, can yield the ion pair (a
methylenic coumarinyl carbocation and the leaving group
anion), whereas heterolysis of the same bond directly affords
the cited ion pair. Once formed, the coumarinyl carbocation can
undergo nucleophilic attack by the solvent to form the final
products (Scheme 3).
All reagents were used as received. Chloromethyl precursors 1a,29
1b,36 1c37 1d,e38 and 5ꢀ(Nꢀtertꢀbutoxycarbonyl)aminolevulinic acid
217 were synthesised as reported elsewhere.
General procedure for the synthesis of ester conjugates 3a-e.
To a solution of the chloromethyl precursor 1aꢀe (1 equiv) in dry
DMF,
potassium
fluoride
(3
equiv),
and
5ꢀ(Nꢀtertꢀ
butoxycarbonyl)aminolevulinic acid (Bocꢀ5ꢀALA) 2 (1 equiv) were
added. The mixture was stirred at room temperature for 24 h. The
solvent was evaporated and the crude residue was purified by silica
gel column chromatography using using ethyl acetate: nꢀhexane 4:6
as eluent (except for 4c which was pure after evaporation). The
fractions containing the desired product were combined and
evaporated.
Conclusions
A series of heterocyclic cages, consisting of more extended
conjugated systems based on coumarin, were used in the preparation
of ester cages of 5ꢀ(NꢀBoc)ꢀaminolevulinic acid and studied for the
controlled delivery of the active molecule by photolysis at selected
wavelengths (254, 300, 350 and 419 nm), monitored by HPLCꢀUV
(6-Methoxy-2-oxo-2H-benzo[h]benzopyran-4-yl)methyl 5-((tert-
butoxycarbonyl)amino)-4-oxopentanoate, 3a. Starting from
chloromethyl precursor 1a (0.024 g, 0.08 mmol), dry DMF (3 mL),
potassium fluoride (0.023 g, 0.26 mmol) and Bocꢀ5ꢀALA 2 (0.019 g,
0.16 mmol), compound 3a was obtained as yellow solid (0.008 g,
0.01 mmol, 20%). mp = 189.2ꢀ190.2 ºC. TLC (ethyl acetate/nꢀ
hexane 4:6): Rf = 0.67. νmax/cmꢀ1 3315, 2925, 1735, 1714, 1687,
1600, 1538, 1475, 1455, 1422, 1386, 1355, 1291, 1198, 1165, 1110,
1087, 1068, 989, 942, 863, 750, 720, 555. δH (400 MHz, CDCl3)
1.46 (s, 9H, C(CH3)3), 2.83 (br s, 4H, αꢀCH2 and βꢀCH2), 4.02 (s,
3H, OCH3), 4.09 (d, J 4.8 Hz, 2H, δꢀCH2), 5.20 (br s, 1H, NH), 5.38
(d, J 1.2 Hz, 2H, OCH2), 6.58 (s, 1H, Hꢀ3), 6.63 (s, 1H, Hꢀ5), 7.66ꢀ
7.71 (m, 2H, Hꢀ8 and Hꢀ9), 8.28 (dd, J 8.0 and 1.2 Hz, 1H, Hꢀ7),
8.53 (dd, J 8.0 and 1.2 Hz, 1H, Hꢀ10). δC (100.6 MHz, CDCl3) 27.61
(αꢀCH2), 28.30 (C(CH3)3), 34.25 (βꢀCH2), 50.23 (δꢀCH2), 55.94
(OCH3), 62.38 (OCH2), 80.01 (C(CH3)3), 95.19 (Cꢀ5), 112.02 (Cꢀ
4a), 112.98 (Cꢀ3), 122.28 (Cꢀ7), 122.44 (Cꢀ10), 124.00 (Cꢀ10a),
1
and H NMR. Overall, the results obtained confirmed the suitability
of the tested photosensitive moieties for the release of 5ꢀ
aminolevulinic acid at the various wavelengths of irradiation. The
timeꢀresolved fluorescence elucidated the presence of different
fluorescing species in the compounds indicative of a multistep
cleavage process. The use of two excitation sources (cleave and
probe) to enable fluorescence to follow the kinetics of the process
and to ascertain optical control over the bond scission appears
promising. For benzocoumarins 3aꢀc the attachment position of the
methoxy and amino substituents influenced the behaviour towards
light of the corresponding cages, and the shortest irradiation times
were obtained for the 6ꢀaminobenzocoumarin 3b, with emphasis on
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 7 of 10
Journal Name
New Journal of Chemistry
View Article Online
ARTICLE
DOI: 10.1039/C6NJ03787A
127.35 (Cꢀ6a), 127.83 (Cꢀ9), 128.59 (Cꢀ8), 145.80 (Cꢀ10b), 148.82 (Cꢀ8a), 126.12 (Cꢀ1’), 127.81 (Cꢀ2’ and Cꢀ6’), 129.05 (Cꢀ3’ and Cꢀ
(Cꢀ4), 152.35 (Cꢀ6), 155.38 (C=O Boc), 160.55 (Cꢀ2, C=O), 171.47 5’), 132.34 (Cꢀ4’), 139.28 (Cꢀ9a), 148.95 (Cꢀ8), 151.96 (Cꢀ4a),
(C=O ester), 204.33 (C=O keto). (ESI) HRMS for C25H28NO8 [M+ + 152.50 (Cꢀ3a), 155.69 (C=O Boc), 160.13 (Cꢀ6, C=O), 164.98 (Cꢀ2),
H]: calculated 470.18154, found 470.18201.
171.63 (C=O ester), 204.09 (C=O keto). (ESI) HRMS for
C27H27N2O8 [M+ + H]: calculated 507.17619, found 507.17617.
(6-Amino-2-oxo-2H-benzo[h]benzopyran-4-yl)methyl
5-((tert-
butoxycarbonyl)amino)-4-oxopentanoate, 3b. Starting from
(6-Oxo-2-(4’-oxo-4H-benzopyran-2’-yl)-6H-benzopyrano[6,7-
chloromethyl precursor 1b (0.060 g, 0.23 mmol), dry DMF (3 mL),
d]oxazol-8-yl)methyl
5-((tert-butoxycarbonyl)amino)-4-
potassium fluoride (0.061 g, 0.69 mmol) and Bocꢀ5ꢀALA 2 (0.050 g, oxopentanoate, 3e. Starting from chloromethyl precursor 1e (0.041
0.23 mmol), compound 3b was obtained as a brown oil (0.043 g, g, 0.11 mmol), dry DMF (3 mL), potassium fluoride (0.028 g, 0.11
0.09 mmol, 42%). TLC (ethyl acetate/nꢀhexane 4:6): Rf = 0.64. mmol) and Bocꢀ5ꢀALA 2 (0.023 g, 0.11 mmol), compound 3e was
νmax/cmꢀ1 3377, 2960, 2930, 1699, 1569, 1510, 1472, 1433, 1394, obtained as a beige solid (0.034 g, 0.06 mmol, 56%). mp = 235.4ꢀ
1253, 1163, 1086, 958, 859, 766, 733. δH (400 MHz, CDCl3) 1.44 (s, 235.3 ºC. TLC (ethyl acetate/nꢀhexane 4:6): Rf = 0.60. νmax/cmꢀ1
9H, C(CH3)3), 2.78ꢀ2.82 (m, 4H, αꢀCH2 and βꢀCH2), 4.07 (d, J 4.8 3427, 3084, 2926, 2854, 1733, 1653, 1573, 1509, 1464, 1440, 1387,
1366, 1328, 1249, 1159, 1127, 1052, 956, 899, 860, 778, 755, 735.
δH (400 MHz, CDCl3) 1.45 (s, 9H, C(CH3)3), 2.80ꢀ2.84 (m, 4H, αꢀ
Hz, 2H, δꢀCH2), 5.23 (d, J 0.8 Hz, 2H, OCH2), 5.32 (br s, 1H, NH),
6.47 (s, 1H, Hꢀ3), 6.60 (s, 1H, Hꢀ5), 7.61ꢀ7.64 (m, 2H, Hꢀ8 and Hꢀ
9), 7.83 (dd, J 8.4 and 1.6 Hz, 1H, Hꢀ10), 8.46 (dd, J 8.0 and 1.6 Hz, CH2 and βꢀCH2), 4.09 (s, 2H, δꢀCH2), 5.26 (br s, 1H, NH), 5.40 (d, J
1H, Hꢀ7). δC (100.6 MHz, CDCl3) 27.56 (αꢀCH2), 28.28 (C(CH3)3), 1.2 Hz, 2H, OCH2), 6.59 (s, 1H, Hꢀ7), 7.36 (s, 1H, Hꢀ3’), 7.52 (dt, J
7.2 and 1.2 Hz, 1H, Hꢀ6’), 7.70 (s, 1H, Hꢀ4), 7.71 (dd, J 8.0 and 1.2
Hz, 1H, Hꢀ8’), 7.81 (dt, J 8.0 and 1.6 Hz, 1H, Hꢀ7’), 8.07 (s, 1H, Hꢀ
9), 8.27 (dd, J 8.0 and 1.2 Hz, 1H, Hꢀ5’). δC (100.6 MHz, CDCl3)
34.20 (βꢀCH2), 50.21 (δꢀCH2), 62.08 (OCH2), 79.95 (C(CH3)3),
100.58 (Cꢀ5), 112.86 (Cꢀ3), 113.06 (Cꢀ4a), 121.09 (Cꢀ7), 123.08 (Cꢀ
10), 123.63 (Cꢀ6a), 125.59 (Cꢀ10a), 127.24 (Cꢀ9), 128.03 (Cꢀ8),
139.15 (Cꢀ6), 144.52 (Cꢀ10b), 149.07 (Cꢀ4), 155.68 (C=O Boc), 27.45 (αꢀCH2), 28.30 (C(CH3)3), 34.22 (βꢀCH2), 50.18 (δꢀCH2),
160.77 (Cꢀ2, C=O), 171.73 (C=O ester), 204.46 (C=O keto). (ESI) 61.72 (OCH2), 79.99 (C(CH3)3), 100.51 (Cꢀ4), 113.24 (Cꢀ3’), 113.73
HRMS for C24H27N2O7 [M+ + H]: calculated 455.18128, found (Cꢀ7), 115.91 (Cꢀ9), 116.02 (Cꢀ8a), 118.55 (Cꢀ8’), 124.56 (Cꢀ4a’),
455.18191.
125.99 (Cꢀ6’), 126.19 (Cꢀ5’), 134.87 (Cꢀ7’), 138.29 (Cꢀ8), 148.57
(Cꢀ9a), 150.42 (Cꢀ2’), 152.35 (Cꢀ4a), 152.46 (Cꢀ3a), 153.20 (Cꢀ8a’),
156.13 (C=O Boc), 157.54 (Cꢀ2), 159.46 (Cꢀ6, C=O), 171.67 (C=O
(7-Amino-2-oxo-2H-benzo[h]benzopyran-4-yl)methyl
5-((tert-
butoxycarbonyl)amino)-4-oxopentanoate, 3c. Starting from ester), 177.29 (Cꢀ4’), 203.98 (C=O keto). (ESI) HRMS for
chloromethyl precursor 1c (0.042 g, 0.16 mmol), dry DMF (3 mL), C30H27N2O10 [M+ + H]: calculated 575.16602, found 575.16419.
potassium fluoride (0.043 g, 0.49 mmol) and Bocꢀ5ꢀALA 2 (0.035 g,
0.16 mmol), compound 3c was obtained as a brown oil (0.034 g,
Synthesis
of
(7-amino-2-oxo-2H-benzo[h]benzopyran-4-
0.08 mmol, 55%). TLC (ethyl acetate/nꢀhexane 4:6): Rf = 0.63. yl)methyl 5-amino-4-oxopentanoate, 4c. Conjugate 3c (0.020 g,
νmax/cmꢀ1 3447, 3377, 2980, 2934, 1699, 1639, 1565, 1510, 1478, 0.05 mmol) was dissolved in dichloromethane/TFA (2:1) (3 mL) and
1441, 1387, 1368, 1332, 1252, 1164, 1101, 1056, 981, 855, 789, the reaction mixture was stirred at room temperature for 1h. The
750, 735, 700, 679. δH (400 MHz, CDCl3) 1.45 (s, 9H, C(CH3)3), solvent was evaporated giving the expected deprotected compound
4c as a brown oil (0.011 g, 0.03 mmol, 68%). TLC (ethyl acetate/nꢀ
hexane 8:2): Rf = 0.23. νmax/cmꢀ1 3437, 3375, 2982, 2937, 2124,
1700, 1635, 1505, 1476, 1439, 1388, 1370, 1330, 1249, 1165, 1099,
1045, 983, 850, 786, 749, 738, 705, 680. δH (400 MHz, DMSOꢀd6)
2.78 (t, J 6.4 Hz, 2H, αꢀCH2), 2.89 (t, J 6.4 Hz, 2H, βꢀCH2), 3.98 (br
s, 2H, δꢀCH2), 5.49 (d, J 1.2 Hz, 2H, OCH2), 6.52 (d, J 1.6 Hz, 1H,
Hꢀ3), 6.88 (dd, J 7.6 and 0.8 Hz, 1H, Hꢀ8), 7.40 (t, J 8.0 Hz, 1H, Hꢀ
9), 7.54 (d, J 8.8 Hz, 1H, Hꢀ5), 7.56 (dd, J 8.0 and 1.2 Hz, 1H, Hꢀ
10), 8.00 (d, J 8.8 Hz, 1H, Hꢀ6). δC (100.6 MHz, DMSOꢀd6) 27.03
2.80ꢀ2.83 (m, 4H, αꢀCH2 and βꢀCH2), 4.09 (d, J 4.8 Hz, 2H, δꢀCH2),
5.24 (br s, 1H, NH), 5.34 (d, J 1.2 Hz, 2H, OCH2), 6.52 (s, 1H, Hꢀ3),
6.94 (dd, J 8.4 and 0.8 Hz, 1H, Hꢀ8), 7.34 (d, J 8.8 Hz, 1H, Hꢀ5),
7.43 (t, J 8.0 Hz, 1H, Hꢀ9), 7.64 (d, J 8.8 Hz, 1H, Hꢀ6), 7.96 (d, J 8.4
Hz, 1H, Hꢀ10). δC (100.6 MHz, CDCl3) 27.54 (αꢀCH2), 28.28
(C(CH3)3), 34.19 (βꢀCH2), 50.23 (δꢀCH2), 61.79 (OCH2), 79.99
(C(CH3)3), 112.18 (Cꢀ4a), 112.33 (Cꢀ3), 121.88 (Cꢀ8), 113.07 (Cꢀ
10), 117.39 (Cꢀ5), 117.67 (Cꢀ6), 124.07 (Cꢀ6a), 124.29 (Cꢀ10a),
127.98 (Cꢀ9), 142.30 (Cꢀ7), 149.53 (Cꢀ4), 151.08 (Cꢀ10b), 155.67
(C=O Boc), 160.53 (Cꢀ2, C=O), 171.72 (C=O ester), 204.15 (C=O (αꢀCH2), 34.25 (βꢀCH2), 46.75 (δꢀCH2), 61.67 (OCH2), 108.66 (Cꢀ
keto). (ESI) HRMS for C24H27N2O7 [M+ + H]: calculated 455.18128, 10), 110.68 (Cꢀ8), 111.13 (Cꢀ3), 112.11 (Cꢀ4a), 117.41 (Cꢀ5), 119.13
found 455.18040.
(Cꢀ6), 123.27 (Cꢀ6a), 123.52 (Cꢀ10a), 128.52 (Cꢀ9), 145.26 (Cꢀ7),
150.19 (Cꢀ10b), 151.29 (Cꢀ4), 159.85 (Cꢀ2, C=O), 171.80 (C=O
ester), 202.84 (C=O keto). (ESI) HRMS for C19H19N2O5 [M+ + H]:
(6-Oxo-2-phenyl-6H-benzopyrano[6,7-d]oxazol-8-yl)methyl
5-
((tert-butoxycarbonyl) amino)-4-oxopentanoate, 3d. Starting from calculated 355.12885, found 355.12875.
chloromethyl precursor 1d (0.020 g, 0.064 mmol), dry DMF (3 mL),
potassium fluoride (0.017 g, 0.19 mmol) and Bocꢀ5ꢀALA 2 (0.014 g,
0.064 mmol), compound 3d was obtained as a beige solid (0.020 g,
0.04 mmol, 62%). mp = 205.3ꢀ206.3 ºC. TLC (ethyl acetate/nꢀ
hexane 4:6): Rf = 0.55. νmax/cmꢀ1 3415, 2956, 2925, 2855, 1718,
1634, 1559, 1490, 1441, 1441, 1398, 1370, 1329, 1291, 1260, 1160,
1141, 1094, 1049, 1021, 989, 946, 918, 875, 844, 813, 777. δH (400
MHz, CDCl3) 1.45 (s, 9H, C(CH3)3), 2.80ꢀ2.83 (m, 4H, αꢀCH2 and
Photolysis general procedure:
A 1 × 10ꢀ4 M methanol/HEPES (80:20) solution of compounds 3aꢀe
and 4c (5 mL) were placed in a quartz tube and irradiated in a
Rayonet RPRꢀ100 reactor at the desired wavelength. The lamps used
for irradiation were of 254, 300, 350 and 419 ± 10 nm. HEPES
buffer solution was prepared in distilled water with HEPES (4ꢀ(2ꢀ
βꢀCH2), 4.07 (d, J 5.2 Hz, 2H, δꢀCH2), 5.36 (br s, 1H, NH), 5.37 (d, hydroxyethyl)ꢀ1ꢀpiperazine ethanesulfonic acid) (10 mM), sodium
J 1.6 Hz, 2H, OCH2), 6.52 (t, J 1.2 Hz, 1H, Hꢀ7), 7.54ꢀ7.61 (m, 4H, chloride (120 mM), potassium chloride (3 mM), calcium chloride (1
Hꢀ4 and Hꢀ3’, Hꢀ4’ and Hꢀ5’), 7.86 (s, 1H, Hꢀ9), 8.26 (dd, J 8.0 and mM) and magnesium chloride (1mM) and pH adjusted to 7.2 with
2.0 Hz, 2H, Hꢀ2’ and Hꢀ6’). δC (100.6 MHz, CDCl3) 27.56 (αꢀCH2), aqueous 1 M sodium hydroxide solution. Aliquots of 100 µL were
taken at regular intervals and analysed by RPꢀHPLC using a
Licrospher 100 RP18 (5 ꢁm) column in a JASCO HPLC system
28.29 (C(CH3)3), 34.21 (βꢀCH2), 50.19 (δꢀCH2), 61.91 (OCH2),
79.93 (C(CH3)3), 99.84 (Cꢀ4), 112.76 (Cꢀ7), 113.97 (Cꢀ9), 114.80
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 7

New Journal of Chemistry
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ARTICLE
VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C6NJ03787A
composed by a PUꢀ2080 pump and a UVꢀ2070 detector with
ChromNav software. The eluent was acetonitrile/water, 75:25 at a
flow rate of 0.8 mL/min, previously filtered through a Millipore,
type HN 0.45 µm filter and degassed by ultraꢀsound for 30 min. The
chromatograms were traced by detecting UV absorption at the
wavelength of maximum absorption for each compound (retention
time: 3a, 6.2; 3b, 4.3; 3c, 4.4; 3d, 6.5; 3e, 7.1; 4c, 4.0 min). The
fluorescence kinetic measurements involved the use of a DeltaFlex
with an additional excitation channel. A DeltaDiode DDꢀ295 was
operated at 100 MHz to provide the “cleavage” source and a DDꢀ
395L was used as a “probe”. A KV370 filter was used on the
emission to block any 295 nm light and the EzTime software was
operated in “histogram streaming” mode, with histograms collected
every two seconds. The intensity was taken from the total number of
counts in each histogram.
12 A. Stenzl, H. Penkoff, E. DajcꢀSommerer, A. Zumbraegel, L. Hoeltl,
M. Scholz, C. Riedl, J. Bugelnig, A. Hobisch, M. Burger, G. Mikuz,
U. Pichlmeier. Cancer, 2011, 117, 938ꢀ947.
13 R. F. V. Lopez, N. Lange, R. Guy, M. V. L. B. Bentley. Adv. Drug
Deliv. Rev., 2004, 56, 77ꢀ94.
14 J. M. Gaullier, K. Berg, Q. Peng, H. Anholt, P. K. Selbo, L. W. Ma,
J. Moan. Cancer Res., 1997, 57, 1481ꢀ1486.
15 J. H. Yoon, H. E. Yoon, O. Kim, S. K. Kim, S. G. Ahn, K. W. Kang.
Lasers Surg. Med., 2012, 44, 76ꢀ86.
16 C. J. Whitaker, S. H. Battah, M. J. Forsyth, C. Edwards, R. W. Boyle,
E. K. Matthews. AntiꢀCancer Drug Des., 2000, 15, 161ꢀ170.
17 G. Berkovitch, D. Doron, A. Nudelman, Z. Malik, A. Rephaeli. J.
Med. Chem., 2008, 51, 7356ꢀ7369.
18 G. Di Venosa, L. Hermida, A. Batlle, H. Fukuda, M. V. Defain, L.
Mamone, L. Rodriguez, A. MacRobert, A. Casas. J. Photochem.
Photobiol. B Biol., 2008, 92, 1–9.
Acknowledgements
19 F. Giuntini, L. Bourre, A. J. MacRobert, M. Wilson, I. M. Eggleston.
J. Med. Chem., 2009, 52, 4026ꢀ4037.
Thanks are due to the Fundação para a Ciência e Tecnologia (FCT,
Portugal) for financial support to the NMR portuguese network
(PTNMR, Bruker Avance III 400ꢀUniv. Minho), FCT and FEDER
(European Fund for Regional Development)ꢀCOMPETEꢀQRENꢀEU
for funds provided through the Chemistry Research Centre of the
20 G. Di Venosa, P. Vallecorsa, F. Giuntini, L. Mamone, A. Batlle, S.
Vanzuli, A. Juarranz, A. J. MacRobert, I. M. Eggleston, A. Casas.
Mol. Cancer Ther., 2015, 14, 440ꢀ451.
University
UID/QUI/0686/2016).
(SFRH/BD/80813/2011) is also acknowledged.
of
Minho
(Ref.
PhD
UID/QUI/00686/2013
and
21 M. Wachowska, A. Muchowicz, M. Firczuk, M. Gabrysiak, M.
Winiarska, M. Wańczyk, K. Bojarczuk, J. Golab. Molecules, 2011,
16, 4140ꢀ4164.
A
grant to A.M.S.S.
22 A. Kasche, S. Luderschmidt, J. Ring, R. Hein. J. Drugs Dermatol.,
References
2006, 5, 353ꢀ356.
23 Y. Hotta, T. Tanaka, H. Takaoka, Y. Takeuchi, M. Konnai. Plant
Growth Regul., 1997, 22, 109ꢀ114.
a Centro de Química, Universidade do Minho, Campus de Gualtar, 4710ꢀ
057 Braga, Portugal.
24 C. A. Rebeiz, K. N. Reddy, U. B. Nandihalli, J. Velu. Photochem.
Photobiol., 1990, 52, 1099ꢀ1117.
b
HORIBA Jobin Yvon IBH Ltd, 133 Finnieston Street, Glasgow G3
8HB, UK
25 Ch. Sasikala, Ch. V. Ramana, P. Raghuveer Rao. Biotechnol. Prog.,
1994, 10, 451ꢀ459.
1
2
B. Krammer, K. Plaetzer. Photochem. Photobiol. Sci., 2008, 7, 283ꢀ
26 L. Bildstein, C. Dubernet, P. Couvreur. Adv. Drug Deliv. Rev., 2011,
63, 3ꢀ23.
289.
N. Fotinos, M. A. Campo, F. Popowycz. Photochem. Photobiol.,
2006, 82, 994ꢀ1015.
27 F. Kratz, I. A. Müller, C. Ryppa, A. Warnecke. ChemMedChem
2008, , 20ꢀ53.
,
3
3
4
K. A. Frei, H. M. Bonel, H. Frick. Br. J. Cancer, 2004, 90, 805ꢀ809.
Q. Peng, K. Berg, J. Moan, M. Kongshaug, J. M. Nesland.
Photochem. Photobiol., 1997, 65, 235ꢀ251.
28 S. E. Jin, H. E. Jin, S. S. Hong. Biomed. Res. Int., 2014, 814208.
29 A. M. S. Soares, S. P. G. Costa, M. S. T. Gonçalves. Amino Acids
2010, 39, 121ꢀ133.
,
,
5
6
P. K. Nootheti, M. P. Goldman. Dermatol. Clin., 2007, 25, 35ꢀ45.
S. Q. Tao, F. Li, L. Cao, R. S. Xia, H. Fan, Y. Fan, H. Sun, C. Jing,
L. J. Yang. Cell Biochem. Biophys., 2015, 73, 701ꢀ706.
E. W. Jeffes, J. L. McCullough, G. D. Weinstein, P. E. Fergin, J. S.
Nelson, T. F. Shull, K. R. Simpson, L. M. Bukaty, W. L. Hoffman,
N. L. Fong. Arch. Dermatol., 1997, 133, 727ꢀ 732.
30 A. M. S. Soares, S. P. G. Costa, M. S. T. Gonçalves. Tetrahedron
2010, 66, 8189ꢀ8195.
31 G. Hungerford, L. Ryderfors, M. J. G. Fernandes, M. S. T.
Gonçalves, S. P. G. Costa. J. Photochem. Photobiol. A: Chem., 2010,
215, 214ꢀ222.
7
32 A. S. C. Fonseca, M. S. T. Gonçalves, S. P. G. Costa. Amino Acids
2010, 39, 699ꢀ712.
,
8
9
T. Hasegawa, Y. Suga, Y. Mizuno, K. Haruna, H. Ogawa, S. Ikeda. J.
Dermatol., 2010, 37, 623ꢀ628.
33 A. M. S. Soares, A. M. Piloto, G. Hungerford, S. P. G. Costa, M. S. T.
Gonçalves. Eur. J. Org. Chem., 2012, 922–930.
T. Dai, Y. Y. Huang, M. R. Hamblin. Photodiagn. Photodyn. Ther.,
2009,
10 O. E. Akilov, S. Kosaka, K. O’Riordan, T. Hasan. Photochem.
Photobiol. Sci., 2007, , 1067ꢀ1075.
11 T. Zhou, L. Shao, S. Battah, C. Zhu, R. Hider, B. Reeder, A. Jabeen,
6, 170ꢀ188.
34 A. M. Piloto, G. Hungerford, S. P. G. Costa, M. S. T. Gonçalves. Eur.
J. Org. Chem., 2013, 34, 7715ꢀ7723.
6
35 A. M. Piloto, G. Hungerford, J. U. Sutter, A. M. S. Soares, S. P. G.
Costa, M. S. T. Gonçalves. J. Photochem. Photobiol. A, 2015, 299
,
S. MacRobert, G. Ren, X. Liang. Med. Chem. Commun., 2016, 7,
44ꢀ53.
1190ꢀ1196.
36 A. M. S. Soares, G. Hungerford, S. P. G. Costa, M. S. T. Gonçalves.
Eur. J. Org. Chem., 2015, 27, 5979ꢀ5986.
8 | J. Name., 2012, 00, 1-3
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ARTICLE
DOI: 10.1039/C6NJ03787A
37 A. M. S. Soares, G. Hungerford, S. P. G. Costa, M. S. T. Gonçalves.
New J. Chem., 2015, 39, 7227ꢀ7233.
38 A. M. S. Soares, G. Hungerford, S. P. G. Costa, M. S. T. Gonçalves.
Dyes Pigments, 2017, 137, 91ꢀ100.
39 F. S. Tjoeng, G. A. Heavner. Synthesis, 1981, 897ꢀ899.
40 J. V. Morris, M. A. Mahaney, J. R. Huber. J. Phys. Chem., 1976, 80
,
969ꢀ974.
41 C. Muller, P. Even, M.ꢀL. Viriot, M.ꢀC. Carré. Helv. Chim. Acta
2001, 84, 3735ꢀ3741.
,
42 P. Klán, T. Solomek, C. G. Bochet, A. Blanc, R. Givens, M. Rubina,
V. Popik, A. Kostikov, J. Wirz. Chem. Rev., 2013, 113, 119ꢀ191.
This journal is © The Royal Society of Chemistry 2012
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Table of Contents (TOC) entry
Light triggering of 5-aminolevulinic acid from fused coumarin ester cages
Ana M. S. Soares, Graham Hungerford, M. Sameiro T. Gonçalves and Susana P. G. Costa
O
H
O
O
N
H
R
HO
N
R
O
O
h
ν
O
MeOH/HEPES
(80:20)
O
+ photo by-products
R = Boc or H
Lightꢀtriggered release of 5ꢀaminolevulinic acid from various fused coumarin ester
cages under UV/vis irradiation in MeOH/HEPES buffer (80:20).
10 | J. Name., 2012, 00, 1-3
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