Photolytic release of bioactive carboxylic acids from fused pyran conjugates

Article abstract of DOI:10.1016/j.dyepig.2017.09.023

New ester cages bearing the coumarin (2H-benzopyran-2-one) skeleton with extended π-systems as phototriggers, for glycine and β-alanine, as models of carboxylic acid bifunctional molecules with biological relevance, were evaluated under photolysis conditions at 254, 300, 350 and 419 nm of irradiation in a RPR-100 photochemical reactor. The processes were followed by HPLC-UV detection and ¹H NMR with collection of kinetic data. The results showed a correlation between the photolysis efficiency and the increasing extension of the conjugation for both glycine and β-alanine, showing that the 7-aminocoumarin afforded the best results at all wavelengths tested. From a study of the time-resolved fluorescence behaviour, these compounds were also found to exhibit more complex fluorescence decay kinetics. This was attributed to the presence of conjugated and non-conjugated coumarin species.

Full text of DOI:10.1016/j.dyepig.2017.09.023

Accepted Manuscript
Photolytic release of bioactive carboxylic acids from fused pyran conjugates
Rafaela Conceição, Graham Hungerford, Susana P.G. Costa, M. Sameiro T.
Gonçalves
PII:
S0143-7208(17)31391-8
10.1016/j.dyepig.2017.09.023
DYPI 6247
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 20 June 2017
Revised Date: 8 September 2017
Accepted Date: 10 September 2017
Please cite this article as: Conceição R, Hungerford G, Costa SPG, Gonçalves MST, Photolytic release
of bioactive carboxylic acids from fused pyran conjugates, Dyes and Pigments (2017), doi: 10.1016/
j.dyepig.2017.09.023.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
Photolytic release of bioactive carboxylic acids from fused pyran conjugates
Rafaela Conceição,a Graham Hungerford,b Susana P. G. Costaa and M. Sameiro T. Gonçalvesa *
aCentre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
bHORIBA Jobin Yvon IBH Ltd, 133 Finnieston Street, Glasgow G38HB, UK
1

ACCEPTED MANUSCRIPT
Photolytic release of bioactive carboxylic acids from fused pyran conjugates
Rafaela Conceição,a Graham Hungerford,b Susana P. G. Costaa and M. Sameiro T. Gonçalvesa *
aCentre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
bHORIBA Jobin Yvon IBH Ltd, 133 Finnieston Street, Glasgow G38HB, UK
HIGHLIGHTS
•
Coumarin (2H-benzopyran-2-one) derivatives with amino, phenyl, 4-methoxyphenyl
and styryl groups at the 7-position, were synthesized and used in the derivatization of
glycine and β-alanine.
•
•
The new ester cages of glycine and β-alanine, selected as models of carboxylic acid
bioactive molecules, revealed to be interesting photo-responsive units.
A remarkable behaviour towards irradiation at higher wavelength (350 and 419 nm)
occurred in conjugates obtained from 4-(chloromethyl)-7-styryl-2H-benzopyran-2-
one.
•
•
Time-resolved fluorescence studies complemented the photolysis results and
proposed mechanism revealing the presence of different fluorescing species by the
determination of decay associated spectra.
This study shows new promising alternative moieties for the development of
photoactivable fluorescent acid prodrugs.
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Photolytic release of bioactive carboxylic acids from fused pyran conjugates
Rafaela Conceição,a Graham Hungerford,b Susana P. G. Costaa and M. Sameiro T. Gonçalvesa *
aCentre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
bHORIBA Jobin Yvon IBH Ltd, 133 Finnieston Street, Glasgow G38HB, UK
e-mail: msameiro@quimica.uminho.pt
Abstract: New ester cages bearing the coumarin (2H-benzopyran-2-one) skeleton with extended π-
systems as phototriggers, for glycine and β-alanine, as models of carboxylic acid bifunctional
molecules with biological relevance, were evaluated under photolysis conditions at 254, 300, 350
and 419 nm of irradiation in a RPR-100 photochemical reactor. The processes were followed by
1
HPLC-UV detection and H NMR with collection of kinetic data. The results showed a correlation
between the photolysis efficiency and the increasing extension of the conjugation for both glycine
and β-alanine, showing that the 7-aminocoumarin afforded the best results at all wavelengths tested.
From a study of the time-resolved fluorescence behaviour, these compounds were also found to
exhibit more complex fluorescence decay kinetics. This was attributed to the presence of conjugated
and non-conjugated coumarin species.
Keywords: Phototriggers; Caging groups; Neurotransmitters; Coumarins; Photolysis.
1. Introduction
2H-Benzopyran-2-one, the IUPAC name of coumarin, is an oxygen heterocycle firstly isolated in
1820 by Voleg from the plant Dypteryx adorata and is at the moment known to be present in a huge
number of natural products [1-3]. Since ancient Egyptian times, coumarins have been known for
their therapeutic properties, namely anticancer, antimicrobial, anesthetic, anti-HIV, anticoagulant
and antioxidant activities. Also, coumarins are used in the cosmetic and food industries, as
fluorescent probes and, more recently, as photocleavable protecting groups (PPGs) [4-9].
PPGs are chemical entities used to temporarily disable the activity of a compound, which is
achieved by a covalent bond between the PPG and a functional group essential to its activity. After
irradiation, the bond is cleaved and the compound activity is restored. For a biological application,
the employed wavelength needs to be as near as possible to the visible radiation wavelength in
order to reduce cellular damage. PPGs can be used to synthesize prodrugs sensitive to light,
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producing inactive therapeutic agents that regain their biological activity after irradiation. These
prodrugs allow the controlled release of drugs; both in time and locally, thus holding the promise of
reduced side effects and toxicity [10-15].
In comparison to one of the most common PPGs, o-nitrobenzyl, coumarins possess higher molar
extinction coefficients at longer wavelengths, higher releasing rates, increased stability and good
fluorescence. This makes them excellent options as PPGs for biological applications, besides their
interest for organic synthesis, and the study of their fluorescence provides a means by which to
assess the photocleavage process [11,16,17]. In addition, some coumarins can be used in two-
photon uncaging, a process where two photons are simultaneous absorbed and provide the
excitation energy required to initiate the photophysical process. This technique enables photolysis
by the absorption of NIR radiation which is less toxic to living systems than UV and presents a
higher tissue penetrating capacity [11-18]. Coumarins have been used in the photorelease of several
bioactive compounds such as amino acids (glycine, β-alanine, GABA, glutamate), secondary
messengers (cGMP, cAMP, diacylglycerol), nucleic acids/oligonucleotides (DNA, mRNA, 8-
bromonucleotids, ATP, ADP, AMP), hormones (progesterone), cholesterol and even drugs
(anisomycin and paclitaxel) [19-29].
Considering that one of the major drawbacks of PPGs is the fact that their maximum absorption
wavelength is at shorter wavelengths, the present work describes the synthesis of a new set of ester
conjugates of coumarins possessing an extended π-system or an amino group at position 7 of the
system. The intention is to bathochromically shift the wavelength of maximum absorption, and
consequently, of photolysis. Glycine and β-alanine were used as models of carboxylic acids, which
besides representing bifunctional molecules used in organic synthesis, possess crucial biological
roles in humans (as neurotransmitters, for example) [40-42].
For this purpose, the caged amino acids were characterized (using UV-Vis and fluorescence
spectroscopy) and irradiated at 254, 300, 350 and 419 nm in a photochemical reactor Rayonet RPR-
100. The release of the active compound was monitored by HPLC-UV detection with collection of
kinetic data and also by 1H NMR.
2. Experimental Section
2.1. Material and instruments
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. Chromatography on silica gel was carried out on Merck
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Kieselgel (230-240 mesh). IR spectra were determined on a BOMEM MB 104 spectrophotometer
using KBr discs. UV/visible absorption spectra (200 – 700 nm) were obtained using a Shimadzu
UV/2501PC spectrophotometer. NMR spectra were obtained on a Bruker Avance III 400 at an
1
13
operating frequency of 400 MHz for H and 100.6 MHz for C using the solvent peak as internal
reference at 25 ºC. All chemical shifts are given in ppm using δH Me4Si = 0 ppm as reference and J
values are given in Hz. Assignments were supported by spin decoupling-double resonance and
bidimensional heteronuclear correlation techniques. Mass spectrometry analyses were performed at
the “C.A.C.T.I. - Unidad de Espectrometria de Masas”, at University of Vigo, Spain. Fluorescence
spectra were collected using a FluoroMax-4 spectrofluorometer and excitation – emission matrices
(EEM’s) were recorded using a FluoroLog 3. Time-resolved fluorescence was measured using a
HORIBA Scientific DeltaFlex equipped with a DeltaDiode (DD-350, emitting at 349 nm) excitation
source. Fluorescence decays were measured at 5 nm increments over the wavelength range 375 nm
to 575 nm. The resultant decays were analysed globally to obtain decay associated spectra (using
EzTime software) or just made use of the decay obtained at 480 nm in the dataset. During the
analysis, an “extra” short-lived component (not plotted in Figure 3) was required and attributed to
scattered excitation. The optical density of the samples used or these fluorescence measurements
was kept to ~0.1 to avoid self-absorption effects. Photolysis measurements were carried out using a
Rayonet RPR-100 chamber reactor equipped with 10 lamps of 254, 300, 350 and 419 ± 10 nm.
HPLC analyses were performed using a Licrospher 100 RP18 (5 µm) column in a JASCO HPLC
system composed by a PU-2080 pump and a UV-2070 detector with ChromNav software. All
reagents were used as received. The synthesis of chloromethyl precursors 1a-e is included in the
supporting information.
2.2. General procedure for the synthesis of conjugates 4 and 5.
The chloromethyl precursors 1a-e were dissolved in dry DMF (3 mL), and potassium fluoride (3
equiv) and N-(tert-butoxycarbonyl)-glycine or N-(tert-butoxycarbonyl)-β-alanine (1 equiv) were
added. The reaction mixture was stirred at room temperature for 24 h. The solvent was removed by
evaporation under reduced pressure and the required conjugate was obtained as a solid. The crude
residue of compounds 4 and 5 was purified by column chromatography using mixtures of increasing
polarity of dichloromethane/methanol (4a-e) or light petroleum/ethyl acetate (5a-e) as eluent.
2.2.1. N-(tert-Butyloxycarbonyl)-glycine (2-oxo-2H-benzopyran-4-yl)methyl ester 4a. Starting from
compound 1a (0.031 g, 1.6 × 10-4 mol) in dry DMF (2 mL), potassium fluoride (0.028 g, 4.8 × 10-4
mol) and N-(tert-butoxycarbonyl)-glycine 2 (0.028 g, 1.6 × 10-4 mol), the ester conjugate 4a was
obtained as an orange solid (0.031 g, 56%). Mp = 115.1-116.6 ºC. Rf = 0.67 (dichloromethane). 1H
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NMR (CDCl3, 400 MHz): δ = 1.47 (s, 9 H, C(CH3)3), 4.07 (d, J 5.6 Hz, 2 H, H-α), 4.98 ( br s, 1 H,
NH), 5.39 (s, 2 H, CH2), 6.51 (s, 1 H, H-3), 7.33 (td, J 8 and 1.2 Hz, 1 H, H-6), 7.39 (dd, J 8 and 0.8
13
Hz, 1 H, H-8), 7.52 (dd, J 8 and 1.6 Hz, 1 H, H-5), 7.58 (td, J 8.8 and 1.6 Hz, 1 H, H-7) ppm. C
NMR (CDCl3, 100.6 MHz): δ = 28.27 (C(CH3)3) 42.41 (C-α), 61.78 (CH2), 80.45 (C(CH3)3)),
113.67 (C-3), 116.99 (C-4a), 117.50 (C-8), 123.41 (C-5), 124.55 (C-6), 132.22 (C-7), 148.25 (C-4),
153.66 (C-8a), 155.70 (C=O Boc), 160.16 (C-2), 169.77 (C=O Gly) ppm. IR (liquid film): ν = 3440,
3058, 2980, 2918, 2850, 1729, 1630, 1608, 1569, 1510, 1452, 1407, 1368, 1266, 1162, 1070, 1006,
937, 862, 738, 703 cm-1. HRMS: m/z (ESI): Found [M++1]: 334.12910; C17H20NO6 requires [M++1]:
334.12925.
2.2.2. N-(tert-Butyloxycarbonyl)-glycine (7-amino-2-oxo-2H-benzopyran-4-yl)methyl ester 4b.
Starting from compound 1b (0.031 g, 1.6 × 10-4 mol) in dry DMF (2 mL), potassium fluoride (0.016
g, 8.0 × 10-5 mol) and N-(tert-butoxycarbonyl)-glycine 2 (0.028 g, 1.6 × 10-4 mol), the ester
conjugate 4b was obtained as an yellow solid (0.038 g, 70%). Mp = 179.6-180.2 ºC. Rf = 0.6
(dichloromethane/methanol 20:1). 1H NMR (DMSO-d6, 400 MHz): δ = 1.38 (s, 9 H, C(CH3)3), 3.83
(d, J 6 Hz, 2 H, H-α), 5.30 (s, 2 H, CH2), 6.00 (s, 1 H, H-3), 6.72 (s, 2 H, NH2), 6.42 (d, J 2 Hz, H-
13
8), 6.55 (dd, J 8.8 and 2 Hz, 1 H, H-6), 7.31-7.36 (m, 2 H, H-5 and NH) ppm. C NMR (DMSO-
d6, 100.6 MHz): δ = 28.12 (C(CH3)3), 42.05 (C-α), 61.43 (CH2), 78.40 (C(CH3)3), 98.58 (C-8),
104.65 (C-3), 105.70 (C-4a), 111.31 (C-6), 125.41 (C-5), 150.56 (C-4), 153.25 (C-8a), 155.57 (C-
7), 155.94 (C=O Boc), 160.54 (C-2), 170.03 (C=O Gly) ppm. IR (KBr 1%): ν = 3383, 3231, 2976,
2363, 1743, 1702, 1616, 1526, 1404, 1279, 1169, 1056, 869, 850, 819, 747 cm-1. HRMS: m/z (ESI):
Found [M+ +1]: 349.14003; C17H21N2O6 requires [M++1]: 349.14010.
2.2.3. N-(tert-Butyloxycarbonyl)-glycine (7-phenyl-2-oxo-2H-benzopyran-4-yl)methyl ester 4c.
Starting from compound 1c (0.037 g, 1.4 × 10-4 mol) in dry DMF (2 mL), potassium fluoride (0.025
g, 4.3 × 10-3 mol) and N-(tert-butoxycarbonyl)-glycine 2 (0.025 g, 1.4 × 10-5 mol), the ester
conjugate 4c was obtained as an yellow solid (0.009 g, 16%). Mp = 129.5-131.6 ºC. Rf = 0.55
1
(dichloromethane/methanol 100:1). H NMR (CDCl3, 400 MHz): δ = 1.46 (s, 9 H, C(CH3)3), 4.08
(d, J 5 Hz, 2 H, H-α), 5.07 (t, J 5 Hz 1 H, NH), 5.41 (s, 2 H, CH2), 6.51 (s, 1 H, H-3), 7.45 (tt, J 7.2
and 1.2 Hz, 1 H, H-4’), 7.51 (tt, J 7.2 and 1.2 Hz, 2 H, H-3’and H-5’), 7.57-7.60 (m, 3 H, H-5, H-6
and H-8), 7.64 (dt, J 7.2 and 1.2 Hz, 2 H, H-2’ and H-6’) ppm. 13C NMR (CDCl3, 100.6 MHz): δ =
28.27 (C(CH3)3), 42.41 (C-α), 61.81 (CH2), 80.46 (C(CH3)3), 113.27 (C-3), 115.51 (C-6), 115.86
(C-4a), 123.38 (C-8), 123.79 (C-5), 127.19 (C-2’ and C-6’), 128.71 (C-4’), 129.14 (C-3’ and C-5’),
138.81 (C-1’), 145.41 (C-7), 148.13 (C-4), 154.10 (C-8a), 155.71 (C=O Boc), 160.31 (C-2), 169.78
(C=O Gly) ppm. IR (liquid film): ν = 3371, 3061, 2978, 2932, 1722, 1617, 1583, 1514, 1452, 1406,
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1368, 1337, 1279, 1252, 1163, 1074, 1055, 1004, 952, 866, 766, 750, 737, 699 cm-1. HRMS: m/z
(ESI): Found [M+ +1]: 410.16042; C23H24NO6 requires [M++1]: 410.16037.
2.2.4. N-(tert-Butyloxycarbonyl)-glycine [7-(4’methoxyphenyl)-2-oxo-2H-benzopyran-4-yl]methyl
ester 4d. Starting from compound 1d (0.037 g, 1.4 × 10-4 mol) in dry DMF (2 mL), potassium
fluoride (0.013 g, 2.2 × 10-4 mol) and N-(tert-butoxycarbonyl)-glycine 2 (0.013 g, 7.0 × 10-5 mol),
the ester conjugate 4d was obtained as a beige solid (0.008 g, 25%). Mp = 142.4-143.7 ºC. Rf = 0.4
(dichloromethane / methanol 100:1). 1H NMR (CDCl3, 400 MHz): δ = 1.46 (s, 9 H, C(CH3)3), 3.87
(s, 3 H, OCH3), 4.07 (d, J 6 Hz, 2 H, H-α), 5.12 (br s, 1H, NH), 5.37 (s, 2 H, CH2), 6.46 (s, 1 H, H-
3), 7.01 (dt, J 6.8 and 2 Hz, 2 H, H-3’ and H-5’), 7.51-7.52 (m, 3 H, H-5, H-6 and H-8), 7.57 (dt, J
6.8 and 2 Hz, 2 H, H-2’ and H-6’) ppm. 13C NMR (CDCl3, 100.6 MHz): δ = 28.25 (C(CH3)3), 42.40
(C-α), 55.38 (OCH3), 61.79 (CH2), 79.03 (C(CH3)), 112.77 (C-3), 114.55 (C-3’ and C-5’), 114.70
(C-6), 115.27 (C-4a), 122.81 (C-8), 123.71 (C-5), 128.29 (C-2’ and C-6’), 131.09 (C-1’), 144.94
(C-7), 148.19 (C-4), 154.14 (C-8a), 155.72 (C=O Boc) 160.24 (C-4’), 160.40 (C-2), 169.78 (C=O
Gly) ppm. IR (liquid film): ν = 3370, 3057, 2975, 2921, 2850, 1721, 1609, 1579, 1526, 1502, 1458,
1406, 1368, 1337, 1300, 1281, 1253, 1163, 1074, 1028, 952, 866, 839, 816, 735, 703 cm-1. HRMS:
m/z (ESI): Found [M+ +1]: 440.17098; C24H26NO7 requires [M++1]: 440.17099.
2.2.5. N-(tert-Butyloxycarbonyl)-glycine (7-styryl-2-oxo-2H-benzopyran-4-yl)methyl ester 4e.
Starting from compound 1e (0.027 g, 9.0 × 10-5 mol) in dry DMF (2 mL), potassium fluoride (0.016
g, 2.7 × 10-4 mol) and N-(tert-butoxycarbonyl)-glycine 2 (0.016 g, 9.0 × 10-5 mol), the ester
conjugate 4d was obtained as a yellow solid (0.023 g, 58%). Mp = 131.3-131.4 ºC. Rf = 0.38
(dichloromethane/methanol 100:1). 1H NMR (CDCl3, 400 MHz): δ = 1.47 (s, 9H, C(CH3)3), 4.07 (d,
J 6.4 Hz, 2 H, H-α), 5.07 (d, J 6.4 Hz , 1 H, NH), 5.37 (s, 2 H, CH2), 6.46 (s, 1 H, H-3), 7.12 (d, J
16.4 Hz, 1 H, H-a), 7.25 (d, J 16.4 Hz, 1 H, H-b), 7.33 (tt, J 7.2 and 1.6 Hz, 1 H, H-4’), 7.40 (tt, J
7.2 and 1.6 Hz, 1 H, H-3´and H-5’), 7.45-7.49 (m, 3 H, H-5, H-6 and H-8), 7.60 (dt, J 7.2 and 1.6
13
Hz, 2 H, H-2’ and H-6’) ppm. C NMR (CDCl3, 100.6 MHz): δ = 28.27 (C(CH3)3), 42.41 (C-α),
61.78 (CH2), 80.43 (C(CH3)), 112.91 (C-3), 114.62 (C-6), 116.04 (C-4a), 122.63 (C-8), 123.63 (C-
5), 126.54 (C-a), 126.93 (C-2’ and C-6’), 128.62 (C-4’), 128.84 (C-3’ and C-5’), 132.33 (C-b),
136.27 (C-1’), 141.73 (C-7), 148.05 (C-4), 154.15 (C-8a), 155.71 (C=O Boc), 160.31 (C-2), 171.61
(C=O Gly) ppm. IR (liquid film): ν = 3368, 2976, 2932, 1722, 1609, 1526, 1407, 1367, 1253, 1168,
1083, 1027, 955, 866, 838, 815, 734 cm-1. HRMS: m/z (ESI): Found [M++1]: 436.17608; C21H19N2O5
requires [M++1]: 436.17605.
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2.2.6. N-(tert-Butyloxycarbonyl)-β-alanine (2-oxo-2H-benzopyran-4-yl)methyl ester 5a. Starting
from compound 1a (0.016 g, 8.0 × 10-5 mol) in dry DMF (2 mL), potassium fluoride (0.017 g, 2.5 ×
10-4 mol) and N-(tert-butoxycarbonyl)-β-alanine 3 (0.016 g, 8.0 × 10-5 mol), the ester conjugate 5a
was obtained as an orange solid (0.011 g, 38%). Mp = 91.4-92.1 ºC. Rf = 0.5 (dichloromethane). 1H
NMR (CDCl3, 400 MHz): δ = 1.45 (s, 9 H, C(CH3)3)), 2.70 (t, J 6 Hz, 2 H, H-α), 3.47 (q, J 6 Hz, 2
H, H-β), 4.98 (br s, 1 H, NH), 5.34 (s, 2 H, CH2), 6.49 (s, 1 H, H-3), 7.33 (td, J 7.2 and 1.2 Hz, 1 H,
H-6), 7.39 (dd, J 8.4 and 0.8 Hz, 1 H, H-8), 7.53 (dd, J 8.4 and 1.6 Hz, 1 H, H-5), 7.58 (td, J 7.2 and
1.2 Hz, 1 H, H-7) ppm. 13C NMR (CDCl3, 100.6 MHz): δ = 28.34 (C(CH3)3), 34.55 (C-α), 36.05 (C-
β), 61.30 (CH2), 79.66 (C(CH3)3), 113.53 (C-3), 117.07 (C-4a), 117.49 (C-8), 123.41 (C-5), 124.52
(C-6), 132.18 (C-7), 148.66 (C-4), 153.65 (C-8a), 155.72 (C=O Boc), 160.24 (C-2), 171.62 (C=O β-
Ala) ppm. IR (liquid film): ν = 3374, 2979, 2932, 1730, 1630, 1607, 1568, 1513, 1452, 1393, 1367,
1318, 1252, 1170, 1082, 934, 860, 737, 704 cm-1. HRMS: m/z (ESI): Found [M++1]: 348.14476;
C18H22NO6 requires [M++1]: 348.14480.
2.2.7. N-(tert-Butyloxycarbonyl)-β-alanine (7-amino-2-oxo-2H-benzopyran-4-yl)methyl ester 5b.
Starting from compound 1b (0.030 g, 1.4 × 10-4 mol) in dry DMF (2 mL), potassium fluoride (0.025
g, 4.3 × 10-3 mol) and N-(tert-butoxycarbonyl)-β-alanine 3 (0.027 g, 1.4 × 10-3 mol), the ester
conjugate 5b was obtained as a beige solid (0.032 g, 65%). Mp = 157.3-158.2 ºC. Rf = 0.54
(dichloromethane/methanol 20:1). 1H NMR (DMSO-d6, 400 MHz): δ = 1.36 (s, 9 H, C(CH3)3), 2.58
(t, J 6 Hz, 2 H, H-α), 3.20 (q, J 6 Hz, 2 H, H-β), 5.22 (s, 1 H, CH2), 5.74 (s, 1 H, H-3), 6.18 (s, 2 H,
NH2), 6.44 (d, J 2 Hz, 1 H, H-8), 6.55 (dd, J 8.4 and 2 Hz, 1 H, H-6), 6.90 (t, J 6 Hz, 1 H, NH), 7.35
(d, J 8.8 Hz, 1 H, H-5) ppm. 13C NMR (DMSO-d6, 100.6 MHz): δ = 28.17 (C(CH3)3), 34.00 (C-α),
36.05 (C-β), 61.27 (CH2), 77.77 (C(CH3)3), 98.60 (C-8), 105.02 (C-3), 105.84 (C-4a), 111.35 (C-6),
125.47 (C-5), 150.63 (C-4), 153.25 (C-8a), 155.50 (C-7), 155.61 (C=O Boc), 160.56 (C-2), 170.83
(C=O β-Ala) ppm. IR (KBr 1%): ν = 3485, 3393, 3349, 3227, 2972, 1748, 1707, 1682, 1643, 1608,
1527, 1448, 1411, 1367, 1325, 1293, 1248, 1186, 857, 844, 806, 781, 746, 708 cm-1. HRMS: m/z
(ESI): Found [M++1]: 363.15569; C18H23N2O6 requires [M++1]: 363.15559.
2.2.8. N-(tert-Butyloxycarbonyl)-β-alanine (7-phenyl-2-oxo-2H-benzopyran-4-yl)methyl ester 5c.
Starting from compound 1c (0.031 g, 1.2 × 10-4 mol) in dry DMF (2 mL), potassium fluoride (0.021
g, 3.6 × 10-3 mol) and N-(tert-butoxycarbonyl)-β-alanine 3 (0.023 g, 1.2 × 10-5 mol), the ester
conjugate 5c was obtained as a beige solid (0.028 g, 55%). Mp = 152.3-153.1 ºC. Rf = 0.61
(dichloromethane / methanol 100:1). 1H NMR (CDCl3, 400 MHz): δ = 1.45 (s, 9 H, C(CH3)3), 2.71
(m, 2 H, H-α), 3.47 (m, 2 H, H-β), 5.01 (br s, 1 H, NH), 5.59 (s, 2 H, CH2), 6.49 (s, 1 H, H-3), 7.43
(tt, J 7.2 and 1.2 Hz, 1 H, H-4’), 7.50 (tt, J 7.2 and 1.2 Hz, 2 H, H-3’ and H-5’), 7.54-7.59 (m, 3 H,
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13
H-5, H-6 and H-8), 7.63 (dt, J 7.2 and 1.2 Hz, 2 H, H-2’ and H-6’) ppm. C NMR (CDCl3, 100.6
MHz): δ = 28.32 (C(CH3)3), 34.54 (C-α), 36.03 (C-β), 61.30 (CH2), 79.62 (C(CH3)3), 113.11 (C-3),
115.44 (C-6), 115.93 (C-4a), 123.32 (C-8), 123.78 (C-5), 127.15 (C-2’ and C-6’), 128.67 (C-4’),
129.10 (C-3’ and C-5’), 138.78 (C-1’), 145.32 (C-7), 148.52 (C-4), 154.07 (C-8a), 155.72 (C=O
Boc) 160.35 (C-2), 171.61 (C=O β-Ala) ppm. IR (liquid film): ν = 3434, 3334, 3078, 2976, 2929,
1743, 1713, 1618, 1546, 1500, 1438, 1409, 1366, 1327, 1246, 1167, 1092, 1066, 1042, 997, 962,
868, 837, 764, 748, 707, 689 cm-1. HRMS: m/z (ESI): Found [M++1]: 424.17608; C24H26NO6 requires
[M++1]: 424.17611.
2.2.9.
N-(tert-Butyloxycarbonyl)-β-alanine
[7-(4’methoxyphenyl)-2-oxo-2H-benzopyran-4-yl]
methyl ester 5d. Starting from compound 1d (0.023 g, 1.2 × 10-4 mol) in dry DMF (2 mL),
potassium fluoride (0.021 g, 3.6 × 10-3 mol) and N-(tert-butoxycarbonyl)-β-alanine 3 (0.023 g, 1.2 ×
10-5 mol), the ester conjugate 5c was obtained as a beige solid (0.028 g, 51%). Mp = 126.9-127.5
1
ºC. Rf = 0.46 (dichloromethane/methanol 100:1). H NMR (CDCl3, 400 MHz): δ = 1.45 (s, 9 H,
C(CH3)3), 2.71 (t, J 6 Hz, 2 H, H-α), 3.47 (q, J 6 Hz, 2 H, H-β), 3.88 (s, 3 H, OCH3), 4.99 (br s, 1 H,
NH), 5.35 (s, 2 H, CH2) 6.46 (s, 1 H, H-3), 7.03 (dt, J 8.8 and 2 Hz, 2 H, H-3’ and H-5’), 7.50-7.56
(m, 3 H, H-5, H-6 and H-8), 7.58 (dt, J 8,8 and 2 Hz, 2 H, H-2’ and H-6’) ppm. 13C NMR (CDCl3,
100.6 MHz): δ = 28.35 (C(CH3)3), 34.56 (C-α), 36.05 (C-β), 55.41 (OCH3), 61.34 (CH2), 79.63
(C(CH3)3), 112.74 (C-3), 114.58 (C-3’ and C-5’), 114.76 (C-6), 115.39 (C-4a), 122.82 (C-8),
123.72 (C-5), 128.32 (C-2’ and C-6’), 131.15 (C-1’), 144.96 (C-7), 148.57 (C-4), 154.18 (C-8a),
155.73 (C=O Boc), 160.26 (C-4’), 160.49 (C-2), 171.65 (C=O β-Ala) ppm. IR (liquid film): ν =
3368, 3056, 2977, 2934, 2840, 2050, 1722, 1610, 1580, 1526, 1444, 1407, 1367, 1302, 1279, 1253,
1170, 1083, 1028, 996, 955, 866, 839, 816,781, 735, 704 cm-1. HRMS: m/z (ESI): Found [M++1]:
454.18664; C25H28NO7 requires [M++1]: 454.18652.
2.2.10. N-(tert-Butyloxycarbonyl)-β-alanine (7-styryl-2-oxo-2H-benzopyran-4-yl)methyl ester 5e.
Starting from compound 1e (0.031 g, 1.1 × 10-3 mol) in dry DMF (2 mL), potassium fluoride (0.018
g, 3.1 × 10-3 mol) and N-(tert-butoxycarbonyl)-β-alanine 3 (0.020 g, 1.1 × 10-4 mol), the ester
conjugate 5c was obtained as a yellow solid (0.011 g, 24%). Mp = 141.2-142.2 ºC. Rf = 0.38
(dichloromethane / methanol 100:1). 1H NMR (CDCl3, 400 MHz): δ = 1.45 (s, 9 H, C(CH3)3), 2.70
(t, J 6 Hz, 2 H, H-α), 3.47 (t, J 6 Hz, 2 H, H-β), 4.98 (br s, 1 H, NH), 5.33 (s, 2 H, CH2), 6.45 (s, 1
H, H-3), 7.12 (d, J 16 Hz, 1 H, H-a), 7.25 (d, J 16 Hz, 1 H, H-b), 7.33 (tt, J 7.2 and 1.2 Hz, 1 H, H-
4’), 7.40 (tt, J 7.2 and 1.2 Hz, 2 H, H-3’ and H-5’), 7.45-7.51 (m, 3 H, H-5, H-6 and H-8), 7.43 (dd,
13
J 7.2 and 1.2 Hz, 2 H, H-2’ and H-6’) ppm. C NMR (CDCl3, 100.6 MHz): δ = 28.30 (C(CH3)3),
34.51 (C-α), 35.99 (C-β), 61.27 (CH2), 79.59 (C(CH3)3), 112.78 (C-3), 114.60 (C-6), 116.09 (C-4a),
9

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122.56 (C-8), 123.59 (C-5), 126.51 (C-a), 126.88 (C-2’ and C-6’), 128.57 (C-4’), 128.80 (C-3’ and
C-5’), 132.27 (C-b), 136.23 (C-1’), 141.66 (C-7), 148.40 (C-4), 154.12 (C-8a), 155.69 (C=O Boc),
160.36 (C-2), 171.60 (C=O β-Ala) ppm. IR (liquid film): ν = 3366, 3059, 2976, 2930, 1720, 1610,
1513, 1450, 1413, 1393, 1367, 1251, 1169, 1082, 1005, 967, 865, 865, 812, 757, 738, 694 cm-1.
HRMS: m/z (ESI): Found [M++1]: 450.19174; C26H28NO6 requires [M++1]: 450.19184.
2.3. General procedure for the synthesis of conjugates 6 and 7.
A solution of conjugates 4b, 4e, 5b or 5e (12-21 mmol) in dichloromethane/trifluoroacetic
acid (2:1) (15 mL) was stirred at room temperature during 30 min. The reactional mixture
was then evaporated to dryness and aqueous 1 M NaOH solution (3 mL) was added and the
pH adjusted to 7. The mixture was extracted with dichloromethane (3 × 3 mL), the organic
phase was dried with anhydrous magnesium sulphate, filtered and evaporated to dryness,
resulting in the required compounds 6b, 6e, 7b or 7e.
2.3.1. Glycine (7-amino-2-oxo-2H-benzopyran-4-yl)methyl ester 6b. Starting from compound 4b
(0.069 g, 2.0 × 10-4 mol), conjugate 6b was obtained as a yellow solid (0.0472 g, 65%). Mp =
1
192.0-193.5 ºC. Rf = 0.18 (dichloromethane / methanol 20:1). H NMR (CD3OD, 400 MHz): δ =
4.05 (s, 2 H, H-α), 5.47 (s, 2 H, CH2), 6.12 (s, 1 H, H-3), 6,53 (d, J 2 Hz, 1 H, H-8), 6.66 (dd, J 8.8
and 2 Hz, 1 H, H-6), 7.39 (d, J 8.8 Hz, 1 H, H-5) ppm. 13C NMR (CD3OD, 100.6 MHz): δ = 41.00
(C-α), 63.98 (CH2), 100.67 (C-8), 106.76 (C-3), 107.95 (C-4a), 113.28 (C-6), 126.11 (C-5), 151.64
(C-4), 154.86 (C-8a), 157.36 (C-7), 164.00 (C-2), 168.27 (C=O Gly) ppm. IR (liquid film): ν =
1757, 1672, 1603, 1416, 1332, 1265, 1188, 1127, 882, 838, 800, 748, 720 cm-1. HRMS: m/z (ESI):
Found [M++1]: 249.08759; C12H13N2O4 requires [M++1]: 249.08767.
2.3.2. Glycine (7-styryl-2-oxo-2H-benzopyran-4-yl)methyl ester 6e. Starting from compound 4e
(0.051 g, 1.2 × 10-4 mol), conjugate 6e was obtained as an orange oil (0.0379 g, 66%). Rf = 0.44
(dichloromethane/methanol 20:1). 1H NMR (DMSO-d6, 400 MHz): δ = 3.15 (s, 2 H, H-α), 4.76 (s, 2
H, CH2), 6.42 (s, 1 H, H-3), 7.28-7.33 (m, 2 H, H-4’and H-a), 7.36-7.41 (m, 4 H, NH2, H-3’and H-
5’), 7.49 (d, 1 H, J 16.8 Hz, H-b), 7.58-7.65 (m, 4 H, H-2’, H-6’, H-6 and H-8), 7.68 (d, J 8 Hz, 1
H, H-5) ppm. 13C NMR (DMSO-d6, 100.6 MHz): δ = 51.70 (C-α), 59.05 (CH2), 110.10 (C-3),
113.81 (C-6), 116.49 (C-4a), 122.52 (C-8), 124.62 (C-5), 126.81 (C-a), 126.93 (C-2’ and C-6’),
128.38 (C-4’), 128.85 (C-3’ and C-5’), 131.59 (C-b), 136.55 (C-1’), 140.97 (C-7), 153.50 (C-8a),
156.38 (C-4), 160.32 (C-2), 171.08 (C=O Gly) ppm. IR (liquid film): ν = 3418, 2925, 2855, 2663,
2362, 1758, 1681, 1612, 1546, 1433, 1329, 1206, 1134, 1076, 1003, 952, 922.2, 886, 839, 801, 722
cm-1. HRMS: m/z (ESI): Found [M++1]: 336.12364; C20H18NO4 requires [M++1]: 336.12358.
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2.3.3. β-Alanine (7-amino-2-oxo-2H-benzopyran-4-yl)methyl ester 7b. Starting from compound 5b
(0.0721 g, 2.1 × 10-4 mol), conjugate 7b was obtained as a yellow solid (0.054 g, 67%). Mp =
1
201.6-203.0 ºC. Rf = 0.18 (dichloromethane / methanol 20:1) H NMR (CD3OD, 400 MHz): δ =
2.93 (t, J 6.4 Hz, 2 H, H-α), 3.33 (t, J 6.4 Hz, 2 H, H-β), 5.47 (s, 2 H, CH2), 6.12 (s, 1 H, H-3), 6.53
13
(d, J 2 Hz, 1 H, H-8), 6,66 (dd, J 8.8 and 2 Hz, 1 H, H-6), 7.39 (d, J 8.8 Hz, 1 H, H-5) ppm. C
NMR (CD3OD, 100.6 MHz): δ = 32.11 (C-α), 36.26 (C- β), 63.14 (CH2), 100.64 (C-8), 107.21 (C-
3), 108.94 (C-4a), 113.82 (C-6), 126.23 (C-5), 152.25 (C-4), 153.05 (C-8a), 157.23 (C-7), 163.94
(C-2), 168.27 (C=O β-Ala) ppm. IR (liquid film): ν = 1738, 1680, 1413, 1329, 1202, 1147, 846,
797, 747, 723 cm-1. HRMS: m/z (ESI): Found [M++1]: 263.10325; C13H15N2O4 requires [M++1]:
263.10327.
2.3.4. β-Alanine (7-styryl-2-oxo-2H-benzopyran-4-yl)methyl ester 7e. Starting from compound 5e
(0.0551 g, 1.2 × 10-4 mol), conjugate 7e was obtained as a yellow solid (0.0403 g, 64%). Mp =
1
205.1-206.2 ºC. Rf = 0.44 (dichloromethane/methanol 20:1). H NMR (DMSO-d6, 400 MHz): δ =
2.84 (t, J 6.8 Hz, 2 H, H-α), 3.11 (t, J 6.8 Hz, 2 H, H-β), 5.43 (s, 2 H, CH2), 6.47 (s, 1 H, H-3), 7.32-
7.34 (m, 2 H, H-4’and H-a), 7.41 (td, J 6.8 and 2.4 Hz, 2 H, H-3’and H-5’), 7.51 (d, 1 H, J 16.8 Hz,
H-b), 7.63-7.68 (m, 4 H, H-2’, H-6’, H-6 and H-8), 7.75-7.79 (m, 3 H, H-5 and NH2) ppm. 13C
NMR (DMSO-d6, 100.6 MHz): δ = 31.43 (C-α), 34.64 (C-β), 61.61 (CH2), 111.91 (C-3), 113.91
(C-6), 115.97 (C-4a), 122.75 (C-8), 125.09 (C-5), 126.78 (C-a), 126.97 (C-2’ and C-6’), 128.50 (C-
4’), 128.87 (C-3’ and C-5’), 131.99 (C-b), 136.46 (C-1’), 141.46 (C-7), 149.70 (C-4), 153.61 (C-
8a), 159.78 (C-2), 169.97 (C=O β-Ala) ppm. IR (liquid film): ν = 1712, 1608, 1452, 1411, 1324,
1201, 1129, 1070, 1010, 974, 901, 867, 831, 801, 760, 720 cm-1. HRMS: m/z (ESI): Found [M++1]:
350.13930; C21H19N2O5 requires [M++1]: 350.13943.
2.4. General photolysis procedure
A 1×10-4
M methanol/HEPES (80:20) solution of compounds 4-7 (mL) were placed in a quartz tube
and irradiated in a Rayonet RPR-100 reactor at the desired wavelength. The lamps used for
irradiation were of 254, 300, 350 and 419 ± 10 nm. The HEPES buffer solution was prepared using
distilled water with HEPES (10 mM), sodium chloride (120 mM), potassium chloride (3 mM),
calcium chloride (1 mM) and magnesium chloride (1 mM). The pH was adjusted to 7.2 by addition
of an aqueous solution on sodium hydroxide 6M.
Aliquots of 100 µL were taken at regular intervals and analysed by RP-HPLC. The eluent was
acetonitrile / water (3:1) for all the compounds, excepting compounds 4b and 5b (acetonitrile /
water (1:1) with 0.1% TFA), 6e and 7e (acetonitrile/water (1:1)), at a flow rate of 0.8 mL/min (4b,
11

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4e, 5a, 5d, 5e, 6b, 7b), 0.6 mL/min for (4c, 4d, 5b, 5c) and 0.4 mL/min (4a), previously filtered
through a Millipore, type HN 0.45 µm filter and degassed by ultra-sound for 30 min. The
chromatograms were traced by detecting UV absorption at the wavelength of maximum absorption
for each compound (retention time: 4a, 6.3; 5a, 4.2; 4b, 6.4; 5b, 9.3; 4c, 6.3; 5c, 8.8; 4d, 10.6; 5d,
7.0; 4e, 7.0; 5e, 11.5; 6b: 13.4; 7b: 4.8 min).
3. Results and Discussion
3.1. Synthesis of amino acid conjugates 4-7
4-(Chloromethyl)-2H-benzopyran-2-one 1a, 7-amino-4-(chloromethyl)-2H-benzopyran-2-one 1b,
4-(chloromethyl)-7-phenyl-2H-benzopyran-2-one 1c, 4-(chloromethyl)-7-(4’-methoxyphenyl)-2H-
benzopyran-2-one 1d and 4-(chloromethyl)-7-styryl-2H-benzopyran-2-one 1e were synthesised by
Pechmann condensations between phenol, ethyl (3-hydroxyphenyl)carbamate, [1,1'-biphenyl]-3-ol,
4'-methoxy-[1,1'-biphenyl]-3-ol, 3-styrylphenol, respectively, and ethyl 4-chloro-3-oxobutanoate,
mediated by aqueous sulfuric acid through a known procedure [43], followed by acidolysis (sulfuric
acid in acetic acid) of ethyl (4-(chloromethyl)-2-oxo-2H-benzopyran-7-yl)carbamate precursor in
the case of compound 1b (see supporting information). Compounds 1a-e were used in the
preparation of ester conjugates of glycine and β-alanine, as representative models of carboxylic
acids, by reacting with N-tert-butyloxycarbonylglycine 2 and N-tert-butyloxycarbonyl-β-alanine 3,
in the presence of potassium fluoride in DMF at room temperature, yielding cages 4a-e and 5a-e,
respectively, in 16 to 70% yields. Therefore, in compounds 4b, 4e, 5b and 5e the N-tert-
butyloxycarbonyl protecting group was removed by acidolysis with trifluoroacetic acid in
dichloromethane giving conjugates 6b, 6e, 7b and 7e bearing the amino acid and the photosensitive
tag (Scheme 1).
1
13
1
All compounds were fully characterized by IR, H and C NMR spectroscopies. H NMR spectra
of conjugates 4-7 showed the amino acid residues, such as the α-CH2 (δ 3.15-4.08 ppm for glycine
and δ 2.58-2.93 ppm for β-alanine), and β-CH2 (δ 3.11-3.47 ppm), in addition to the heterocycle
methylene group visible for all conjugates (δ 4.76-5.59 ppm). The newly formed ester linkages were
established by their 13C NMR spectra signals of the carbonyl group, at about δ 168.27-171.65 ppm.
The confirmation of the cleavage of the N-tert-butyloxycarbonyl-protecting group at the amino
acids was also supported by 13C NMR, with the disappearance of spectra signals of the
corresponding carbonyl group in 6b, 6e, 7b and 7e, which occurred at δ 155.61 or 155.94 ppm in
conjugates 4b, 4e, 5b and 5e. The IR spectra of compounds 4-7 displayed stretching vibration
bands of the ester carbonyl groups from 1712 to 1758 cm-1.
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<Scheme 1>
3.2. Evaluation of the photophysical properties of amino acid conjugates 4-7
With the aim of finding the parameters required for monitoring the photolytic process, as well as to
evaluate the effect of the fluorophore structure in different solvents, such as diethyl ether,
dichloromethane, acetonitrile, dimethyl sulfoxide, methanol and methanol/HEPES buffer (80:20),
characterization by UV/visible absorption was carried out. Furthermore, fluorescence emission data
was also collected in methanol and methanol/HEPES buffer (80:20). Degassed 10-4 M solutions of
ester conjugates 4-7 and the corresponding coumarins were measured. The corresponding data:
absorption (λabs) and emission maxima (λem), molar extinction coefficients (as log ε) and relative
fluorescence quantum yields (ΦF) are reported in Table 1. Relative fluorescence quantum yields
were calculated using 9,10-diphenylanthracene in ethanol (ΦF 0.95) [44], with the fluorescence
standard excited at the maximum absorption wavelength for each compound.
Concerning the absorption data of conjugates 4-7, variation of the solvent in general did not
significantly affect the wavelength of maxima absorption; however, as it was expected, the increase
of the π system extension resulted in a bathochromic shift (λabs 4a < 4c < 4d < 4e and 5a < 5c < 5d
< 5e). Despite the higher conjugation of systems possessing aromatic substituents at the coumarin
skeleton (compounds 4c-e and 5c-e), comparatively to the amino group (compounds 4b and 5b) the
former presented higher λabs in dimethyl sulfoxide, methanol and methanol/HEPES buffer (80:20),
in the range 353-364 nm.
<Table 1>
To investigate the fluorescence behaviour of the compounds excitation-emission matrices (EEM’s)
were obtained; with the emission intensity scanned for different excitation wavelengths. The
outcome for representative compounds (1, 4 and 5; b, d and e - in methanol/ HEPES), showing a
contour surface of fluorescence intensity for the different excitation / emission wavelengths, are
given in Figure 1. These fluorescence contour surfaces indicate that the steady state fluorescence
behaviour of the compounds is quite similar, with an emission maximum close to 480 nm with an
optimal excitation around 360 nm.
<Figure 1 >
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3.3. Photolysis studies of amino acid conjugates 4-7
Comparison of the sensitivity towards UV/visible radiation of glycine and β-alanine ester
conjugates 4-7, bearing different substituents at position 7 of the heterocycle, was carried out by
exposing solutions of the mentioned compounds in methanol/HEPES buffer (80:20) solution in a
Rayonet RPR-100 reactor at 254, 300, 350 and 419 nm. The course of the photocleavage 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 3 runs. The determined irradiation time 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, which suggested a first order
reaction, obtained by the linear least squares methodology for a straight line. The corresponding rate
constants were calculated and are presented in Table 2. The photochemical quantum yields (Φphot
)
were calculated based on half-lives (t1/2), extinction coefficient ( ) and the incident photon flux (I0),
ε
which was determined by potassium ferrioxalate actinometry [45]. The calculated photochemical
quantum yields indicated that the photocleavage process was not as efficient as desirable, probably
due to the dissipation of part of the absorbed energy via fluorescence pathways that compete with
the photochemical reaction.
<Table 2>
It was expected that the bathochromic shift in the maximum absorption wavelengths, related to the
increase of the π conjugated system due to the presence of aromatic substitution at position 7 of the
benzopyran, would result in an increase in the efficiency of the photolysis at longer wavelengths,
leading to shorter irradiation times. The obtained results confirmed this expectation, since
photocleavage at 350 and 419 nm of conjugates 4e and 5e occurred fast (57 and 109 min at 350 nm;
318 and 1359 min at 419 nm), in comparison with conjugates 4a, 4c, 4d, 5a, 5c and 5d.
Furthermore, at these wavelengths the release of N-tert-butyloxycarbonyl glycine 2 and β-alanine 3
required shorter irradiation times than the corresponding free amino acids from cages 6b and 7b.
Nonetheless, the best results of photolysis obtained at all the wavelengths (254, 300, 350 and 419
nm) considered in the present study, including at 419 nm were related to conjugates 4b (5-24 min)
and 5b (7-85 min), possessing the electron donating amino group at position 7 of the heterocycle.
Although the importance of all the fluorescent caging groups evaluated, the use of 7-amino-4-
(chloromethyl)-2H-benzopyran-2-one 1b is the most feasible for release of the amino acids at 419
14

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nm both for organic synthesis and caging applications, owing to the short irradiation times and
lowest possible side reactions concerning the functionalities of the molecule. Coumarinyl esters are
thought to cleave through an ionic mechanism that involves both homolytical or heterolytical
fission of the O-CH2 bond, although the latter is energetically favoured. The homolytic cleavage of
the O-C bond, followed by electron transfer, can yield the ion pair (a methylenic coumarin
carbocation and the leaving group anion), whereas the heterolytic cleavage of the O-C bond directly
affords the already mentioned ion pair. Once formed, the methylenic coumarin carbocation can
undergo nucleophilic attack by the solvent to form the final products. The ion pair may also
recombine to the starting material [46-47]. In our previous work, after irradiation at the various
wavelengths of irradiation, by NMR monitoring a new set of signals were identified in the aromatic
region with slight shifts of the signals from those of the starting conjugates, which were in
agreement with the proposed coumarin by-products, by NMR and mass spectrometry. In recent
literature and in terms of potential toxicity issues, there are various reports on the use of coumarin
derivatives in photodelivery applications that have undergone biological validation [48-52].
In order to better understand the process, time-resolved fluorescence measurements were made
exciting at 349 nm, with the emission monitored at 480 nm. The time-resolved decays are shown in
Figure 2 and in all cases the fluorescence was multi-exponential in nature indicating the presence of
(at least) two excited states. Compound 1b exhibited a dominant (>90%) decay time of ~5 ns,
resulting in an average decay time of 4.8 ns. The other b compounds gave average decay times that
were significantly less (4b, 1.36 ns; 5b, 2.32 ns; 6b, 1.75 ns; 7b, 1.84 ns). This was because of the
presence of other (non-radiative) decay pathways as expected since the photocleavage of the
conjugate would be expected to proceed by the production of an ion pair, which would then either
recombine or lead to the photoproducts. Thus, because of the use of the fluorescent coumarin
moiety, the time-resolved behaviour can report on this process. The d and e compounds, however,
did not display any major difference in their relative decay times. Average decay times were; 1d,
3.2 ns; 4d, 2.99 ns; 5d, 3.12 ns; 1e, 2.12 ns; 4e, 3.09 ns; 5e, 2.81 ns. This may indicate that the
conjugate is having any significant influence on the photophysical properties of the coumarin
moiety. During these experiments, it is assumed that any photocleavage of the conjugate is not
significant.
To further characterise these compounds the b compounds were selected and from the time-
resolved datasets (decays every 5 nm, spanning 375 nm to 575 nm) decay associated spectra
determined. This involves a global analysis of the time-resolved decays, linking common decay
times for all decays, and then plotting the pre-exponential factor for a decay time (weighted by that
decay time) against wavelength. This helps elucidate both the energy (wavelength) and kinetic of
the excited states, hence species, present. The results from this form of analysis are presented in
15

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Figure 3. The lifetimes obtained are similar to those from the single curves analysis, however the
presence of a sub-nanosecond contribution is now elucidated. Since these spectra are weighted by
the decay time they are representative of the contribution to the steady state emission. For 1b the
emission is dominated by the spectra associated with the longer-lived emission. For 4b, 5b and 7b
however the major contribution is that between ~1.3 ns to 2.3 ns, although similar in spectral
appearance. This would indicate that in the latter compounds the decay time has been reduced
because of the presence of non-radiative decay pathways. For compound 6b the emission is
dominated by the longer-lived emission, which because of the decay properties of 1b would be
tempting to attribute to the photocleaved coumarin moiety. Because of the spectral separation it is
clear that overall at least three different species are present in the conjugated compounds. It would
be expected that conjugated, ion pair and photocleaved coumarin moiety would be present, with the
longer-lived decay associated with the photocleaved species and the shortest decay probably
associated with the conjugated form.
In addition to the HPLC/UV monitoring of the photolytic release of glycine and β-alanine, as the
1
expected products of the photolysis, was also followed by H NMR in a methanol-d4/D2O (80:20)
solution. This methodology was particularly important for conjugates 6b and 7b, due to difficulties
in the monitoring by HPLC-UV. NMR monitoring was carried out with 2.50 × 10-2 M solutions,
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.
As representative examples, upon irradiation at 350 nm of solutions of N-(tert-butyloxycarbonyl)
conjugates of glycine 4b and β-alanine 5b and of glycine 6b and β-alanine 7b conjugates, a
decrease in the intensity of the signals was observed and this was related to the amino acid in the
conjugates form namely the α-CH2 (δ ≈ 4.00-4.10 ppm, 4b and 6b; δ ≈ 3.30-3.40 ppm, 5b and 7b)
and β-CH2 (δ ≈ 2.70-3.00 ppm, 5b and 7b), as well as the singlet for the benzylic-type CH2 of the
heterocycle (δ ≈ 5.40-5.60 ppm). The N-Boc glycine 2 and β-alanine 3 released from conjugates 4b
and 5b, and free glycine or β-alanine released from conjugates 6b and 7b, gave rise to a new set of
signals with the same multiplicity at lower chemical shift, namely α-CH2 (δ ≈ 3.80-3.70 ppm, 4b
and 6b; δ ≈ 3.30 ppm, 5b and 7b) and β-CH2 (δ ≈ 2.50-2.70 ppm, 5b and 7b) (Figure 4). Based on
the results obtained by 1H NMR, it was possible to corroborate the release of the amino acids from
these conjugates in methanol/HEPES buffer (80:20) solutions at higher concentrations.
< Figure 4 >
4. Conclusion
16

ACCEPTED MANUSCRIPT
The comparative photorelease of glycine and β-alanine, as representative models of carboxylic acid
bioactive molecules, from 4-(methylene)-2H-benzopyran-2-one unsubstituted and substituted with
different substituents at the 7-position of the heterocyclic system, such as amino, phenyl, 4-
methoxyphenyl and styryl groups, under irradiation at variable wavelengths (254, 300, 350 and 419
nm) was carried out. A remarkable behaviour towards irradiation at higher wavelength (350 and
419 nm) occurred in conjugates obtained from 4-(chloromethyl)-7-styryl-2H-benzopyran-2-one,
however the best results were related to 4-(chloromethyl)-7-phenyl-2H-benzopyran-2-one, with
release of glycine and β-alanine in shorter irritation times (ca 8 min at 350 nm; 5 and 85 min, at 419
nm, respectively), which can be considered in practicable applications. The photolysis observations
were also complemented by time-resolved fluorescence, which indicated the presence of different
fluorescing species elucidated by the determination of decay associated spectra.
Acknowledgements
Thanks are due to Fundação para a Ciência e Tecnologia (FCT) and FEDER (European
Fund for Regional Development)-COMPETE-QREN-EU for financial support through the
Chemistry Research Centre of the University of Minho (Ref. UID/QUI/00686/2013 and
UID/ QUI/0686/2016). The NMR spectrometer Bruker Avance III 400 is part of the
National NMR Network and was purchased within the framework of the National Program
for Scientific Re-equipment, contract REDE/1517/RMN/2005 with funds from POCI 2010
(FEDER) and FCT.
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CAPTIONS
Scheme 1. Synthesis of ester cages of glycine and β-alanine 4-7.
Table 1: UV-visible data (λabs and log ε) for compounds 1a-e and conjugates 4-7 in diethyl ether,
dichloromethane, acetonitrile, dimethyl sulfoxide, methanol and methanol/HEPES buffer (80:20).
a in nm.
Table 2. Irradiation times (tirr in min), rate constants (k, ×10-2 min-1) and photochemical quantum
yields (ΦP, × 10-3) for the photolysis of conjugates 4-7 at 254, 300, 350 and 419 nm in
methanol/HEPES buffer (80:20) solution
Figure 1. EEMs for selected compounds in methanol/ HEPES. The relative intensity is shown in a
rainbow colour scale with red most intense, blue (black) least intense. The excitation wavelengths
are plotted on the y-axis, with emission on the x-axis.
Figure 2. Time-resolved fluorescence decays for selected compounds in methanol/ HEPES. The
compounds were excited at 349 nm with the emission monitored at 480 nm.
Figure 3. Decay associated spectra for selected compounds in methanol/ HEPES. The excitation
wavelength was 349 nm.
Figure 4. Partial 1H NMR spectra in methanol-d4/D2O (80:20) of the photolysis at 350 nm of 2.50 ×
10-2 M solutions of N-Boc conjugates of glycine 4b and β-alanine 5b: a) before irradiation; b) after
irradiation for 90 min; and c) after irradiation for 150 min; and glycine conjugate 6b and β-alanine
conjugate 7b: a) before irradiation; b) after irradiation for 150 min; and c) after irradiation for 270
min.
22

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SCHEMES
Scheme 1
23

ACCEPTED MANUSCRIPT
TABLES
Table 1
24

ACCEPTED MANUSCRIPT
Methanol/HEPES
Diethyl ether
Dichloromethane
Acetonitrile
Dimethyl sulfoxide
Methanol
buffer (80:20)
Cpd
a
a
a
a
a
a
λabs
λabs
λabs
λabs
λabs
λabs
log ε
3.50
4.14
4.14
4.12
4.21
3.72
4.24
4.13
4.03
4.09
3.69
4.19
4.23
4.22
4.30
2.76
3.13
2.52
2.72
log ε
3.34
4.19
4.01
4.09
4.13
3.59
4.13
4.02
3.99
4.05
3.53
4.06
4.07
4.20
4.33
2.60
3.36
2.67
3.68
log ε
3.37
4.18
4.05
4.11
4.06
3.67
4.11
4.00
4.00
4.02
3.6
log ε
3.29
4.23
3.97
4.16
4.00
3.66
4.11
4.03
4.06
3.90
3.51
4.17
4.17
4.20
4.17
4.00
3.69
3.95
4.24
log ε
3.43
4.21
4.09
4.23
4.15
3.69
4.20
4.11
4.04
3.97
3.66
4.12
4.11
4.25
4.24
4.01
3.61
3.97
4.27
log ε
3.29
3.98
4.13
4.14
4.18
3.52
4.18
4.08
3.91
3.98
3.54
4.14
4.08
4.11
4.17
4.05
3.83
4.01
4.25
310
346
327
335
349
299
341
326
329
345
305
341
324
336
347
335
333
333
344
314
347
331
342
356
314
343
329
338
354
314
342
328
339
354
335
338
336
353
308
350
324
338
349
310
348
323
332
348
310
348
323
335
348
345
335
347
349
316
365
325
343
358
312
364
326
336
353
310
362
326
338
353
356
342
355
351
310
361
327
339
352
309
353
326
337
348
308
356
326
336
350
356
341
356
352
310
357
329
342
353
305
353
323
335
352
308
356
323
340
354
361
347
361
354
1a
1b
1c
1b
1e
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6b
6e
7b
7e
4.11
4.00
4.22
4.16
3.65
3.61
3.91
4.10
25

ACCEPTED MANUSCRIPT
Table 2
Cpd
254 nm
k
300 nm
350 nm
419 nm
k
tirr
tirr
83
k
tirr
k
tirr
Φphot
Φphot
Φphot
Φphot
a
a
a
a
a
a
77 3.9
0.41
3.6
0.18
-
-
-
-
-
-
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6b
7b
12 23.7 0.63
24
12.5 0.15
8
36.3 0.41
4.9
1610
382
5.3
0.2
0.8
0.8
0.06
0.003
0.02
47 6.7
105 2.8
83 3.6
84 3.6
0.17
0.08
0.14
0.37
128
189
110
80
2.4
1.6
2.7
3.7
0.03 421 0.8 0.005
0.04 268 1.1
0.03
0.13
0.05
0.19
57 4.9
318
0.02
a
a
a
a
a
a
-
-
-
-
-
-
15 19.7 0.54
248 1.3 0.03
16
18.0 0.30
2.2 0.03 421 0.8 0.007
7
42.8 0.60
85
3.4
0.05
137
14945
0.02 0.0002
a
a
a
a
a
a
887 0.3 0.009 3778 0.08 0.001
-
-
-
-
-
-
175 1.8
100 3.2
171 1.8
0.04
0.05
0.03
178
73
1.7
4.2
2.6
0.02 109 2.7
0.03 71 4.3
0.03 123 2.4
0.03
0.04
0.03
1359
0.2
0.003
a
a
a
-
-
-
114
775
0.38 0.004
a No cleavage was detected up to 8h of irradiation.
26

ACCEPTED MANUSCRIPT
FIGURES
Figure 1
400
380
400
400
380
360
340
320
380
1b
4b
5b
5d
5e
360
340
320
360
340
320
300
280
300
280
300
280
300
300
300
400
500
500
500
300
400
500
300
300
300
400
500
EM Wavelength( nm )
EM Wavelength( nm )
EM Wavelength( nm )
400
380
360
340
320
400
400
380
360
340
320
380
360
340
320
1d
1e
4d
4e
300
280
300
280
300
280
400
300
400
500
400
500
EM Wavelength( nm )
EM Wavelength( nm )
EM Wavelength( nm )
400
380
360
340
320
400
380
400
380
360
340
320
360
340
320
300
280
300
280
300
280
400
300
400
500
400
500
EM Wavelength( nm )
EM Wavelength( nm )
EM Wavelength( nm )
27

ACCEPTED MANUSCRIPT
Figure 2
1
0.1
1b
4b
5b
6b
7b
IRF
0.01
1E-3
1E-4
1
0
10
20
30
40
50
time / ns
1d
4d
5d
IRF
0.1
0.01
1E-3
1E-4
0
10
20
30
40
time / ns
1
0.1
1e
4e
5e
0.01
1E-3
1E-4
0
10
20
30
40
time / ns
28

ACCEPTED MANUSCRIPT
Figure 3
10
8
1b
2.57ns
4.98ns
6
4
2
0
400
450
500
550
wavelength / nm
16
14
12
10
8
207ps
1.32ns
5.05ns
5b
336ps
2.31ns
4.1ns
4b
10
8
6
4
2
0
x 10
6
x10
4
x10
2
x10
0
400
450
500
550
400
450
500
550
wavelength / nm
wavelength / nm
14
12
10
8
256ps
1.58ns
5.22ns
6b
7b
1.43ns
4.75ns
15
10
5
x10
x10
6
4
2
0
0
400
450
500
550
400
450
500
550
wavelength / nm
wavelength / nm
29