Aminobenzocoumarinylmethyl esters as photoactive precursors for the release of butyric acidby Ana M. S. Soares, Graham Hungerford, Susana P. G. Costa, M. Sameiro T. Gonçalves

New J. Chem.


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This journal is?The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem.

Cite this:DOI: 10.1039/c5nj00699f

Aminobenzocoumarinylmethyl esters as photoactive precursors for the release of butyric acid?

Ana M. S. Soares,a Graham Hungerford,b Susana P. G. Costaa and

M. Sameiro T. Gon?alves*a

The evaluation of the photorelease of a carboxylic acid drug, using butyric acid as a representative model, was carried out by using 7-amino-4-chloromethyl-2-oxo-2H-naphtho[1,2-b]pyran, an aminobenzocoumarin, and its mono- and di-methylated or ethylated derivatives. This study was intended to improve the release of butyric acid from benzocoumarins by the addition of an amino group to the heterocycle by applying the knowledge of second-generation coumarinylmethyl-based photoremovable protecting groups. Photolysis studies were performed on the resultant 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 showed that these new fluorescent aminobenzocoumarins are superior to all the previously tested benzocoumarins with the same or different ring fusions. The photophysics of the compounds was characterised by both steady state and time-resolved methods.


The use of light of an appropriate wavelength can liberate, in a spatially and temporally controlled release, synthetic or biologically relevant molecules from their light-sensitive conjugated inactive precursors, covalently bonded to the functional group.

In recent years, photosensitive groups have been widely applied in the release of phosphates,1 thiols,2 amines,3 alcohols,3,4 and carboxylic acids.4 The use of fluorescent photolabile protecting groups enables the visualization, quantification, and the follow-up of spatial distribution, localization, and depletion of the active compound through the monitoring of the fluorescent caged precursor using fluorescence techniques.5?10 The broad range of applications drives the development of new photolabile groups with enhanced properties to be suitable for biological purposes. These properties include larger molar extinction coefficients with higher photolysis efficiency above 350 nm, to allow fast cleavage and release of the corresponding functionality and the use of longer wavelengths to minimise side reactions or cell damage.8?10 Nevertheless, among the considerable number of light-sensitive groups that have been reported, only a few cleave in a practical time by irradiation at wavelengths longer than 400 nm.11 (Coumarin-4-yl)methyl derivatives (trivial name ofmethyl-2-oxo-2H-benzo[1,2-b]pyran), introduced by Givens and

Matuszewski in the release of phosphate esters in 1984,9 display attractive properties, namely large molar absorption coefficients (e) at longer wavelengths, extending into the visible region (400? 500 nm) and fast release rates from their excited singlet state.

These features have motivated interest in their usage, especially for bioapplications11,12 and offset their low photochemical quantum yields, reduced aqueous solubility and the inconvenience of strong fluorescence and competitive absorption from the coumarin photolysis byproducts.

Structural modifications performed by several researchers, particularly concerning the substituents at C-6 and C-7, revealed that the first generation of coumarylmethyl-based phototriggers possessing hydroxyl or methoxy substituents at C-7 displayed high release rates and hydrolytic stability. The introduction of an amino group at C-7 can be considered as a second-generation of coumarinylmethyl-based protecting groups, resulting in an improvement of the photophysical and photochemical properties of the cage, moving the absorption maxima to 350?400 nm and exhibiting the highest photochemical quantum yields among the analogues. The extension of the aromatic ring of coumarins, resulting in polyaromatic analogues, has also led to the enhancement of the fluorophoric properties.13?19 Benzocoumarin chromophores were introduced for the first time by our research group in 2006: 1-chloromethyl-3-oxo-3H-naphto[2,1-b]pyran was used in the caging of N-benzyloxycarbonyl and p-toluenesulfonyl phenylalanine, as model carboxylic compounds.13 a Centro de Qu??mica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga,

Portugal. E-mail: b HORIBA Jobin Yvon IBH Ltd, 133 Finnieston Street, Glasgow G3 8HB, UK ? Electronic supplementary information (ESI) available: 1H and 13C NMR spectra of compounds 1, 2, 3a,b and 4a,b; absorption spectra of compounds 2, 3a,b and 4a,b, EEM and decay associated spectra of compound 2 in MeOH/HEPES (80 : 20) and the difference between frontier orbitals for compounds 3a,b and 4a,b; UV/Vis absorption and fluorescence spectra of compounds 1, 2, 3a,b and 4a,b in ethanol; and HPLC chromatograms for the photolysis of compounds 2, 3a,b and 4a,b. See

DOI: 10.1039/c5nj00699f

Received (in Montpellier, France) 24th March 2015,

Accepted 1st July 2015

DOI: 10.1039/c5nj00699f



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New J. Chem. This journal is?The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015

Later, we reported a comparative study using various fluorophores of aromatic, namely naphthalene and pyrene, and heteroaromatic nature, such as oxobenzopyran derivatives, undertaken to evaluate their performance as photolabile groups in the release of N-benzyloxycarbonyl-g-aminobutyric acid.14 Relying on the preliminary encouraging results obtained, we applied the 3-oxo-3H-naphtho[2,1-b]pyranylmethyl group in the caging of the carboxylic acid of other neurotransmitter amino acids under the same conditions as previously mentioned,15 and their two-photon absorption properties were studied for the first time.16

A new benzocoumarin-4-yl-methyl moiety, (4-(chloromethyl)6-methoxy-2-oxo-2H-naphtho[1,2-b]pyran or 4-(hydroxymethyl)-6methoxy-2-oxo-2H-naphtho[1,2-b]pyran), with a different benzene ring fusion was designed and evaluated as a phototrigger using the same class of compounds (neurotransmitter amino acids) as targets, with the caging through ester or carbamate linkages to the carboxylic acid or amine functions, respectively.18 Furthermore, the cages obtained from this 2-oxo-2H-naphtho[1,2-b]pyran showed absorption in ethanol at about 470 nm, whereas 3-oxo-3H-naphtho[2,1-b]pyranylmethyl absorbed in the range of 446?449 nm. This resulted in a considerable enhancement in relation to our previously fluorescent fused heterocycles studied as photoremovable protecting groups. The convenience of this family of benzocoumarins as a photolabile protecting group, combined with the improved photophysical and photochemical properties reported in coumarins by the introduction of an amino group at C-7, has directed this work to evaluate the performance of aminobenzocoumarin derivatives in the release of butyric acid. This short-chain fatty acid is related to the disruption of cell proliferation and induction of apoptosis, modification of cell morphology and alteration of gene expression. However, the presence of a carboxylic acid group can result in poor absorption from the gastrointestinal tract because of lipophilicity/solubility issues.20?22 The use of aminobenzocoumarins also represents an effort towards the improvement of the results previously reported by the authors concerning the photorelease of butyric acid from prodrugs based on fluorescent benzocoumarin derivatives with or without a similar ring fusion.23