Photorelease of glycine and β-alanine from (7-bromocoumarin-4-yl)methyl cages

: The synthesis of a new 7-bromo-4-(chloromethyl)-coumarin to be used as cage for the release of bioactive molecules is presented. Ester cages of two neurotransmitter amino acids, glycine and β-alanine, were also synthetized. These neurotransmitters were chosen as models due to their biological relevance. The glycine and β-alanine ester conjugates, in methanol/HEPES buffer (80:20), were irradiated at 254, 300, 350 and 419 nm in a Rayonet RPR-100 photochemical reactor and the photolysis process was followed by HPLC with UV detection. These results were compared with those previously obtained by our research group when 4-chloromethyl-coumarin was used in the photorelease of glycine and β-alanine.


Introduction
In recent years, remarkable research in photocleavable protecting groups (PPGs) has given rise to a variety of structures suitable for masking a broad range of chemically and biologically relevant molecules.These groups can be easily removed by irradiation at a suitable wavelength, releasing the desired active compound.Notice that for bioapplications the chosen wavelength must be harmless to cells, ideally as close as possible to the visible spectrum. 1[4][5] Among the most interesting PPGs are coumarin (trivial designation for 2H-benzopyran-2-one) derivatives that generally present high molar extinction coefficients at long wavelengths, good stability and fast release rates.7][8] So far, coumarin derivatives have been used as PPGs for phosphates, carboxylates, sulphates, sulfonates, diols and carbonyls. 9th the goal of evaluating the efficiency of 7-bromo-4-(chloromethyl)-coumarin as phototrigger for amino acid neurotransmitters glycine and β-alanine, the corresponding ester conjugates were synthetized.Their irradiation at different wavelengths (254, 300, 350 and 419 nm) in a Rayonet RPR-100 photochemical reactor, in methanol/HEPES buffer (80:20) was carried out.The photolysis process was followed by HPLC with UV detection.
Compounds 1-3 were characterized by the usual techniques, as well as by UV-visible absorption and fluorescence spectroscopies in methanol/HEPES buffer (80:20).For UV-visible absorption studies solutions of 1×10 -4 M were prepared, maximum absorption wavelengths (λabs) and molar extinction coefficient (ε) were obtained for each compound.
Then, taking into account the Lambert-Beer law, solutions of the compounds were diluted to a maximum absorbance of 0.1, and the emission spectra were traced, exciting each compound at its λabs.
The maximum emission wavelength (λem) and the Stokes' shift (∆) were obtained.In addition, each compound's fluorescence relative quantum yield (F) was calculated using 9,10-diphenylanthracene as standard. 10The UV-Vis absorption and fluorescence data for compounds 2 and 3 is presented in Table 1.Conjugates 2 and 3 displayed λabs and λem at about 315 and 390 nm, respectively, which represents a considerably high Stokes' shift (75 nm), with low F values (< 0.1), as it is expected due to the presence of bromine that can act as a fluorescence quencher because of the heavy atom effect (Figure 1). 11th the goal of evaluating the 7-bromo-4-(chloromethyl)coumarin 1 as possible phototrigger for neurotransmitter amino acids, glycine and β-alanine were used as models.Photolysis tests for conjugates 2 and 3 were performed in a Rayonet RPR-100 photochemical reactor at 254, 300, 350 and 419 nm using solutions of 1×10 -4 M in methanol/HEPES buffer (80:20).The disappearance of the conjugates was monitored by HPLC with UV detection at each conjugate's maximum absorption wavelength (λmax) until only 5% of conjugate was present in solution.In Table 1 the photolysis results (irradiation time, tirr, rate constant, k, and photochemical quantum yield, phot) for conjugates 2 and 3 are presented, in comparison with the results previously presented by our research group for unsubstituted coumarin conjugates 4 and 5 (Figure 2). 12It was possible to verify that conjugates 2 and 3 present lower tirr and higher k than conjugates 4 and 5 at all tested wavelengths.More precisely, at 254 and 300 nm, for conjugates 4 and 5, tirr is 3 to 4 times higher than that of conjugates 2 and 3, whereas at 350 nm no consumption of conjugates 4 and 5 was seen after 8 hours of irradiation.The results for conjugates 2, 3, 4 and 5 at 254 nm are graphically represented as a plot of irradiation time (min) versus ln A (A being the area determined in the chromatogram) (Figure 3).
Although photolysis occurred at 350 nm for conjugates 2 and 3, the obtained tirr were too long to be practical for application at this wavelength.Notice that photolysis of conjugates 2 -5 was also tested at 419 nm, however photolysis was not detected for none of the conjugates.

Conclusions
In this work a new 7-bromo-4-(chloromethyl)coumarin 1 cage for model amino acids glycine and βalanine was synthesized.The photolysis of the corresponding N-(tert-butoxycarbonyl) glycine and βalanine ester conjugates 2 and 3 at 254 and 300 nm showed higher rate constants and lower irradiation times comparatively to the related glycine and β-alanine ester conjugates 4 and 5 (obtained from 4chloromethylcoumarin).

Table 1 :
UV/vis absorption and fluorescence data for conjugates 2 and 3.