A novel photoresponsive benzoquinolone: evaluation of the release of model amino acids

A photoresponsive benzoquinolone was conjugated with two model amino acids at their C-terminus, affording the corresponding ester conjugates. These ester conjugates were submitted to photocleavage studies in different mixtures of methanol/HEPES buffer solution at different wavelengths of irradiation (300, 350 and 419 nm), which confirmed the quantitative release of the model molecules in short irradiation times.


Introduction
Photoresponsive moieties have proven useful in biochemical and biophysical studies with biomolecules that require spatial and temporal resolution of light-controlled processes. In order to develop more efficient photoactive units, with fast and clean cleavage, it is desirable that their absorption is shifted towards longer wavelengths (> 400 nm), which can be accomplished by substituent tailoring at the photoactive unit or by careful choice of the solvent for the photolytic process. Shifting absorption to higher wavelength also minimises side reactions due to competing absorption of radiation by other chromophores in the molecule under study. 1 Quinolone derivatives were recently reported for the first time by us for the protection of carboxylic acids, in the form of ester conjugates, by using an amino acid as model biomolecule. 2 This nitrogen heterocycle, structurally related to coumarin, a well-known photocleavable protecting group [3][4][5] assured fast cleavage of the ester bond between the amino acid and the heterocycle. Considering our current research interests which includes the design of new oxygen and nitrogen heterocycles, as well as polycyclic aromatics, and their application as photocleavable protecting groups for the amino and carboxylic functions, 2,6-10 we now report our efforts to further optimize the photolytic process at longer wavelengths by using a polycyclic nitrogen heterocycle in an attempt to accomplish larger shifts of the maximum wavelength of absorption in the UV/Vis, that can influence the outcome of the photocleavage reaction. In this work, a novel benzoquinolone was used as photolabile protecting group of two model amino acids, alanine and glutamic acid.
The stability to irradiation of the ester bond between model amino acids and the novel benzoquinolone was evaluated in a photochemical reactor at 300, 350 and 419 nm using different mixtures of methanol/HEPES buffer solutions. The photolysis at different wavelengths was monitored by HPLC-UV detection and kinetic data was obtained.

one-1-yl) methyl ester, 3a
Compound 2 (0.025 g, 0.09 mmol) was dissolved in dry N,N-dimethylformamide (3 mL). Nbenzyloxycarbonyl-L-alanine 1a (1 equiv, 0.020 g, 0.09 mmol) and potassium fluoride (3 equiv, 0.016 g, 0.27 mmol) were added to the mixture. The reaction mixture was stirred at room temperature for 2 days. The mixture was filtered and the solvent was removed by rotary evaporation. The obtained crude solid was purified by silica gel column chromatography using dichloromethane/methanol

General photolysis procedure
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). Considering the results in Table 2, it was found that higher water content had a positive influence in the cleavage rates, resulting in shorter irradiation times. Such behaviour might be explained by the nature of the photocleavage mechanism that could resemble that of the coumarin system, involving an ionic pair formed by homolytical (followed by electron transfer) or heterolytical cleavage of the ester O-CH2 bond. 13 Fast cleavage was possible by irradiation at 300 and 350 nm whereas at 419 nm the irradiation times were too long to be useful for practical applications.

Conclusions
The synthesis of ester conjugates using a novel nitrogen heterocycle, a benzoquinolone derivative, was described. This novel benzoquinolone proved to be useful for application as photocleavable protecting group, with very short irradiation times at 300 and 350 min (between 7 and 15 min) being necessary for the complete release of the model amino acids. The best results obtained show that the amount of water in the solvent mixture influences the photolysis rates, cleaving faster in higher water content.