pH Effect on the Photochemistry of 4-Methylcoumarin Phosphate Esters: Caged-Phosphate Case Study Andre ´ Vidal Pinheiro, †,‡ A. Jorge Parola, † Pedro V. Baptista, ‡ and J. C. Lima* ,† REQUIMTE, Departamento de Quı ´mica, Faculdade de Cie ˆncias e Tecnologia, UniVersidade NoVa de Lisboa, 2829-516 Caparica, Portugal and CIGMH, Departamento de Cie ˆncias da Vida, Faculdade de Cie ˆncias e Tecnologia, UniVersidade NoVa de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal ReceiVed: April 5, 2010; ReVised Manuscript ReceiVed: October 23, 2010 There are numerous reports of coumarin ester derivatives, in particular phosphate esters, as photocleavable cages in biological systems. Despite the comprehensive analysis of the photocleavage mechanism, studies of 4-methylcoumarin caged phosphates and/or nucleotides were always performed at constant pH. In this work, we present the study of the pH effect on the photochemistry of (7-diethylaminocoumarin-4-yl)methyl phosphate (DEACM-P). Fluorescence and photocleavage quantum yields, as well as the fluorescence decay times were measured as a function of the pH. It was found that the pH produces significant changes in the overall photochemical quantum yield of DEACM-P, and the observed changes are complementary to those obtained from the fluorescence quantum yield. Deprotonation of DEACM-HPO 4 - to yield DEACM-PO 4 2- , produces a decrease in the photochemical quantum yield (from 0.0045 to 0.0003) and an increase in the fluorescence quantum yield (from 0.072 to 0.092). Moreover, from the analysis of the decay times, we have also found that hydroxyl ion is not only relevant, but it is mechanistically involved in the photoreaction of DEACM-HPO 4 - . Introduction Controlled temporal and spatial release of biomolecules from photolabile precursors, commonly known as caged molecules, has become increasingly valuable for cell and molecular biology studies. The rationale of using caged compounds is straight- forward: the molecule of interest is rendered biologically inactive (caged) by chemical modification with a protecting group that can be removed with light by irradiation with a suitable wavelength. 1,2 This promotes the release of the biologically active molecule, generating a time-controlled burst in concentra- tion with tight spatial control. 3 Among the several caging groups reported in the literature, 4,5 coumarin derivatives present a great potential for biological application due to their absorption bands with high extinction coefficients in the visible region of the spectrum, which minimizes the risk of damaging nucleic acids or proteins upon irradiation. In addition, a careful choice of the coumarinic core substitution pattern allows one to fine-tune the absorption and emission maxima in the visible region. 6 There are numerous reports of coumarin derivatives used in biological systems, such as caged peptides, 7 caged phosphates for acidi- fication of membranes, 8 caged glutamate, 9 gene activation using caged RNA, 10 and caged cyclic nucleotides release for activation of cyclic nucleotide-gated (CNG) channels. 11,12 The first interpretation of the photocleavage mechanism of 4-methylcoumarin esters was suggested by Furuta, 9 where he considered to occur from the triplet state, through a homolytic bond cleavage. Bendig and co-workers 6 excluded the hypothesis of triplet origin due to the absence of phosphorescence at 77 K. The homolytic cleavage was also excluded since no products were detected resulting from hydrogen abstraction by radicals. It is now generally accepted that the photocleavage of coumarin phosphate esters involves photoheterolysis of the C-O ester bond and subsequent ion-pair separation in a polar solvent, followed by trapping of the coumarinylmethyl carbocation by water. 6 Further studies have shown that the coumarin alcohol photoproduct is, in some cases, formed in the subnanosecond time-range, which is the fastest photodeprotection rate reported so far. 13 In 2007, in a broader study regarding the characterization of coumarin cages (Cm) with different leaving groups (A), it was proposed that, upon coumarin excitation to S1 excited state, a significant weakening of the Cm-A ester leads to the formation of 1 [Cm + A - ] ion pair. 14 Stabilization of the carbocation Cm + is favored by strong electrodonating substituents in the coumarin moiety, while stabilization of the leaving anions A - is better accomplished if they present low basicity. Transient ion pair stabilization results in the increase of the observed rate of product formation and also in photochemistry efficiency, since recombination of the ion pair is disfavored. Despite the comprehensive analysis of the photocleavage mechanism, the photochemical studies of 4-methylcoumarin caged phosphates and nucleotides were always performed at constant pH, inde- pendently on the number of prototropic species in equilibrium. Among the species present in solution, it is important to clarify which ones are responsible for the photochemistry and evaluate the impact of pH in the photorelease quantum yield, mainly since the microenvironments inside cells might be difficult to predict in the case of in vivo applications. Experimental Methods General Information. All chemicals were purchased from Sigma Aldrich in the highest purity available and used without * To whom correspondence should be addressed. Tel: +351 212 948 575. Fax: +351 212 948 550. E-mail: lima@dq.fct.unl.pt. † REQUIMTE, Departamento de Quı ´mica, Faculdade de Cie ˆncias e Tecnologia, Universidade Nova de Lisboa. ‡ CIGMH, Departamento de Cie ˆncias da Vida, Faculdade de Cie ˆncias e Tecnologia, Universidade Nova de Lisboa. J. Phys. Chem. A 2010, 114, 12795–12803 12795 10.1021/jp103045u  2010 American Chemical Society Published on Web 11/19/2010