Two-Photon Uncaging with Fluorescence Reporting: Evaluation of the o-Hydroxycinnamic Platform Nathalie Gagey, † Pierre Neveu, †,‡ Chouaha Benbrahim, † Bernard Goetz, # Isabelle Aujard, † Jean-Bernard Baudin, † and Ludovic Jullien* ,† Contribution from the E Ä cole Normale Supe ´ rieure, De ´ partement de Chimie, UMR CNRS-ENS-UniVersite ´ Paris 6 8640 PASTEUR, 24, rue Lhomond, 75231 Paris Cedex 05, France, E Ä cole Normale Supe ´ rieure, Laboratoire de Physique Statistique, UMR CNRS-ENS-UniVersite ´ Paris 6 and Paris 7 8550, 24, rue Lhomond, 75231 Paris Cedex 05, France, and E Ä cole Normale Supe ´ rieure, De ´ partement de Chimie, UMR CNRS-ENS-UniVersite ´ Paris 6 8642; 24, rue Lhomond, 75231 Paris Cedex 05, France Received March 29, 2007; E-mail: ludovic.jullien@ens.fr Abstract: This paper evaluates the o-hydroxycinnamic platform for designing efficient caging groups with fluorescence reporting upon one- and two-photon excitation. The model cinnamates are easily prepared in one step by coupling commercial or readily available synthons. They exhibit a large one-photon absorption that can be tuned in the near-UV range. Uncaging after one-photon excitation was investigated by 1 H NMR, UV-vis absorption, and steady-state fluorescence emission. In the whole investigated series, the caged substrate is quantitatively released upon photolysis. At the same time, uncaging releases a strongly fluorescent coproduct that can be used as a reporter for quantitative substrate delivery. The quantum yield of double bond photoisomerization leading to uncaging after one-photon absorption mostly lies in the 10% range. Taking advantage of the favorable photophysical properties of the uncaging coproduct, we use a series of techniques based on fluorescence emission to measure the action uncaging cross sections with two-photon excitation of the present cinnamates. Exhibiting values in the 1-10 GM range at 750 nm, they satisfactorily compare with the most efficient caging groups reported to date. Noticeably, the uncaging behavior with two-photon excitation is retained in vivo as suggested by the results observed in living zebrafish embryos. Reliable structure property relationships were extracted from analysis of the present collected data. In particular, the careful kinetic analysis allows us to discuss the relevance of the o-hydroxycinnamic platform for diverse caging applications with one- and two-photon excitation. Introduction The spatio-temporal delivery pattern of precise given amounts of chemicals is of major importance in chemistry and biology. Syringes have been used to achieve this goal at the microscopic level. However such microinjections, by their invasive nature, are not appropriate if the targeted organism has to remain intact. Given the excellent intrinsic localization in two-photon excita- tion, 1 photoactivation has been envisioned to provide an attractive noninvasive alternative: the “optical microsyringe’’. 2-5 In the corresponding approach, a focused light pulse is used to release within an organism perfused with a solution of a biologically nonactive precursor the desired chemical after two- photon photoactivation (or uncaging). 6 A large enough two-photon action uncaging cross section is a key factor to satisfactorily implement the preceding two- photon photoactivation strategy in sensitive samples. In fact, the cross section should be as large as possible to reduce the illumination duration and power needed to release a given amount of effector without generating detrimental effects. For instance, it was claimed to have to exceed 0.1 or 10 Goeppert- Mayer (GM; 1 GM ) 10 -50 cm 4 s/photon) for biological applications. 3,7 In fact, the 1 GM range has already been reached, 3,8,9 and the corresponding caging groups have been successfully used in biological systems. Nevertheless the improvement of the efficiency of a photolabile protecting group upon two-photon excitation remains difficult, and it is therefore useful to gain reliable structure property relationships. Indeed we still lack general rules to predict the cross section for two- † UMR CNRS-ENS-Universite ´ Paris 6 8640 PASTEUR. ‡ UMR CNRS-ENS-Universite ´ Paris 6 and Paris 7 8550. # UMR CNRS-ENS-Universite ´ Paris 6 8642. (1) Xu, C.; Zipfel, W.; Shear, J. B.; Williams, R. M.; Webb, W. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10763-10768. (2) Cambridge, S. B.; Davis, R. L.; Minden, J. S. Science 1997, 277, 825- 828. (3) Furuta, T.; Wang, S. S. H.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E. M.; Denk, W.; Tsien, R. Y. Proc. Nat. Acad. Sci. U.S.A., 1999, 96, 1193-1200. (4) Ando, H.; Furuta, T.; Tsien, R. Y.; Okamoto, H. Nat. Genet. 2001, 28, 317-325. (5) Momotake, A.; Lindegger, N.; Niggli, E.; Barsotti, R. J.; Ellis-Davies, G. C. R. Nat. Methods 2006, 3, 35-40. (6) Mayer, G.; Heckel, A. Angew. Chem., Int. Ed., 2006, 45, 4900-4921. (7) Kiskin, N.; Chillingworth, R.; McCray, J. A.; Piston, D.; Ogden, D. Eur. Biophys. J. 2002, 30, 588-604. (8) Lu, M.; Fedoryak, O. D.; Moister, B. R.; Dore, T. M. Org. Lett. 2003, 5, 2119-2122. (9) Gagey, N.; Neveu, P.; Jullien, L. Angew. Chem., Intl. Ed. 2007, 46, 2467- 2469. Published on Web 07/21/2007 9986 9 J. AM. CHEM. SOC. 2007, 129, 9986-9998 10.1021/ja0722022 CCC: $37.00 © 2007 American Chemical Society