selective approach utilizes IR light and multiphoton excitation
(MPE),5 which confines the messenger activation to the focus
of the laser beam, ∼1 (µm)3. As an added advantage,
chromophores with sensitivity to MPE tend to be highly
sensitive to single-photon excitation, decreasing irradiation
times and enabling the use of low-intensity light sources in
conventional applications of caged compounds.
Photolabile protecting groups that possess sufficiently large
two-photon absorbance cross-sections, δu, for biological
applications exist.6 For example, 6-bromo-7-hydroxycou-
marin-4-ylmethyl (1, Bhc, Figure 1) has been utilized as a
in many biological effectors, especially drugs. Synthetically
useful photoremovable protecting groups for carbonyls such
as N,N-dimethylhydrazones13 require the generation of singlet
oxygen, while others require a triplet sensitizer, as in the
case of dithioacetals.14 Both methods would be incompatible
with biological systems. o-Nitrophenylethylene glycol de-
rivatives 415 and 616 release carbonyl compounds upon
exposure to 350 nm light in organic solvents (Scheme 1).
Scheme 1. o-Nitrophenylethylene Glycols as Photoremovable
Protecting Groups for Aldehydes and Ketones
Similarly, polymer-supported o-nitrophenylethylene glycols17
offer photoremovable protection to aldehydes, releasing them
after exposure to a visible-light mercury lamp for 7 h. These
protecting groups require significant synthetic adaptation for
physiological use, and they would still suffer from very poor
sensitivity to MPE.
As part of our program to develop applications for two-
photon-sensitive photoremovable protecting groups, we
required a caging agent capable of releasing ketones inside
living cells, tissues, and animals. We rationalized that acetals
and ketals of 6-bromo-4-(1,2-dihydroxyethyl)-7-hydroxy-
coumarin (Bhc-diol-acetal/ketal, 7) would be capable of
liberating aldehydes and ketones upon single- or two-photon
photolysis under simulated physiological conditions18 (Scheme
2). Our success in this endeavor is somewhat surprising
because saturated alcohols cannot be released directly from
Bhc; X must be a good leaving group (Figure 1).9,19
Apparently, trapping the zwitterion 8, a possible intermediate
suggested by Bendig et al.,20 with solvent (H2O or -OH)
followed by dissociation to Bhc-diol (9) and the carbonyl
Figure 1. Two-Photon Releasing Groups with Biological Utility.
multiphoton-sensitive caging agent for neurotransmitters,7
DNA and RNA,8 diols,9 and an inhibitor of nitric oxide
synthase.10 Recently, we reported the synthesis, photochem-
istry, and potential use of 8-bromo-7-hydroxyquinoline (2,
BHQ) as a caging group for biological effectors possessing
carboxylate groups.11 MNI-glutamate (3) has been shown
to release glutamate upon two-photon excitation in sufficient
quantities to be useful for investigating the function of
glutamate receptors.12
There exists a lack of physiologically useful caging groups
for ketones and aldehydes, functional groups that are found
(5) Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73-76.
Xu, C.; Webb, W. W. In Topics in Fluorescence Spectroscopy: Nonlinear
and Two-Photon-Induced Fluorescence; Lakowicz, J., Ed.; Plenum Press:
New York, 1997; Vol. 5, pp 471-540. Denk, W.; Piston, D. W.; Webb,
W. W. In Handbook of Biological Confocal Microscopy; Pawley, J. B.,
Ed.; Plenum Press: New York, 1995; pp 445-458. Williams, R. M.; Piston,
D. W.; Webb, W. W. FASEB J. 1994, 8, 804-813. Xu, C.; Zipfel, W.;
Shear, J. B.; Williams, R. M.; Webb, W. W. Proc. Natl. Acad. Sci. U.S.A.
1996, 93, 10763-10768.
(6) δu is a measure of the sensitivity of a chromophore to two-photon
photolysis. To be biologically useful, it should exceed 0.1 GM (Goeppert-
Mayer, 10-50 cm4 s photon-1). See ref 7.
(7) Furuta, T.; Wang, S. S.-H.; Dantzker, J. L.; Dore, T. M.; Bybee, W.
J.; Callaway, E. M.; Denk, W.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 1193-2000.
(8) Ando, H.; Furuta, T.; Tsien, R. Y.; Okamoto, H. Nat. Genet. 2001,
28, 317-325.
(13) Friedrich, E.; Lutz, W.; Eichenauer, H.; Enders, D. Synthesis 1977,
893-894.
(14) Takahashi, T. T.; Nakamura, C. Y.; Satoh, J. Y. J. Chem. Soc., Chem.
Commun. 1977, 680. McHale, W. A.; Kutateladze, A. G. J. Org. Chem.
1998, 63, 9924-9931.
(15) He´bert, J.; Gravel, D. Can. J. Chem. 1974, 52, 187-189. Gravel,
D.; Hebert, J.; Thoraval, D. Can. J. Chem. 1983, 61, 400-410.
(16) Blanc, A.; Bochet, C. G. J. Org. Chem. 2003, 68, 1138-1141.
(17) Aurell, M. J.; Boix, C.; Ceita, M. L.; Llopis, C.; Tortajada, A.;
Mestres, R. J. Chem. Res., Synop. 1995, 452-453.
(18) KMOPS buffer: 100 mM KCl and 10 mM MOPS titrated to pH
7.2 with KOH.
(19) Schade, B.; Hagen, V.; Schmidt, R.; Herbich, R.; Krause, E.;
Eckardt, T.; Bendig, J. J. Org. Chem. 1999, 64, 9109-9117.
(20) On the basis of studies of (7-methoxycoumarin-4-yl)methyl-caged
phosphates, carboxylates, and sulfonates, Bendig et al. proposed a photo
SN1 reaction (solvent-assisted photoheterolysis) mechanism. See ref 19.
(9) Lin, W.; Lawrence, D. S. J. Org. Chem. 2002, 67, 2723-2726.
(10) Montgomery, H. J.; Perdicakis, B.; Fishlock, D.; Lajoie, G. A.;
Jervis, E.; Guillemette, J. G. Bioorg. Med. Chem. 2002, 10, 1919-1927.
(11) Fedoryak, O. D.; Dore, T. M. Org. Lett. 2002, 4, 3419-3422.
(12) Matsuzaki, M.; Ellis-Davies, G. C. R.; Nemoto, T.; Miyashita, Y.;
Iino, Y.; Kasai, H. Nature Neurosci. 2001, 4, 1086-1092.
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