Sensitized 1‑Acyl-7-nitroindolines with Enhanced Two-Photon Cross Sections for Release of Neurotransmitters

Precise photochemical control, using two-photon excitation
(2PE),
of the timing and location of activation of glutamate is useful for
studying the molecular and cellular physiology of the brain. Antenna-based
light harvesting strategies represent a general method to increase
the sensitivity to 2PE of otherwise insensitive photoremovable protecting
groups (PPGs). This was applied to the most commonly used form of
“caged” glutamate, MNI-Glu. Computational investigation
showed that a four- or six-carbon linker attached between the 4-position
of thioxanthone (THX) and the 4-position of the 5-methyl derivative
of MNI-Glu (MMNI-Glu) would position the antenna and PPG close to
one another to enable Dexter energy transfer. Nine THX-MMNI-Glu conjugates
were prepared and their photochemical properties determined. Installation
of the THX antenna resulted in a red shift of the absorption (λmax = 385–405 nm) along with increased quantum yield
compared to the parent compound MNI-Glu (λmax = 347
nm). The THX-MMNI-Glu conjugate with a four-carbon linker and attachment
to the 4-position of THX underwent photolysis via 1PE at 405 and 430
nm and via 2PE at 770 and 860 nm, yielding glutamate. The two-photon
uncaging action cross section (δu) was 0.11 and 0.29
GM at 770 and 860, respectively, which was greater than for MNI-Glu
(0.06 and 0.072 GM at 720 and 770 nm, respectively). The THX sensitizer
harvested the light via 2PE and transferred its resulting triplet
energy to MMNI-Glu. Release of glutamate through 2PE at 860 nm from
the compound (100 μM) activated iGluSnFR, a genetically encoded,
fluorescent glutamate sensor, on the surface of cells in culture,
portending its usefulness in studies of neurophysiology in acute brain
slice.