in acetic anhydride to give 4 (19% yield ), which was deprotected
by TFA treatment to give MDNI-glu.{
The quantum yield for uncaging of MNI-glu in water at pH 7 is
3
0
.085. HPLC analysis of a photolyzed solution of MNI-glu and
MDNI-glu (mixed in a 2 : 1 ratio, ensuring equal absorptivities)
revealed that the latter is about 5.5 times more photosensitive than
the former, implying that the additional nitro group at the
5-position of MDNI-glu has a dramatic effect on the decay of the
key intermediate 5 (Fig. 2, this reaction mechanism is well
4
established). The presence of an electron-withdrawing group at
the 5-position promotes deprotonation at C–2, preventing
reversion of 5 to 1, enhancing the quantum yield of N–O bond
4
scission to give the desired glutamate.
We have previously determined that the 2-photon cross section
for uncaging of MNI-glu in physiological buffer is 0.06 GM (GM
250
is 10
4 21 2
cm s photon ). We have tested the effect of the 5-nitro
Fig. 3 Absorption spectrum of MDNI-glu (orange), and time-course of
conversion to photoproduct 6.
group on this physico-chemical property. We utilized the direct
output from a mode-locked Ti:sapphire laser (10 W Verdi-Mira,
Coherent) to perform 2-photon uncaging. The beam from the
Mira laser has a diameter of approximately 0.8 mm and an energy
of 900 mW at 730 nm. Irradiation of an equimolar solution (pH 7.4
and 300 mOsm) of MNI-glu and MDNI-glu for 3–4 hours in a
cuvette (total volume of 20 mL) revealed that the 2-photon cross
section for uncaging of MDNI-glu is about 0.06 GM. We could
detect no hydrolysis of either caged glutamate by HPLC during
this period.
astrocyte cell body (see Fig. 4: cells were loaded with fluo-4 AM,
and imaged using laser-scanning confocal microscopy. The period
for each image frame was 1.4 s, and the frames were at 9 s
intervals. Uncaging (at the red dot in frame 1) was immediately
before the 2nd frame. The scale bar, lower right, is 30 mm and the
images are 8 bit depth).
In this report we introduce a new photochemical protecting
group for carboxylates (MDNI) and have used this moiety to cage
L-glutamate. MDNI-glu (1) has a superior combination of
physico-chemical properties when compared to all other previous
synthesized caged glutamates (Table 1). Photorelease of glutamate
The absorption spectrum of MDNI-glu is shown in Fig. 3
(orange line). The caged compound undergoes smooth photolysis
to the indole photoproduct (6), as shown by the clean isobestic
point. The identity of 6 was confirmed by NMR and MS.
The properties of several caged glutamates are summarized in
Table 1. It can be seen from these data that MDNI-glu makes
more efficient use of incident light (i.e. the product w.e) than all
other caged glutamates.
2+
i
from MDNI-glu evoked rapid increases in [Ca ] in living brain
slices, demonstrating the effectiveness of the caged compound for
neurobiological experiments. We believe that photolysis of MDNI-
caged neurotransmitters will permit much improved 4-dimensional
mapping of excitatory neuroreceptors on living cells (both in brain
When a solution of MDNI-glu (200 mM) was applied to acutely
isolated brain slices from mouse hippocampus, no stimulation of
glutamate receptors could be detected by confocal imaging of
9
slices and in vivo) with 2-photon uncaging at energies much lower
2,6,9,10
than were previously required.
MNI-glu requires 5–10 mW
2
2
+
2+
of 720 nm light to mimic quantal release. MDNI-glu will require
approximately 2.5-fold less energy in the image plane to produce
similar quantities of neurotransmitter. Since 2P-induced photo-
intracellular Ca concentration ([Ca ]
solution with a focused laser (continuous wave Ar, 30 mW at
51–364 nm, 3 mm diameter, for 30 ms) evoked rapid increase in
i
). Irradiation of this
3
1
1
2+
damage is known to be highly non-linear, use of this new
photosensitive neurotransmitter for 2-photon uncaging in brain
slices and in vivo should provide access to a whole panoply of long-
term experiments.
i
[Ca ] via activation of metabotropic glutamate receptors on the
This work was supported by grants from the NIH (to GED and
PGH), NSF, McKnight Endowment Fund for Neuroscience and
the Human Frontiers Science Organisation (to GED).
Table 1 Summary of the properties of caged glutamates
Extinction
coefficient
2-photon
cross section
(730 nm)/ GM
Quantum
yield (w)
(e)(350 nm)/
cm
2
M
1
21
Chromophore
w.e
MDNI
MNI
NI
0.47
0.06
0.06
No datum
0.95
,0.001
No datum
8600
4300
2700
17300
500
4042
366
116
329
75
0.085
0.043
0.019
0.15
5
6
Bhc
CNB
pHP
7
8
0.08
200
16
Fig. 2 Photorelease of glutamate from MDNI-glu.
This journal is ß The Royal Society of Chemistry 2005
Chem. Commun., 2005, 3664–3666 | 3665