Fluorescent neuroactive probes based on stilbazolium dyes

Article abstract of DOI:10.1039/c0ob00849d

A set of spectrally diverse stilbazolium dyes was identified in an uptake assay using cultured brainstem and cerebellum cells isolated from e19 chicks. Pretreatment of cells with indatraline, a monoamine reuptake inhibitor, allowed identification of dyes

Full text of DOI:10.1039/c0ob00849d

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Organic &
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Chemistry
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PAPER
Fluorescent neuroactive probes based on stilbazolium dyes†
Adrienne S. Brown, Lisa-Marie Bernal, Teresa L. Micotto, Erika L. Smith‡ and James N. Wilson*
Received 7th October 2010, Accepted 13th December 2010
DOI: 10.1039/c0ob00849d
A set of spectrally diverse stilbazolium dyes was identified in an uptake assay using cultured brainstem
and cerebellum cells isolated from e19 chicks. Pretreatment of cells with indatraline, a monoamine
reuptake inhibitor, allowed identification of dyes that may interact with monoamine transporters. Two
structurally related, yet spectrally segregated, probes, (E)-1-methyl-4-[2-(2-naphthalenyl)ethenyl]-
pyridinium iodide (NEP+, 3A) and (E)-4-[2-(6-hydroxy-2-naphthalenyl)ethenyl]-1-methyl-pyridinium
iodide (HNEP+, 4A), were selected and further investigated using HEK-293 cells selectively expressing
dopamine, norepinephrine or serotonin transporters. HNEP+ was selectively accumulated via
catecholamine transporters, with the norepinephrine transporter (NET) giving the highest response;
NEP+ was not transported, though possible binding was observed. The alternate modes of interaction
enable the use of NEP+ and HNEP+ to image distinct cell populations in live brain tissue explants. The
preference for HNEP+ accumulation via NET was confirmed by imaging uptake in the absence and
presence of desipramine, a norepinephrine reuptake inhibitor.
Introduction
recently demonstrated a coumarin-based probe as substrates of
vesicular MAT. The cationic dye, 4-[4-(dimethyl amino)styryl]-N-
methylpyridinium iodide (ASP+, Fig. 1), a fluorescent analog of
the neurotoxin MPP+, serves as substrate for multiple transporter
types. ASP+ was utilized as a probe of OCTs by Pietruck and
Monoamine transporters (MATs) play critical regulatory roles
removing neurotransmitters from synaptic clefts and extracellular
1,2
milieu. The serotonin transporter (SERT), dopamine trans-
porter (DAT) and norephinephrine transporter (NET) are the
targets of antidepressants and related pharmacotherapies. The
side effects of many psychoactive drugs underscore the multitude
of roles these neurotransmitters fulfill as well as the complexity
11
coworkers, while DeFelice showed that ASP+ is transported via
16,17
DAT, NET, and to a lesser extent SERT.
The stilbazolium
core of ASP+ represents a structurally broad class of fluorophores
20–26
with tunable optical properties.
We anticipated that varying the
3–6
of their expression and regulation. The development of new
classes of drugs remains a high priority with dual- and triple-
acting inhibitors receive increasing interest. Concurrent with the
search for new pharmacotherapies is a drive for improving tools
for screening and imaging, particularly with respect to in vivo or ex
identity of the electron donating vinylarene or electron accepting
aryl cation components would impart different modes of cellular
uptake while simultaneously introducing spectral diversity. For
example, Rosania et al. demonstrated that members of a library of
styryl-pyridinium dyes exhibited selectivity for certain subcellular
7
,8
9
–11
vivo assessment of transporter dynamics.
Fluorescence-based,
20
compartments. Probes with selectivity for a particular MAT
functional assays in which probes accumulate intracellularly are
of special interest as they avoid the use of radioactive materials.
Furthermore, they can identify inhibitors with novel binding
would be useful for monitoring the activity or level of expression
of a specific transporter. Ideally, a ‘tool kit’ of probes could
be employed to target specific cell types (i.e. dopaminergic vs.
12
modes that may be overlooked in competitive binding assays.
Fluorescent substrates for MATs and organic cation trans-
1
1,13–19
porters (OCTs) have been described by several groups.
13
Hadrich reported fluorescent substrates targeting neuroblas-
tomas based on guanidine, cocaine and nisoxetine while Sames
19
Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral
Gables, FL, 33124, USA. E-mail: jnwilson@miami.edu; Fax: +1 305 284
4
571; Tel: +1 305 284 2619
Electronic supplementary information (ESI) available: H NMR,
C NMR spectra), 3D reconstructions (as .mov files). See DOI:
1
†
13
1
‡
0
0.1039/c0ob00849d
Present address: Princeton University, 2457 Frist Center, Princeton, NJ,
8544, USA.
Fig. 1 The structure three stilbazolium dyes: ASP+, a known MAT and
OCT substrate, NEP+ (3A) and HNEP+ (4A), described herein.
2
142 | Org. Biomol. Chem., 2011, 9, 2142–2148
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serotonergic, astrocytes vs. neurons). Herein we describe our
efforts to identify additional neuroactive probes from a family
of stilbazolium dyes utilizing a cellular uptake assay, chemical
inhibitors of MATs as well as directly imaging uptake of the
fluorescent reporters in cell culture and live embryonic chick brain
explants.
Results and discussion
Probe synthesis
Fig. 2 Normalized absorption (left) and emission spectra (right) of
selected library members (10 mM MeOH solutions). Absorption maxima
vary from the near UV to blue; all dyes are compatible with 405 nm laser
exciation. Emission ranges across the entire visible spectrum with large
N-methyl-picolinium iodide or 1,4-dimethylquinolium iodide
27,28
(
A–D) were exposed to aryl aldehydes (1–8) under Knoevenagel
conditions (Scheme 1) for a total of 32 reaction combinations
(
i.e. 1A–8D); several members of this library have previously
(
150+ nm) Stokes shifts for several dyes.
20–26
been described.
The cationic pyridinium or quinolium func-
tionality is likely to mimic the protonated ethylamine of the
native substrates. The arylaldehydes serve two roles: first, they
impart chemical diversity that may lead to preferential uptake by
specific transporters or cell types. Second, the aryl functionalities
selected serve as electron rich substituents that may modulate the
absorption and emission wavelengths. For screening purposes, the
reaction isolates were utilized without further purification as the
expected products were achieved with >90% purity in many cases
by simply decanting the supernatant liquid; reactions involving
component B resulted in low yields of the desired products.
Compounds exhibiting high activity in cellular uptake assays, i.e.
The dyes possess large Stokes shifts of 120 to 165 nm. Compounds
containing the quinolium functionality, D, exhibit longer absorp-
tion and emission wavelengths than the corresponding pyridinium
analogs, e.g. 2A vs. 2D (lmax, em = 490 and 540 nm, respectively).
Benzo-fusion produces a red shift in the case of 2A vs. 5A, as
well. Introduction of electron donating groups also results in a
bathochromic shift as in the case of 4A, the hydroxy substituted
homolog of 3A shifting the emission maxima approximately
80 nm. Epifluorescent imaging of the probes can be accomplished
using conventional filtersets such as those for DAPI and GFP.
All of the dyes can be excited using a 405 nm laser, enabling
confocal imaging without the need for UV specific optics. The
broad range of emission wavelengths allows for selective detection
of appropriately paired dyes (i.e. 2A or 3A in combination with 4A
2
A, 2D, 3A, 3D, 4A, 5D, 7A and 7D (vide infra), were selected
1
13
for purification and characterization by H-NMR, C-NMR, IR,
HRMS, UV-vis and fluorescence spectroscopy (see Experimental).
-
1
or 5D). Molar absorptivities (e) ranged from 29,000 to 55,000 M ,
-
1
cm with quantum yields of photoemission (Uem) ranging from
.20 to 0.01 (data taken in methanol). While compounds with low
0
U
em are of limited utility, the overall brightness (e · Uem) of probes
utilized in imaging experiments (3A and 4A, see below) compares
favorably with other fluorescent probes such as acridine orange,
Hoescht 33258 and DAPI.
Cellular uptake and inhibition studies
The native fluorescence of the probes enables direct detection of
their uptake in dissociated e19 chick brain cells in a convenient 96-
microwell plate format; this method allows us to screen for activity
against multiple cell types and transporters including MATs and
15,18,29,30
OCTs without the use of radioactive compounds.
Solutions
of 1A–8D were prepared from the reaction isolates to produce
a probe concentration of approximately 100 mM; incubation
time was 10 min. The fluorophore solution was removed, fresh
media added and the plate immediately analyzed by a microwell
plate reader. Given the broad range of absorption and emission
wavelengths, excitation varied from 355 nm to 500 nm with
emission collected at +100 nm to +150 nm. Twelve of the thirty-
two compounds screened were found to associate with the cultured
cells in this initial assay (Fig. 3) based on an increase in emission
intensity of 3¥ compared to untreated cells. Components A and
D are well represented, while only two examples of B and no
compounds possessing C, 6 or 8 were detected in this screen. This
may be due to a combination of factors including low uptake, low
Scheme 1 Reaction conditions and building blocks used to produce the
library of fluorescent arylene-vinylene probes.
Optical spectroscopy
UV-vis spectroscopy of 2A, 2D, 3A, 4A, 5D, and 7A in methanol
reveals absorption maxima ranging from 358 nm for 2A to 441 nm
for 5D (Fig. 2); the peaks lack vibronic structure, characteristic
of intramolecular charge transfer absorption expected for donor–
acceptor systems. Emission maxima range from 475 nm, in the
case of 2A, to 623 nm (5D) spanning much of the visible spectrum.
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Live cell and tissue imaging
The two brightest compounds inhibited by indatraline, 3A and
4A, were selected for examination in live brain explants as well as
cell lines expressing specific MATs in order to evalute their use as
probes of MAT function. HEK-293 cells stably expressing hDAT,
5,16,31
hNET and hSERT were provided by Prof. R. Blakely
and
cultured on 35 mm dishes with a coverslip imaging window for
confocal microscopy (see Experimental). Baseline images for each
cell type were taken to establish the level of cell autofluorescence,
then stock solutions of probe were introduced to produce final
concentrations ranging from 10 mM to 100 mM; laser intensity
and gain were not altered for subsequent images captured at 10 s
intervals. Representative images are shown in Fig. 5 together with
intensity profiles captured from cell bodies. Time course images
reveal that 4A is rapidly accumulated by HEK-hNET and to
a lesser extent by HEK-hDAT. Based on the time dependent
intensity profiles, the response towards HEK-hSERT cells does
not differ substantially from control untransfected HEK cells.
Inspection of images obtained for DAT and SERT cells reveals
Fig. 3 Fluorescent probes identified in uptake assay utilizing dissociated
whole brain cells harvested from e19 chicks; excitation wavelength key at
bottom, emission was collected at +100 to +150 nm. Conditions detailed
in Experimental.
brightness, poor reaction yields or high cytotoxicity resulting in
cell detachment and removal during the washing step. Overall, a
small fraction of the reactions proved to produce compounds with
high brightness (e ¥ U) and high bioactivity. However, it is likely
that many more neuroactive stilbazolium with attractive optical
properties could be identified in larger libraries or through rational
design.
From this initial assay we are only able to determine if a
compound associates with the cultured cells; it is not clear if
a particular probe is actively transported into cells or simply
interacts with the cell membrane. The use of the MAT reuptake
inhibitor, indatraline, provides some insight into what mechanisms
of cellular uptake are at play. Dissociated brain cells were
pretreated with indatraline then the dye solutions as described
above. The emission response of several probes was affected by
indatraline pretreatment, including 2D, 3A, and 4A suggesting that
at least one mode of cellular uptake for these compounds is via a
MAT. In contrast, probes 2A, 3D, 5D, 7A and 7D maintained high
responses in spite of indatraline pretreatment (Fig. 4), indicating
these probes either simply interact with the cell membrane, or
are accumulated via other transporters, i.e. OCTs. The remaining
combinations possessed weak responses or low overall intensity
and were not characterized further.
Fig. 5 The contrasting behaviour of 3A and 4A towards HEK293 cells
expressing DAT, NET or SERT. 4A preferentially accumulates in cells
expressing NET as revealed by time-lapse fluorescence microscopy; panel
A, 10 s (immediately after introduction of 4A), panel B 150 s (scale bar is
10 mm). By contrast, 3A did not accumulate in cells expressing DAT, NET
or SERT, though binding may occur at DAT or SERT (panel C, 10 s) with
emission intensity decreasing with time due to photobleaching (D, 150 s).
Time-dependent intensity plots are shown in E, F.
Fig. 4 Relative uptake based on emission intensities of stilbazolium dyes
in e19 chick whole brain cultures in the absence and presence of indatraline,
a monoamine reuptake inhibitor (mean of six experiments; error bars show
SEM).
2
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3
2
that 4A may be binding to these transporters as fluorescent halos
distributed than 4A, though both dyes are limited to discreet
cell populations tracing the cerebellar gyri. This distribution
is consistent with the behavior of these probes towards MAT-
expressing HEK293 cells. The wider distribution of 3A may be
due to binding to multiple transporter types including MATs,
were observed, however, transport does not appear to occur in the
case of SERT and at a much slower rate (and delayed onset) in the
case of DAT. By contrast, 3A does not appear to be transported
via DAT, NET or SERT. 3A may bind to cells expressing
these transporters, however, we did not observe an increase of
emission intensity within the cell bodies and instead a slow loss
of fluorescence due to photobleaching was observed (Fig. 5). The
time dependent imaging of 4A reveals a preference for NET, similar
34
OCTs or cationic aminoacid transporters (CATs), while 4A may
be limited to NET or DAT expressing catecholaminergic cells.
The spectral segregation of 3A and 4A enables their simultaneous
use to identify cells that differentially accumulate each probe.
Image overlays from 100 mm sagittal (Fig. 6A) and transverse (Fig.
16,17
to ASP+.
However in contrast to ASP+, 4A does not appear to
33
be transported via SERT making this a selective catecholaminergic
probe with a high level of discrimination between DAT and NET.
We next examined 3A and 4A in live brainstem and cerebellum
sections to determine their distribution in a complex, multicellular
environment. Fig. 6 (A,B) shows cerebellum sections treated
individually with 3A and 4A; 3A appears to be more widely
6B) sections reveal that both probes are associated with cells of
chick mesencephalon and metencephalon. The fluorophores were
excited at 405 nm; the emission of 3A (in green) was collected
between 425 and 500 nm, the emission of 4A (in red) was collected
from 575 to 650 nm with areas of overlap appearing as yellow.
3A is widely distributed in both the cerebellum and pons while
4A appears in distinct cell groupings along the cerebellar gyri and
around the midline of the brainstem especially in multiple tract-
like sructures. The chick brainstem possesses dense clusters of
35,36
37
chatecholaminergic
and serotonergic cells that project into
the cerebellum and front brain. We observed both perikarya
and fibers stained with 4A in this region with several discreet
cell clusters brightly illuminated (Fig. 6C). Pretreatment of brain
sections with desipramine, a norepinephrine reuptake inhibitor,
limited the accummulation of 4A (Fig. 7), consistent with the
results observed for hNET-expressing HEK-293 cells. Visual
inspection of the images reveals a distinct difference between
desipramine treated (Fig. 7B) and untreated (Fig. 7A) tissue that
Fig. 6 Confocal microscopy images of live e19 chick brain explants
treated with 3A (green) and 4A (red). 3D overlays of sequential transverse
cerebellum sections (panels A,B scale bar: 100 mm) demonstrate that 4A
is largely accumulated in discreet cell bodies while the distribution of 3A
is more diffuse. Transverse sections of the metencephalonic brain stem
reveal discrete clusters of cells that accumulate 4A (C, scale bar: 10 mm)
visible both in the cell bodies and processes; 3A association with the same
cells is minimal (D). The spectral segregation of 3A and 4A enables their
simultaneous to image cells that differentially accumulate each probe in the
metencephalon (E, F, scale bar: 50 mm); while 3A is broadly distributed,
Fig. 7 Ex vivo assessment of 4A uptake in the absence and presence of
the norepinephrine reuptake inhibitor, desipramine. Sequential sections
of e19 chick cerebellum were treated with 4A alone (40 mM, 10 min;
panel A, scale bar is 100 mm) or pretreated with desipramine (10 mM,
3 min) then 4A (B). In the absence of desipramine, 4A accumulates in cell
bodies and processes while desipramine pretreatment severely attenuates
uptake leaving only diffuse distribution. Histogram of pixel intensity (C)
for desipramine dependent 4A uptake; mean intensity (D), 5 sections, error
bars show SEM. Laser intensity and detector gain were kept constant while
obtaining images.
4
A is localized to distinct clusters and tracts.
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correlates with image analysis: While individual cell bodies and
processes are visible in cells exposed to 4A, pretreatment with
desipramine limits the overall emission intensity (Fig. 7D) and the
lack of accumulation to discreet structures or cells is evident in
intensity histograms (Fig. 7C).
necessary to induce precipitation. The supernatant liquid was
decanted, the product rinsed, then collected over filter paper,
dried under vacuum and stored in a dessicator. For screening
purposes, the crude reaction isolate was used directly. Compounds
2A, 2D, 3A, 3D, 4A, 5D, 7A and 7D, identified from reaction
mixtures that demonstrated high activity (vide supra) were further
purified by crystallization from MeOH; their characterization data
is presented below.
Conclusions
We have identified a fluorescent stilbazolium dye, HNEP+, that
is a highly selective NET substrate suitable as an imaging agent
and reporter of transporter function both in vitro and ex vivo.
(
E)-4-[2-(4-Hydroxyphenyl)ethenyl]-1-methylpyridinium iodide
(
2A). IR vmax: 831, 985, 1020, 1112, 1168, 1261, 1470, 1514, 1571,
1589, 3048, 3402 cm . H NMR (500 MHz, DMSO): d 3.81 (3H,
s), 4.22 (3H, s), 7.03 (2H, d, J = 8.5 Hz), 7.36 (1H, d, J = 16.5 Hz),
.71 (2H, d, J = 8.5 Hz), 7.98 (1H, d, J = 16.5 Hz), 8.16 (2H,
d, J = 6.5 Hz), 8.81 (2H, d, J = 6.0). C NMR (125.74 MHz,
DMSO): d 47.24, 55.93, 115.12, 121.16, 123.48, 128.25, 130.46,
41.02, 145.32, 153.31, 161.64. Uem = 0.011, e = 46,000 M , cm .
HRMS (ES+) calcd for C15
E = 1.8 ppm). MP: 210 C (dec.) Yield: 62 mg (34%)
-
1
1
38
Given the complex modes of transporter regulation, the ability
to monitor transporter function in tissue explants (or in vivo)
may lead to improved assays and identification of new classes
or improved selectivity of reuptake inhibitors. Our results support
the notion that the MATs are capable of transporting molecular
reporters that mimic certain aspects of the parent substrates. The
necessity for dyes possessing high brightness (e · Uem) is apparent,
as only HNEP+ and NEP+ proved useful for in vitro and ex vivo
imaging. We have demonstrated that subtle changes in structure
lead to pronouced differences in behavior towards MATs: no
accumulation of NEP+ was observed for DAT, NET or SERT
expressing HEK293 cells. We anticipate that identified probes
herein, as well as additional neuroactive fluorophores based on
the styryl-pyridinium core, will compliment ASP+ in fluorescence
based assays and imaging applications.
7
13
-
1
-1
1
+
H
16NO : 226.1232 Found: 226.1228
◦
(
(
E)-4-[2-(4-Methoxyphenyl)ethenyl]-1-methylquinolium iodide
(
2D). IR vmax: 753, 776, 824, 834, 978, 1020, 1168, 1224, 1257,
-1
1
1
4
8
8
566, 3024 cm . H NMR (500 MHz, DMSO): d 3.85 (3 H, s),
.52 (3 H, s), 7.09 (2H, d, J = 8.5 Hz), 7.98 (2 H, d, J = 9.0 Hz),
.02 (1 H, t, J = 7.5 Hz), 8.18–8.27 (3 H, m), 8.42 (1 H, d, J = 8.5),
.45 (1 H, d, J = 6.5 Hz), 9.06 (1 H, d, J = 8.5 Hz), 9.30 (1 H, d,
J = 6.5). C NMR (125.74 MHz, DMSO): d 45.01, 55.97, 115.07,
13
116.01, 117.62, 119.79, 126.64, 126.95, 128.71, 129.56, 131.36,
Experimental
135.38, 139.23, 143.47, 148.27, 153.35, 162.01. U = 0.012, e =
em
-
1
-1
+
3
3,000 M , cm . HRMS (ES+) calcd for C19
H
18NO : 276.1388
Materials and methods
◦
Found: 276.1385 (E = 1.1 ppm). MP: 271 C (dec.) Yield: 87 mg
(
43%)
Reagents were obtained from Sigma Aldrich and TCI America
and used without further purification. ACS reagent grade sol-
(
E)-1-Methyl-4-[2-(2-naphthalenyl)ethenyl]pyridinium
iodide
1
13
vents were obtained from EMD Chemicals. H and C NMR
spectra were obtained on a Bruker 500 MHz spectrometer using
tetramethylsilane TMS as the internal standard and DMSO as
the solvent. Absorbance spectra were recorded on a Perkin–Elmer
Lambda 35 UV-vis spectrometer; emission spectra were recorded
(
3A). IR nmax: 746, 813, 823, 863, 873, 966, 1180, 1192, 1517,
-1
1
1
7
8
8
4
1
614, 3023 cm . H NMR (500 MHz, DMSO): d 4.27 (3H, s),
.58–7.61 (2H, m), 7.67 (1H, d, J = 16.0 Hz), 7.95–8.03 (4H, m),
.16 (1H, d, J = 16.5 Hz), 8.21 (1H, s), 8.27 (2H, d, J = 5.5 Hz),
.88 (2H, d, J = 6.0 Hz). C NMR (125.74 MHz, DMSO): d
7.43, 124.01, 124.11, 124.16, 127.46, 127.96, 128.25, 129, 129.24,
13
on a Perkin–Elmer LS55 Fluorometer. For determination of Uem
,
solutions were prepared to an optical density of 0.05 or less in
MeOH to minimize inner filter effects. Perylene in cyclohexane
30.01, 133.29, 133.39, 134.19, 141.01, 145.61, 152.86. Uem = 0.18,
-1
-1
+
e = 32,000 M , cm . HRMS (ES+) calcd for C18
Found: 246.1278 (E = 2.0 ppm). MP: 279–281 C (dec.). Yield:
1
H
16N : 246.1283
39
was used as a reference for quantum yields.
◦
All procedures and experiments involving embryonic chick
derived cells and tissue are in compliance with University of Miami
Institutional Animal Care & Use Committee.
21 mg (65%)
(E)-1-Methyl-4-[2-(2-naphthalenyl)ethenyl]quinolinium iodide
(
3D). IR nmax: 752.09, 826.83, 958.06, 1111.73, 1300.92, 1532.52,
-
1 1
Synthesis
1566.79, 1593.56, 3014.8, 3416.7 cm . H NMR (500 MHz,
DMSO): d 4.56 (3H, s), 7.61 (2H, t, J = 3.5 Hz), 7.98–8.1 (4H, m)
2
1,24,26
21,24,26
22
The syntheses of compounds 1A–D,
2A–D,
3A
8
.29 (3H, m) 8.32 (1H, d, J = 16.0 Hz), 8.45 (2H, m), 8.56 (1H, d,
23
and 6A have been previously described. In a typical reac-
tion, 0.5 mmol of N-methylpicolinium iodides (A–C) or 1, 4-
dimethylquinolinium iodide was placed in a 20 mL scintillation
vial equipped with a microstribar, followed by 0.55 mmol (1.1 eq)
of the appropriate aldehyde, methanol (10 mL) and 3 drops of
piperidine. The reaction vessel was capped, stirred and heated at
5 C for 2–3 h. The reaction mixture was allowed to reach room
temperature at which point most compounds precipitated; com-
pounds not observed to precipitate were kept at 4 C overnight.
13
J = 6.1 Hz), 9.11 (1H, d, J = 8.1 Hz), 9.40 (1H, d, J = 6.0 Hz).
C
NMR (125.74 MHz, DMSO): d 45.22, 116.73, 119.73, 120.52,
1
1
1
24.91, 126.70, 126.98, 127.33, 128.01, 128.17, 128.97, 128.99,
29.70, 130.84, 133.25, 134.12, 135.41, 135.58, 139.10, 143.15,
-1
-1
48.43, 152.71. Uem = 0.08, e = 29,000 M , cm . HRMS (ES+)
+
calcd for C22
H
18N : 296.1434 Found: 296.1436 (E = 0.7 ppm).
◦
6
MP: 239 (dec.) Yield: 106 mg, 51%.
◦
(E)-4-[2-(6-Hydroxy-2-naphthalenyl)ethenyl]-1-methyl-pyridini-
um iodide (4A). IR nmax: 660, 805, 824, 868, 961, 1154, 1174,
Acetic acid was added to those reactions involving aldehydes with
hydroxyl groups. In some cases, the addition of ethyl acetate was
-
1
1
1271, 1480, 1604, 3036, 3159 cm . H NMR (500 MHz, DMSO):
2
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d 4.24 (3H, s), 7.14 (1H, d, J = 8.5 Hz), 7.17 (1H, s), 7.54 (1H,
d, J = 16.5 Hz), 7.78 (1H, d, J = 8.5 Hz), 7.83–7.85 (2H, m),
were read on a Perkin–Elmer LS55 Fluorometer equipped with
a plate reader attachment; the excitation and emissions set based
on the optical properties of each compound. A second round
of screening exposed cells to the reuptake inhibitor, indatraline,
with an incubation period of 10 min prior to exposure to the
compounds. The inhibitor was added to 200 mL of media to reach
a concentration of 100 mM, followed by probe addition and rinsing
steps described above.
8
8
.07 (1H, s), 8.11 (1H, d, J = 16.0 Hz), 8.21 (2H, d, J = 7.0 Hz),
.83 (2H, d, J = 7.0 Hz), 10.13 (1H, s). C NMR (125.74 MHz,
13
DMSO): d 47.28, 109.58, 119.92, 122.33, 123.64, 124.34, 127.50,
1
27.87, 130.18, 130.55, 130.91, 136.17, 141.61, 145.41, 153.14,
-
1
-1
157.50. Uem = 0.106, e = 38,000 M , cm . HRMS (ES+) calcd
+
for C14
H
14NO : 262.1232 Found: 262.1235 (E = 1.1 ppm) MP:
◦
◦
2
86–290 C, 270 C color change (yellow→orange). Yield: 88 mg
(
45%)
Midbrain and hindbrain isolation
(
E)-4-[2-(6-Methoxy-2-naphthalenyl)ethenyl]-1-methylquinolini-
um iodide (5D). IR vmax: 749, 758, 813, 823, 855, 969, 1168, 1191,
The mesencephelon, metencephalon and myelencephalon were
isolated from e19 chick embryos and placed in an aluminium
foil mold. The tissue was submerged in 2.5% low melting point
agarose gel (Invitrogen LMP Agarose, Ultrapure #5517–014) and
placed on ice to solidify. A Polaron H1200 Vibrating Microtome
was used to section the brain into approximately 100 mm slices.
Once the gel was solidified, the block was removed from the foil
mold and excess agarose gel was removed with a razor blade. The
block was then positioned on the stage of the microtome using
instant dry glue and allowed to dry before it was submerged in
Dulbecco’s Phosphate Buffered Saline solution (DPBS, Mediatech
-
1
1
1518, 1614, 3020 cm . H NMR (500 MHz, DMSO): d 3.92 (3H,
s), 4.54 (3H, s), 7.23 (1H, dd), 7.41 (1H, d, J = 2.0 Hz), 7.91 (1H,
t), 7.94 (1H, t), 8.06 (1H, t, J = 2.0 Hz), 8.22 (1H, d, J = 9.0 Hz),
8
8
6
1
1
1
.21–8.32 (3H, m), 8.38 (1H, d, J = 15.5 Hz), 8.44 (1H, d, J = 9),
.52 (1H, d, J = 6.5 Hz), 9.09 (1H, d, J = 8.5 Hz), 9.34 (1H, d, J =
1
3
.5 Hz). C NMR (125.74 MHz, DMSO): d 45.12, 55.88, 106.85,
16.42, 119.34, 119.83, 119.87, 125.52, 126.75, 126.96, 128, 128.7,
29.67, 130.77, 130.94, 131.45, 135.43, 136.08, 139.22, 143.70,
-
1
-1
48.44, 153.05, 159.19. Uem = 0.11, e = 30,000 M , cm . HRMS
+
(
ES+) calcd for C23
H
20NO : 326.1545 Found: 326.1544 (E = 0.3
◦
◦
ppm). MP: 269–272 C (dec.) Yield: 61 mg (27%).
#21-031-CV) at 0 C. To obtain sagittal section the anterior
side of the cube was positioned facing up. Transverse slices were
taken in 100 mm increments starting at the anterior edge of
the mesencephalon and continuing posteriorly to the hindbrain.
Brain regions were determined using the stereotaxic atlas of the
(
E)-4-[2-(5-Chloro-1H-indol-3-yl)ethenyl]-1-methylpyridinium
iodide (7A). IR vmax: 672, 748, 810, 821, 860, 967, 1185, 1421,
593, 1607, 3027, 3174 cm . H NMR (500 MHz, DMSO): d 4.17
3H, s), 7.23 (1H, d, J = 8.5 Hz), 7.27 (1H, d, J = 16.5 Hz), 7.50
1H, d, J = 8.5 Hz), 8.05 (1H, s), 8.13 (2H, d, J = 6.5 Hz), 8.20 (1H,
s), 8.23 (1H, d, J = 17.0 Hz), 8.70 (2H, d, J = 6.5 Hz), 12.05 (1H, s).
C NMR (125.74 MHz, DMSO): d 46.81, 113.60, 114.51, 117.94,
19.93, 122.28, 123.26, 126.30, 126.65, 133.16, 135.64, 136.24,
44.70, 154.43. Uem = 0.018, e = 40,000 M , cm . HRMS (ES+)
calcd for C16
-
1 1
1
(
(
32
chick brain however, exact coordinate positions varied due to
differences in developmental stages. Slices from the mesencephelon
and metencephalon were retrieved using an inoculating loop and
placed onto an 8 chamber glass vessel tissue culture treated glass
slide (Cultureslide, BD Falcon, 354118) with 500 mL of NM.
Sagittal slices were obtained in the same manner. Slices from the
sagittal midline were retrieved and placed onto the cover slip slides.
The brain sections were then treated with the fluorescent probes.
Probes were added to the media to reach a concentration of 100
mM and allowed to incubate for 10 min. The tissue slices were
then rinsed using an 18 gauge aspirating needle to remove the
media. 50 mL of media was then added back to the tissue. The
chamber compartments were then removed and the tissue was
covered with a 24 ¥ 60 mm micro cover glass (micro cover glass,
VWR#48393252) and sealed using clear nail polish.
1
3
1
1
-
1
-1
+
H
14ClN
2
: 269.0846 Found: 269.0851 (E = 1.9 ppm).
◦
◦
MP: 279–281 C (dec.), 230 C color change (yellow→orange),
Yield: 60 mg (30%).
(
E)-4-[2-(5-Chloro-1H-indol-3-yl)ethenyl]-1-methylquinolium
iodide (7D). IR vmax: 671.00, 751.88, 893.10, 1024.17, 1050.02,
112.44, 1134.70, 1221.66, 1309.30, 1394.64, 1515.93, 1589.25,
161.8 cm . H NMR (500 MHz, DMSO): d 4.43 (3H, s), 7.23
1H, d, J = 7.0 Hz), 7.49 (1H, d, J = 8.0 Hz), 7.98 (2H, m), 8.19
2H, m), 8.29 (1H, d, J = 8.5 Hz), 8.48 (3H, m), 8.94 (2H, d,
J = 8.0 Hz), 9.12 (1H, d, J = 5.5 Hz). C NMR (125.74 MHz,
DMSO): d 44.70, 114.14, 114.28, 114.47, 114.51, 119.43, 119.53,
1
3
(
(
-
1
1
1
3
123.23, 125.92, 126.44, 126.71, 127.66, 128.97, 132.77, 135.03,
Confocal imaging
1
35.86, 137.61, 139.20, 147.2, 154.00. Uem = 0.008, e = 41,000
-
1
-1
+
Imaging was performed on a Leica SP5 confocal microscope
housed within the UM Biology Imaging Core Facility. A 405 nm
diode laser was used as the excitation source. XYZ-Scans were
collected with 2 mm sections. Images were analyzed using ImageJ
M , cm . HRMS (ES+) calcd for C20
H
16ClN : 319.1002 Found:
2
◦
3
19.1007 (E = 1.5 ppm). MP: 285 C (dec.) Yield: 69 mg (31%).
Microwell plate preparation and microwell plate assays
1
.41 software (NIH, USA). 3D reconstructions of the sections
Brains were isolated from e19 chick embryos and plated on 96-well
displayed in Fig. 6 are available as separate files.†
18
plates following a previously reported procedure. Plates incubate
for 72 h before screening. For the initial screening, fluorescent
probes were added in 200 mL neuronal media (NM, Sciencell 1521
containing basal medium, neuronal growth supplement, and peni-
cillin/streptomycin supplement) to obtain final concentrations of
Acknowledgements
We thank Prof. K. Tosney, L. White and A. Hayward (UM,
Biology) for helpful discussions and assistance with live tissue
preparations.
100 mM and incubated for 10 min. Plates were then rinsed and
00 mL NM was added and left in each well. The microwell plates
2
This journal is © The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2142–2148 | 2147

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148 | Org. Biomol. Chem., 2011, 9, 2142–2148
This journal is © The Royal Society of Chemistry 2011