Isomerically Pure Tetramethylrhodamine Voltage Reporters

Article abstract of DOI:10.1021/jacs.6b05672

We present the design, synthesis, and application of a new family of fluorescent voltage indicators based on isomerically pure tetramethylrhodamines. These new Rhodamine Voltage Reporters, or RhoVRs, use photoinduced electron transfer (PeT) as a trigger for voltage sensing, display excitation and emission profiles in the green to orange region of the visible spectrum, demonstrate high sensitivity to membrane potential changes (up to 47% ΔF/F per 100 mV), and employ a tertiary amide derived from sarcosine, which aids in membrane localization and simultaneously simplifies the synthetic route to the voltage sensors. The most sensitive of the RhoVR dyes, RhoVR 1, features a methoxy-substituted diethylaniline donor and phenylenevinylene molecular wire at the 5′-position of the rhodamine aryl ring, exhibits the highest voltage sensitivity to date for red-shifted PeT-based voltage sensors, and is compatible with simultaneous imaging alongside green fluorescent protein-based indicators. The discoveries that sarcosine-based tertiary amides in the context of molecular-wire voltage indicators prevent dye internalization and 5′-substituted voltage indicators exhibit improved voltage sensitivity should be broadly applicable to other types of PeT-based voltage-sensitive fluorophores.

Full text of DOI:10.1021/jacs.6b05672

Communication
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Isomerically Pure Tetramethylrhodamine Voltage Reporters
Parker E. Deal,† Rishikesh U. Kulkarni,† Sarah H. Al-Abdullatif,† and Evan W. Miller*,†,‡,§
‡
§
†Department of Chemistry, Department of Molecular & Cell Biology, and Helen Wills Neuroscience Institute, University of
California, Berkeley, California 94720, United States
S
* Supporting Information
molecular wire along with a sulfonated silicon-rhodamine
ABSTRACT: We present the design, synthesis, and
application of a new family of fluorescent voltage
indicators based on isomerically pure tetramethylrhod-
amines. These new Rhodamine Voltage Reporters, or
RhoVRs, use photoinduced electron transfer (PeT) as a
trigger for voltage sensing, display excitation and emission
profiles in the green to orange region of the visible
spectrum, demonstrate high sensitivity to membrane
potential changes (up to 47% ΔF/F per 100 mV), and
employ a tertiary amide derived from sarcosine, which aids
in membrane localization and simultaneously simplifies the
synthetic route to the voltage sensors. The most sensitive
of the RhoVR dyes, RhoVR 1, features a methoxy-
substituted diethylaniline donor and phenylenevinylene
molecular wire at the 5′-position of the rhodamine aryl
ring, exhibits the highest voltage sensitivity to date for red-
shifted PeT-based voltage sensors, and is compatible with
simultaneous imaging alongside green fluorescent protein-
based indicators. The discoveries that sarcosine-based
tertiary amides in the context of molecular-wire voltage
indicators prevent dye internalization and 5′-substituted
voltage indicators exhibit improved voltage sensitivity
should be broadly applicable to other types of PeT-based
voltage-sensitive fluorophores.
fluorophore. Although some members of the VF dye family
display high voltage sensitivity (ΔF/F of 48−49% per 100 mV
change),6 their excitation and emission profiles in the cyan to
green range limit their application alongside many common
optical tools like green fluorescent protein (GFP) and Ca2+-
sensitive GFPs (GCaMPs).10 BeRST 1 partially solves this
problem of spectral overlap by the use of the far-red/near-
infrared Si-rhodamine, but its voltage sensitivity is lower
compared with the VF dyes (24% ΔF/F per 100 mV) and the
synthetic route to BeRST 1 is low-yielding because of the
inclusion of a sulfonic acid functional group on the meso-aryl ring
of BeRST 1. We hoped to develop a voltage-sensing scaffold that
would retain the high sensitivity of the VF dye series, expand the
spectrum of colors available for high-fidelity voltage sensing, and
circumvent the inclusion of the sulfonate group that makes
synthetic efforts challenging. Toward this end, we disclose the
design and synthesis of the Rhodamine Voltage Reporter
(RhoVR, “rover”) family of tetramethylrhodamine (TMR)-
based PeT voltage indicators. We have synthesized four new
RhoVR dyes, which display excitation and emission profiles
spectrally distinct from those of both VF and BeRST-type dyes,
feature voltage sensitivities ΔF/F of 3−47% per 100 mV, and
make use of an ortho-tertiary amide instead of a sulfonate to
achieve membrane localization.
TMR-based voltage indicators were synthesized in isomeri-
cally pure form from the 4′- and 5′-bromo TMR derivatives
(Scheme 1). We generated isomerically pure rhodamines by
condensation of dimethylaminophenol with the corresponding
2-carboxybenzaldehydes 3 and 5 (SI Scheme 1), which were
prepared in 79% and 88% yield over a two-step radical
bromination followed by hydrolysis of either commercially
available 5-bromophthalide 4 or 6-bromophthalide 2 (prepared
from commercially available phthalide 1 in 62% yield; SI Scheme
1).11,12 Separation of isomers at the 6-bromophthalide stage
obviates the need to separate isomers at the rhodamine stage:
addition of dimethylaminophenol occurs exclusively at the
electrophilic aldehyde carbonyl.13 Condensation of dimethyla-
minophenol with carboxybenzaldehyde in propionic acid in the
presence of catalytic PTSA13 gave the 4′- and 5′-bromoTMR
derivatives in 35% (6) and 57% (7) yield, respectively, after
purification by silica gel chromatography (SI Scheme 2).
Subsequent Pd-catalyzed Heck coupling with substituted
styrenes 8 and 9 (SI Scheme 3) gave the TMR-based voltage
sensors in 41−55% yield following silica gel purification.
Formation of an N-methylglycine-derived tertiary amide
ells expend a large amount of energy to maintain an
Cunequal distribution of ions across the plasma membrane,
resulting in a transmembrane voltage or potential (Vm).1 Fast
changes in Vm are responsible for the distinct cellular physiology
of neurons and cardiomyocytes, and mounting evidence points
to the importance of Vm in shaping fundamental cellular
processes, such as differentiation, migration, and division, across
a number of cell types.2 Traditionally, changes in Vm have been
monitored using electrodes, which are highly invasive and limited
in throughput.3 Broadly applicable and sensitive optical methods
to track Vm would expand our capacity to disentangle the
contributions Vm makes to human health and disease.4
We have recently undertaken a program to design and apply
small-molecule fluorescent dyes that use photoinduced electron
transfer (PeT) as a molecular switch to optically monitor changes
in Vm. The parent family of sensors includes VoltageFluor (VF)
dyes (SI Table 1)5−7 and makes use of PeT8 through a
phenylenevinylene molecular wire to modulate the fluorescence
intensity of a sulfonofluorescein-based reporter in a Vm-
dependent fashion. More recently, we disclosed the development
of Berkeley Red Sensor of Transmembrane potential (BeRST 1)
(SI Table 1),9 which features the shared phenylenevinylene
Received: June 2, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.6b05672
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Communication
Scheme 1. Synthesis of Isomerically Pure Rhodamine Voltage Sensors
mediated by HATU gave the tert-butyl ester-protected voltage
sensors in >70% yield, which exist as mixtures of rotamers around
the sarcosinyl−amide bond, as determined by variable-temper-
ature (VT) NMR analysis (SI Figure 1). TFA-catalyzed
deprotection of the tert-butyl ester gave the final voltage sensors
in 7−18% yield over two steps after reversed-phase HPLC
purification.
Each RhoVR displayed absorption profiles centered at 564−
565 nm (ε = 70 000 to 87 000 M−1 cm−1 (SI Figure 2). A strong
secondary absorption band near 400 nm indicated the presence
of the phenylenevinylene molecular wire (SI Figure 2). Emission
from all of the RhoVRs was centered at 586−588 nm (Φ = 0.89−
9.2%; Table 1). The low Φ values may indicate variable levels of
PeT within the compounds.
range from −100 to +100 mV in 20 mV increments from a
holding potential of −60 mV provided voltage sensitivities of 3−
47%, depending on the dye (Table 1, Figure 1c,d, and SI Figure
6). Compound 21, which we dubbed RhoVR 1, emerged as the
most voltage-sensitive dye (Table 1). Dyes bearing methoxy
substitution on the aniline (17 and 21) showed improved voltage
sensitivity relative to unsubstituted anilines (26% (17) vs 3%
(16) and 47% (21, RhoVR 1) vs 7% (20)), as observed for
fluorescein-based voltage indicators.6 We also observed that
voltage indicators derived from 5′-substituted rhodamines were
both more voltage-sensitive (47% (21, RhoVR 1) vs 26% (17)
and 7% (20) vs 3% (16)) and brighter in cells (SI Figure 3).
Studies are underway to probe the nature of this difference.
Given the high sensitivity and brightness of RhoVR 1 in cells, we
chose this dye to evaluate in subsequent experiments. RhoVR 1
shows photostability comparable to that of VF2.1.Cl (SI Figure
3e).
When bath-applied to cultured rat hippocampal neurons,
RhoVR1 gave distinct membrane-associated staining. The clear
membrane staining, coupled with the high voltage-sensitivity of
RhoVR 1 enabled the detection of spontaneously firing action
potentials with an average ΔF/F of 15% (N = 27 spikes) and
signal-to-noise ratios (SNRs) ranging from 10:1 to 20:1 that were
largely dependent on the illumination intensity used (1.7 to 3.1
W/cm2) (Figure 2, cells 2−5). To confirm that the spiking events
we observed arose from action potentials, we treated actively
firing cultures with tetrodotoxin (TTX), a sodium channel toxin
that inhibits action potential firing, and observed no spiking
activity after TTX treatment (SI Figure 7).
Table 1. Properties of RhoVRs
ε/M−1 cm−1
a
ΔF/F
SNR
b
a
b
compd
(λmax/nm)
Φ (λmax/nm)
(100 mV)
(100 mV)
16
17
20
21
75000 (565)
70000 (565)
77000 (564)
87000 (564)
0.036 (586)
0.092 (588)
3
0.2%
26 3%
1%
47 3%
19:1
37:1
0.0089 (586)
0.045 (588)
7
96:1
160:1
a
b
PBS, pH 7.2, 0.1% SDS. Voltage-clamped HEK cells.
All of the sarcosine-substituted RhoVRs (500 nM, imaged in
dye-free HBSS) localized well to the plasma membrane of HEK
cells, as determined by fluorescence microscopy (Figure 1a,b and
SI Figures 3−5). Control experiments conducted with TMR
derivatives lacking the sarcosine amide (i.e., free carboxylates
10−13) showed strong internal membrane staining (Figure 1a).
The dramatic change in cellular localization is consistent with our
hypothesis that inclusion of a charged tertiary amide at the ortho
position of the pendant aryl ring prevents the formation a neutral
spirocycle and subsequent cellular dye uptake. The use of
carboxylate derivatives drastically simplifies the synthetic route to
make long-wavelength voltage indicators.
RhoVR 1 represents the most sensitive red-shifted PeT-based
voltage indicator to date: VF2.1(OMe).Cl has a voltage
sensitivity of 49%6 but emission at 536 nm, while BeRST 1 has
bathochromic emission at 680 nm but a voltage sensitivity of only
24%.9 Given the success of far-red voltage dyes like BeRST 1 in
integrating multiple functional signals simultaneously, we
wondered whether we might be able to perform two-color
imaging simultaneously with RhoVR 1 despite the tighter optical
overlap between rhodamines and GFP-based chromophores.
Expression of cytosolic enhanced GFP (eGFP) in HEK cells
stained with RhoVR 1 did not result in significant bleed-through
The voltage sensitivity of each RhoVR was assessed in HEK
cells using patch-clamp electrophysiology in whole-cell voltage-
clamp mode. Hyper- and depolarizing voltage steps spanning a
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Figure 1. Cellular characterization of rhodamine-based voltage indicators. (a, b) Confocal fluorescence images of HEK cells stained with (a) compound
13 (no sarcosine amide) or (b) RhoVR 1 (compound 21, with sarcosine amide). Incorporation of the tertiary amide based on sarcosine results in a clear
enhancement of membrane-localized fluorescence (panel a vs b). The fluorescence of membrane-localized RhoVR 1 is voltage-sensitive. The scale bar is
10 μm. (c) Plot of the fractional change in fluorescence vs time for 100 ms hyper- and depolarizing steps ( 100 mV in 20 mV increments) from a holding
potential of −60 mV for single HEK cells under whole-cell voltage-clamp mode. (d) Plot of % ΔF/F vs final membrane potential summarizing data from
nine separate cells, revealing a voltage sensitivity of approximately 47% per 100 mV. Error bars are SEM.
4) and clearly resolve spikes in neurons expressing eGFP in single
trials (Figure 2b−d, neuron 1).
Encouraged by these results, we next sought to image voltage
and Ca2+ dynamics simultaneously14 by exciting the specimen
with green and blue light and splitting the resulting emission to
capture rhodamine and GFP fluorescence in parallel (SI Scheme
4). Brief rises in intracellular [Ca2+] (Ca2+ transients), on the
order of hundreds of milliseconds, are used as a surrogate for
neuronal membrane depolarizations and are often monitored
with green-fluorescent Ca2+-sensitive fluorophores like the
genetically engineered GCaMPs.10 We expressed GCaMP6s,
on account of its excellent sensitivity to Ca2+ released in response
to single action potentials,10 in cultured hippocampal neurons
and incubated these neurons with RhoVR 1 (500 nM). Using an
image splitter (SI Scheme 4) to separate emitted photons
according to wavelength, we recorded Ca2+ transients via
GCaMP6s fluorescence and voltage spikes via RhoVR 1
fluorescence. Under these conditions, hippocampal neurons
again displayed significant amounts of spontaneous spiking
activity in multiple distinct neurons, as detected by GCaMP6s
(Figure 3a, green) and RhoVR 1 (Figure 3a, magenta).
The sensitivity of RhoVR 1 to these action potentials was
similar to values obtained in experiments recorded at a single
wavelength alone (ΔF/F per spike = 9.5%, SNR = 12:1, N = 70
spikes; Figure 3a,e−g). Typically, one neuron expressed high
levels of GCaMP6s fluorescence in a field of view (Figure 3d),
which enabled us to directly compare transient rises in Ca2+ with
Figure 2. (a−c) Imaging of spontaneous neuronal activity with RhoVR
fast voltage spikes in the same neuron (Figure 3a). In all cases,
the fast voltage spike clearly precedes the subsequent Ca2+
transient. RhoVR 1 displays comparable or better ΔF/F values
for single spikes relative to the corresponding Ca2+ transients in
the same cell (Figure 3a and SI Movie 1). Because of the
inherently fast kinetics of Vm relative to Ca2+, monitoring Vm
directly via RhoVR 1 enables resolution and precise timing of
spikes occurring in quick succession from multiple cells (Figure
3f−h), which would be impossible using more traditional
approaches such as Ca2+ imaging or single-cell electrophysiology.
In summary, we have presented the design, synthesis, and
application of four members of a new class of voltage-sensitive
dyes based on tetramethylrhodamine. All of the new sensors
display excitation and emission profiles greater than 550 nm,
good photostability, and varying degrees of voltage sensitivity in
patch-clamped HEK cells. The best of the new voltage indicators,
RhoVR 1, displays a voltage sensitivity ΔF/F of 47% per 100 mV,
rivaling the sensitivity of the fluorescein-based VF2.1(OMe).H
(ΔF/F = 49% per 100 mV)6 and surpassing that of our most red-
1 and eGFP. Rat hippocampal neurons (DIC, a) were stained with 500
nM RhoVR (fluorescence, b). A small number of neurons transiently
expressed eGFP (fluorescence, c). The scale bar is 20 μm. (d−f) The
spontaneous activity of the neurons in panels (a) and (b) were recorded
optically at 500 Hz using RhoVR 1. The activity of each neuron (1−5,
panel a) is displayed as a trace of fluorescence intensity vs time (d). The
boxed regions in (d) are shown on an expanded scale in (e). Asterisks
indicate traces expanded in (f).
of eGFP fluorescence into the rhodamine channel and did not
substantially diminish the voltage sensitivity of RhoVR-1 in
voltage-clamped HEK cells (47 3% without vs 45 1% with
eGFP; SI Figure 8). Furthermore, cytosolic eGFP in cultured
neurons did not attenuate our ability to track action potentials in
spontaneously firing neurons in culture (Figure 2). Indeed, when
imaging spontaneous activity in cultured rat hippocampal
neurons, we were able to clearly distinguish distinct spiking
events arising from neighboring cells (Figure 2b,d, neurons 3 and
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Journal of the American Chemical Society
Communication
been the typical substitution pattern for our previous molecular-
wire voltage sensors and many PeT-based analyte sensors.15−17
Experiments are underway to probe the nature of this voltage
sensitivity enhancement as well as to apply this substitution
pattern to future generations of voltage-sensitive dyes and other
sensing platforms.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b05672.
■
S
Synthetic details and additional data (PDF)
Movie of RhoVR 1 and GCaMP6s fluorescence (AVI)
AUTHOR INFORMATION
Corresponding Author
*evanwmiller@berkeley.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge generous support from the University
of California, Berkeley, the Hellman Foundation, the March of
Dimes (5-FY16-65), the Alzheimer’s Association (2016-NIRG-
394290), and the NIH (R00 NS07581). R.U.K. was supported in
part by an NIH Chemical Biology Training Grant (T32
GM066698). Confocal imaging was performed at the CRL
Molecular Imaging Center, UC Berkeley, supported by the
Helen Wills Neuroscience Institute. We thank Dr. Alison Walker
and Pei Liu for expert technical assistance with neuronal cultures
and Dr. Hasan Celik for help with VT-NMR studies.
Figure 3. Simultaneous two-color imaging of voltage and Ca2+ in
hippocampal neurons using RhoVR 1 and GCaMP6s. (a) The green
trace shows the relative change in fluorescence from Ca2+-sensitive
GCaMP6s, while the magenta trace depicts relative fluorescence
changes in RhoVR 1 fluorescence from neuron 1 in (b). The inset
shows an expand time scale of the boxed region. (b) DIC image of
neurons expressing GCaMP6s and stained with RhoVR 1. (c)
Fluorescence image showing membrane localization of RhoVR 1
fluorescence from neurons in (b). (d) Fluorescence image of neurons in
(b) showing GCaMP6s fluorescence. Scale bar is 20 μm. (e) Traces
showing the activity of each neuron in (b−d), displayed as the fractional
change in voltage-sensitive RhoVR 1 fluorescence vs time. (f−h)
Regions of the traces in (e) are shown on an expanded time scale to
compare the spike timing of imaged neurons.
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shifted probe, BeRST 1 (ΔF/F = 24% per 100 mV).9 Although
the excitation and emission spectra of RhoVR 1 are blue-shifted
relative to those of BeRST 1, the TMR optical profile still
provides ample spectral separation to perform two-color imaging
alongside other optical probes, such as GFP and the GCaMP
family of sensors. Using a combination of GCaMP6s and RhoVR
1, we simultaneously imaged Ca2+ transients and membrane
potential depolarizations in cultured hippocampal neurons,
establishing that RhoVR 1 and related compounds will be useful
for parsing the roles of Ca2+and Vm in living cells.
Taken together, these data demonstrate the utility of
sarcosine-substituted rhodamines dyes for voltage sensing in
living cells. The incorporation of the 2′-carboxylate simplifies the
synthetic route to long-wavelength voltage sensors by avoiding
highly polar sulfonates, which complicate purification, and
subsequent modification with sarcosine prevents the internal-
ization that plagues unfunctionalized xanthene-based voltage
indicators. Inclusion of the free carboxylate on sarcosine provides
a convenient handle for subsequent functionalization and
localization to genetically encoded protein partners or delivery
agents. Furthermore, expansion of PeT-based voltage indicators
to include rhodamines offers a new optical channel for use in
voltage sensing and demonstrates the versatility and generality of
a PeT-based approach to voltage sensing. We were pleasantly
surprised to find that the 5′-substituted rhodamines showed
greater voltage sensitivity than the 4′-substituted dye, which has
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DOI: 10.1021/jacs.6b05672
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