Phosphonofluoresceins: Synthesis, Spectroscopy, and Applications

Article abstract of DOI:10.1021/jacs.1c01139

Xanthene fluorophores, like fluorescein, have been versatile molecules across diverse fields of chemistry and life sciences. Despite the ubiquity of 3-carboxy and 3-sulfonofluorescein for the last 150 years, to date, no reports of 3-phosphonofluorescein exist. Here, we report the synthesis, spectroscopic characterization, and applications of 3-phosphonofluoresceins. The absorption and emission of 3-phosphonofluoresceins remain relatively unaltered from the parent 3-carboxyfluorescein. 3-Phosphonofluoresceins show enhanced water solubility compared to 3-carboxyfluorescein and persist in an open, visible light-absorbing state even at low pH and in low dielectric media while 3-carboxyfluoresceins tend to lactonize. In contrast, the spirocyclization tendency of 3-phosphonofluoresceins can be modulated by esterification of the phosphonic acid. The bis-acetoxymethyl ester of 3-phosphonofluorescein readily enters living cells, showing excellent accumulation (>6x) and retention (>11x), resulting in a nearly 70-fold improvement in cellular brightness compared to 3-carboxyfluorescein. In a complementary fashion, the free acid form of 3-phosphonofluorescein does not cross cellular membranes, making it ideally suited for incorporation into a voltage-sensing scaffold. We develop a new synthetic route to functionalized 3-phosphonofluoresceins to enable the synthesis of phosphono-voltage sensitive fluorophores, or phosVF2.1.Cl. Phosphono-VF2.1.Cl shows excellent membrane localization, cellular brightness, and voltage sensitivity (26% ΔF/F per 100 mV), rivaling that of sulfono-based VF dyes. In summary, we develop the first synthesis of 3-phosphonofluoresceins, characterize the spectroscopic properties of this new class of xanthene dyes, and utilize these insights to show the utility of 3-phosphonofluoresceins in intracellular imaging and membrane potential sensing.

Full text of DOI:10.1021/jacs.1c01139

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Phosphonofluoresceins: Synthesis, Spectroscopy, and Applications
Joshua L. Turnbull, Brittany R. Benlian, Ryan P. Golden, and Evan W. Miller*
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ABSTRACT: Xanthene fluorophores, like fluorescein, have been
versatile molecules across diverse fields of chemistry and life
sciences. Despite the ubiquity of 3-carboxy and 3-sulfonofluor-
escein for the last 150 years, to date, no reports of 3-
phosphonofluorescein exist. Here, we report the synthesis,
spectroscopic characterization, and applications of 3-phosphono-
fluoresceins. The absorption and emission of 3-phosphonofluor-
esceins remain relatively unaltered from the parent 3-carboxy-
fluorescein. 3-Phosphonofluoresceins show enhanced water sol-
ubility compared to 3-carboxyfluorescein and persist in an open,
visible light-absorbing state even at low pH and in low dielectric
media while 3-carboxyfluoresceins tend to lactonize. In contrast,
the spirocyclization tendency of 3-phosphonofluoresceins can be
modulated by esterification of the phosphonic acid. The bis-acetoxymethyl ester of 3-phosphonofluorescein readily enters living cells,
showing excellent accumulation (>6x) and retention (>11x), resulting in a nearly 70-fold improvement in cellular brightness
compared to 3-carboxyfluorescein. In a complementary fashion, the free acid form of 3-phosphonofluorescein does not cross cellular
membranes, making it ideally suited for incorporation into a voltage-sensing scaffold. We develop a new synthetic route to
functionalized 3-phosphonofluoresceins to enable the synthesis of phosphono-voltage sensitive fluorophores, or phosVF2.1.Cl.
Phosphono-VF2.1.Cl shows excellent membrane localization, cellular brightness, and voltage sensitivity (26% ΔF/F per 100 mV),
rivaling that of sulfono-based VF dyes. In summary, we develop the first synthesis of 3-phosphonofluoresceins, characterize the
spectroscopic properties of this new class of xanthene dyes, and utilize these insights to show the utility of 3-phosphonofluoresceins
in intracellular imaging and membrane potential sensing.
available with simple modifications to the terminal and
bridgehead atoms of the xanthene fluorophore core. Replace-
ment of the terminal oxygen atoms with substituted nitrogen
results in another class of xanthene fluorophores: rhodamines.
Spectral properties of rhodamines can be fine-tuned by varying
the amine substitution,16 for example, four-membered
azetidines substantially improve brightness and photostabil-
ity.17 Replacement of the 10′ oxygen atom with carbon,18,19
silicon,20−23 phosphorus,24−26 or sulfur27 red-shifts excitation
and emission wavelengths, avoiding issues related to photo-
toxicity andautofluorescence and improving tissue penetration
for in vivo imaging.
INTRODUCTION
■
Since the original synthesis of fluorescein in 1871,1 this
versatile molecule remains one of the most widely utilized
fluorophores in biology, medicine, and chemical biology.
Fluorescein labeled antibodies were the first immunofluor-
escent stains,2 and reagents such as fluorescein isothiocyanate,
or FITC, remain ubiquitous for the preparation of fluorescent
biological conjugates.3 One of the few fluorophores FDA-
approved for use in humans, sodium fluorescein, has found
clinical uses in ophthalmology4,5 and more recently, neuro-
surgery.6 A staple in the chemical biology community,
countless fluorescein-derived probes and sensors have been
reported over the years.7−9 Design of such probes often take
advantage of the ability to modulate fluorescence by
substitution at the phenolic oxygen, control of spirocyclization,
or changes in the rate of photoinduced electron transfer
(PeT).8,10−12 For example, appending BAPTA, a calcium
chelator, to fluoresceins is a common strategy that makes use
of PeT to modulate fluorescence and has enabled probing of
intracellular calcium dynamics in neurons and brain tissue with
high sensitivity and spatiotemporal resolution.13−15
By comparison, substitution of the pendant carboxylate of
fluoresceins has remained relatively underexplored. Substitu-
tion at the 3 position restricts free rotation of the meso ring,
improving the fluorescence quantum yield.10 The chemical
Received: January 29, 2021
Published: April 2, 2021
The wide utility of fluorescein and related xanthene dyes is
due, in part, to their high brightness and wide range of colors
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a
identity of the substitution at the 3 position can have profound
effects on the properties of fluoresceins and related xanthenes.
Fluoresceins spirocyclize at low pH or in low dielectric
mediums to a nonfluorescent lactone, and this is often the basis
for design of fluorogenic probes.12 Substitution of the 3-
carboxylate for hydroxymethyl, aminomethyl, or mercapto-
methyl nucleophiles tunes the spirocyclization equilibrium of
various rhodamines.28−30 This led to the development of
spontaneously blinking fluorophores for single molecule
localization microscopy.28 Sulfono-fluoresceins (Scheme 1),
Scheme 2. Synthesis of Phosphonofluoresceins
a
For X = H the following conditions were used: H3PO4 (85%), 130
°C, 2.5 days.
Scheme 1. Unique Properties of Phosphonofluorescein
remove the generated ethyl bromide. This approach enabled
facile access to up to 10 g quantities of 2 in one step from
simple starting reagents. Hydrolysis to phosphonic acid 3 was
performed in concentrated HCl under refluxing conditions to
give the free acid in 87% yield. While the diethyl ester
precursor (2) could be carried directly into the condensation
with resorcinols to make 3-phosphonofluoresceins (with
hydrolysis of diethylphosphonate occurring in situ), we
observed improved yields and simpler purification when
using free phosphonic acid (3). Condensation of 3 with the
corresponding resorcinol in neat methanesulfonic acid gave
dihalogenated phosphonofluoresceins 4 and 5. Nonhalogen-
ated phosphonofluorescein (6) could also be prepared via this
route, but cleaner conversion was observed with 85%
phosphoric acid at the expense of a longer reaction time.
The purification of crude phosphonofluoresceins was
difficult, owing to the high polarity of the water-soluble dyes.
Careful trituration of the crude reaction isolate with mixtures
of cold methanol/isopropanol or methanol/acetonitrile
enabled isolation of pure dyes (Supporting Information
(SI)). This approach negated the need for costly and low
throughput reverse phase chromatographic techniques (such as
HPLC) but came at the cost of reduced isolated yields, ranging
from 6 to 17%. It is notable that this synthetic route provides
similar yields to those observed with the analogous sulfonated
derivatives (SI) and low yields/nontrivial purifications are
commonly encountered with such reactions.
Spectroscopic Characterization of 3-Phosphonofluor-
escein. To examine the influence of phosphonate substitution
on fluorescein, we evaluated the spectroscopic properties of 3-
phosphonofluoresceins (4−6) compared to those of traditional
3-carboxy- and 3-sulfono- fluoresceins. In 0.1 M NaOH(aq)
2′,7′-dichloro-3-phosphonofluorescein (pF.Cl, 4) absorbs at
498 nm and emits at 517 nm (Table 1, Figure S1a),
demonstrating a very slight hypsochromic shift relative to
2′,7′-dichloro-3-carboxyfluorescein (502 nm/523 nm, Table 1,
Figure S1d).40 The sulfono derivative, 2′,7′-dichloro-3-
sulfonofluorescein (sF.Cl), however, has a slight bathochromic
shift and absorbs at 509 nm and emits at 526 nm (Table 1,
Figure S1e). These small shifts are likely due to slight inductive
differences from the meso ring. Since the meso ring and the
xanthene are orthogonal to one another, there should be
minimal ground state interactions between the two. As a result,
the Stokes shift, extinction coefficients, and quantum yields
show only minor variability across the series (Table 1). The
photostability of 4 is similar to the 3-carboxy and 3-sulfono
analogs (Figure S1g−i). One large change is the improved
water solubility; pF.Cl (4) is almost twice as soluble as 2′,7′-
dichloro-3-carboxyfluorescein and slightly less soluble than
sF.Cl (Table 1).
bearing a sulfonate at the 3 position, do not spirocyclize and
have improved water solubility: properties that have been
instrumental in the design of voltage sensitive fluorophores
(VF dyes).31−34
While fluoresceins with acidic substituents at the 3 position
have been ubiquitous in the literature for some time (3-
sulfonofluorescein was reported a mere 13 years35 after 3-
carboxyfluorescein1), we were somewhat surprised to find no
report describing 3-phosophono-fluorescein, which possesses
the biologically relevant acidic phosphonate group (Scheme 1).
We were curious to explore this unreported class of
fluoresceins and envisioned that 3-phosphonofluoresceins
might have unique properties compared to 3-carboxyfluor-
esceins. For example, the two acidic sites on phosphonates, the
first pKa above sulfonic acid but below carboxylic acid, and a
second pKa near physiological pH,36 might provide oppor-
tunities for functionalization. The persistent ionization of
phosphonates at physiological pH might also enhance water
solubility compared to 3-carboxyfluoresceins. Here, we report
the first synthesis of 3-phosphonofluoresceins, characterize the
spectral properties of this new class of fluorophore, and exploit
the properties of 3-phosphonofluoresceins for two orthogonal
live-cell imaging applications.
RESULTS AND DISCUSSION
■
Synthesis of 3-Phosphonofluoresceins. Substituting the
carboxylate functionality of fluoresceins poses an inherent
synthetic challenge. Attempts to displace fluoride from 2-
fluorobenzaldehyde with triethylphosphite, in a fashion
analogous to the synthesis of 3-sulfonofluorescein precur-
sors,32,37 resulted in no reaction. However, Ni-mediated
catalysis38,39 enabled access to arylphosphonic ester 2 from
2-bromobenzaldehyde 1 in 65% yield (Scheme 2). A slight
excess of triethyl phosphite is required due to undesired
oxidation of the phosphite to the corresponding phosphate at
elevated temperature. A weak nitrogen flow is also prudent to
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Table 1. Properties of Fluoresceins
a
b
b
b,
c
b
d
Dye
3-substituent
X
λmax / nm
λem / nm
ε / M‑1cm‑1
Φfl
Solubility
fluorescein
2′,7′-dichloro-3-carboxy-fluorescein
2′,7′-dichloro-3-sulfonofluorescein (sF.Cl)
2′,7′-dichloro-3-phosphonofluorescein (pF.Cl, 4)
2′,7′-difluoro-3-phosphonofluorescein (pF.F, 5)
3-phosphonofluorescein (pF.H, 6)
-CO2H
-CO2H
-SO3H
-PO3H2
-PO3H2
-H
-Cl
-Cl
-Cl
-F
491
502
509
498
488
487
514
523
526
517
508
508
88000
86000
87000
88000
84000
75000
0.92
0.94
0.89
0.90
0.94
0.99
1
3.1
1.8
-PO3H2
-H
a
b
c
d
See Scheme 2. In 0.1 M NaOH(aq). At max absorption. Measured in PBS relative to 2′,7′-dichlorofluorescein
Figure 1. Spectroscopic characterization of the pH dependence of dichlorofluoresceins. Normalized absorbance spectra and corresponding plots of
normalized absorbance vs pH at λmax for carboxy- (a, b), sulfono- (d, e) and phosphono- (g, h) dichlorofluoresceins. Spectra were recorded in 10
mM buffered solutions (see SI) containing 150 mM NaCl from pH 2.4 (red) to 9.8 (magenta), and intermediate values of 3.1, 3.8, 4.3, 4.8, 5.3, 5.5,
6.1, 6.6, 7.1, 7.5, 8.2, 8.7, and 9.2 at a dye concentration of 2 μM. Titration curves fit to sigmoidal dose response curves (solid black) enabled pKa
determination (dashed red). Error bars represent SEM for n = 3 independent determinations and if not visible are smaller than the marker.
Summary of pH equilibria with determined pKa values for carboxy- (c), sulfono- (f), and phosphono- (i) dichlorofluoresceins.
pH Titration of 3-Phosphonofluoresceins. Fluorescein
can exist in cationic, neutral, anionic, or dianionic forms,
making absorption and fluorescence of fluoresceins strongly
pH dependent.41,42 Halogenated (2′,7′- dichloro or difluoro)
fluoresceins have lower phenolic pKa values than the
corresponding unhalogenated fluorescein, making dichloro-
and difluoro- fluoresceins less sensitive to biologically relevant
pH fluctuations.40,43 In order to assess the effect of meso
substitution on the pH sensitivity, we titrated 3-carboxy, 3-
sulfono, and 3-phosphono- dichlorofluoresceins from pH 2.3
to 9.8 (Figure 1a,d,g). Transition to the dianion can be
monitored by measuring the increase in absorption at λmax with
respect to increasing pH (Figure 1b,e,h). The phenolic pKa of
all three dichlorofluoresceins is 4.5, suggesting substitution at
the 3 position has little effect on formation of the dianion
(Figure 1c,f,i).
The pKa values for nonhalogenated, 3-phosphonofluorescein
(6, pKa = 6.4) and fluorinated 3-phosphonofluorescein (5, pKa
= 4.8) also closely match the pKa value of the analogous 3-
carboxy analog (Figures S2 and S3).43 Unique to 3-
phosphonofluoresceins, after deprotonation to the dianion, a
5 nm hypsochromic shift is observed as pH continues to
increase (Figure 1g, Figure S2a,c). This likely results from
formation of a trianion (Figure 1i) due to the extra acidic site
on the phosphonate, and quantification of this shift with 4
(pF.Cl) reveals a pKa of 7.8−in the typical range of acidities for
aryl phosphonic acids.34
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Cyclization Tendency of 3-Phosphonofluoresceins. In
the neutral form, carboxy fluoresceins spirocyclize to a
colorless, nonfluorescent lactone (Figure 1c) whereas 3-
sulfono-fluoresceins do not (Figure 1d−f). Both sulfono- and
phosphono-dichlorofluoresceins have a clear isosbestic point
between pH 2.3 and 6.8 (Figure 1d,g), resulting from the
interconversion of the anionic quinoid and dianion. The same
isosbestic point is not observed with 3-carboxy dichlorofluor-
escein, as absorption continuously decreases with pH due to
spirocyclization to the neutral lactone at low pH (Figure 1a).
The observation that carboxy-fluoresceins spirocyclize whereas
sulfono- and phosphono-fluoresceins do not can be rational-
ized by the difference in pKa values. The 3-substituents on the
latter two are strongly acidic, with pKa values lower than
protonation of the xanthene to the cationic form (Figure S3c),
and thus the neutral form favors an open zwitterion. The
carboxylate, however, has a higher pKa, so a significant portion
of the neutral form exists as a closed lactone.41 Fluorescein can
also spirocyclize in low dielectric media and thus does not
absorb light in the visible region (Figure S4a,b, red trace). In
low dielectric media, both 3-phosphono- (Figure S4d−f) and
3-sulfono-fluoresceins (Figure S4c) possess absorbance profiles
similar to the protonated xanthene in the open form. Moving
from high to low dielectric, we observe an apparent increase in
the pKa of the phenolic oxygen but no tendency to spirocyclize
into the colorless lactone.
Cell Permeability and Retention. Acetoxy methyl (AM)
ethers are commonly employed to deliver anionic fluorophores
and small molecules into cells.44 The high pKa (∼13) of the
formaldehyde hydrate leaving group provides chemical and
hydrolytic stability, therefore AM ether hydrolysis relies on
endogenous cellular esterases.45 In the context of fluorescein,
this uncaging process is fluorogenic; hydrolysis of the first AM
ether releases the dye from its closed, colorless lactone form,
and hydrolysis of the second AM ether provides the negatively
charged phenolate responsible for strong fluorescence. This
fluorogenicity has resulted in the widespread use of fluorescein
AMs as cell viability reagents and has enabled the intracellular
delivery of numerous fluorescein-derived probes.45,46 Despite
their widespread use, carboxy fluoresceins are rapidly effluxed
out of cells, hindering the long-term imaging of live cells.47−49
We considered whether the improved water solubility and
lower pKa of 3-phosphonofluoresceins would improve intra-
cellular retention, since this charged group must be masked in
some way in order to cross the lipid bilayer. While 3-sulfono-
fluoresceins also possess high water solubility and low pKa
values, sulfonic esters are potent electrophiles, making them
inherently unstable and difficult to chemically mask for
intracellular delivery.50 Phosphonates are commonly masked
with biologically labile protecting groups, such as AM esters, to
deliver phosphonate containing molecules into cells.51−53 This
approach is commonly incorporated into the design of
nucleotide (or nucleoside phosphate) prodrugs.54,55
Scheme 3. Synthesis of Phosphonofluorescein Acetoxy
Methyl Esters and Ethers
nm and emission profiles characteristic of a singly alkylated
xanthene ether (Figure S5a,b). No hydrolysis was observed
after several hours of incubation in HBSS at 37 °C (Figure S6).
Incubation in strong base or in the presence of porcine liver
esterase (PLE), however, resulted in a ∼30x fold increase in
fluorescence (Figure S6 and S5a,b), demonstrating effective
release of the free dye and behaving in the same way as the
analogous carboxy−fluorescein AM (Figure S5i,j). While the
closed, 3-carboxy fluorescein AMs absorb no visible light in
HBSS (Figures S5e−h), 4-AM closed, with an absorption
maximum at 443 nm, displays a spectrum (Figure S5c,d)
similar to that of the cationic species observed at low pH
(Figure S2b), suggesting the 4-AM closed is in an open,
zwitterionic form. Subsequent incubation of 4-AM closed at
37 °C in HBSS for 2 h results in a loss of any visible absorption
(Figure S5d) along with a decrease in m/z corresponding to
loss of a single AM group (Figure S7). This suggests that in
this form, the phosphono ester of 4-AM closed is prone to
facile hydrolysis (Figure S8). The 31P NMR chemical shift of
4-AM closed is significantly downfield (30.3 ppm, Spectrum
S23) relative to 4-AM open (14.3 ppm, Spectrum S20), likely
a result of increased electrophilicity, and thus confers
decreased hydrolytic stability of 4-AM closed.
Unsurprisingly, 4, which contains no AM esters, is cell
impermeable and no uptake into HEK293T (HEK) cells was
observed by fluorescence microscopy after 20 min of
incubation (Figure 2a,d). The same is true for 4-AM closed,
suggesting the phosphonoester is rapidly hydrolyzed and the
resulting negative charge precludes the ability to diffuse across
the cell membrane (Figure 2b,e, Figure S8). The strong cellular
fluorescence from cells treated with 4-AM open indicates a
high degree of cell permeability followed by fluorogenic
uncaging (Figure 2c,f). Compared to 3-carboxyfluorescein AM
derivatives, 4-AM open has between 3.5 to 6−fold increase in
cellular fluorescence intensity compared to FCl and F-AMs
(both open and closed) at 500 nM in HEK cells (Figure 3a,d),
enabling the phosphono derivative to be used at much lower
concentrations. In fact, we saw reasonable fluorescence
intensity at 100 nM concentrations, whereas carboxy
fluoresceins are often loaded in the μM range.43
Treatment of 3-carboxy−fluoresceins with bromomethyl
acetate in the presence of Ag(I) in MeCN results
predominantly in the closed, nonfluorescent, lactonized form
with two phenolic AM ethers, although a small amount of the
open AM ether/ester can be isolated.45 Owing to the tendency
to not spirocyclize, the opposite selectivity was observed with
pF.Cl (4), where the open form (4-AM open) was the major
product, and the cyclized form (4-AM closed) was the minor
product (Scheme 3). In buffered saline (Hank’s balanced salt
solution, HBSS), 4-AM open has absorption centered at 467
We postulated the increased cellular fluorescence intensity
of 3-phosphonofluoresceins may result from improved cellular
retention compared to 3-carboxyfluoresceins. Serial washing of
cells loaded with FCl-AM (2′,7′-dichloro-3-carboxyfluores-
cein) results in a dramatic loss of fluorescence, and after 3
washes, cellular fluorescence levels are 8% of original
intensities (Figure 3b,c). Conversely, with 4-AM open, cellular
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Figure 2. Cell permeability of phosphonofluoresceins. Widefield
fluorescence (a−c) and DIC (d−f) images of HEK cells stained with
500 nM 4 (a,d), 4-AM closed (b,e), and 4-AM open (c,f) for 20 min
at 37 °C. Cells were washed once with HBSS prior to imaging. Scale
bar is 50 μM.
fluorescence intensity levels remain at 89% of the original
values, even after 3 washes (Figure 3b,c), thereby demonstrat-
ing an almost 70 fold increase in fluorescence intensity
compared to cells stained with FCl-AM. The enhanced cellular
retention of 3-phosphonofluoresceins expands the scope of use
for prolonged imaging in living cells.
We then incubated HEK cells labeled with FCl-AM closed,
4-AM open, and calcein AM (a multiply carboxylated
fluorescein derivative with excellent cellular retention)49,56 in
DMEM for up to 60 min prior to imaging. Carboxy fluorescein
was rapidly effluxed, and no fluorescence was seen after the 0
min time point, whereas phosophonofluorescein appeared to
efflux at a much slower rate, and almost half the intracellular
fluorescence was retained after an hour (Figure S10b). Anionic
transporters such as multidrug resistant-associated proteins
(MRP) have been implicated in the efflux of fluorescein from
cells, and MRP inhibitors such as MK-571 improve retention
and accumulation of dyes.57−59 Incubation of stained HEK
cells in DMEM containing MK-571 (50 μM) reduces the rate
of dichlorofluorescein efflux after 15 min (Figure S10d);
however, almost all cellular fluorescence was still lost after 60
min (Figure S10c). On the contrary, phosphonofluorescein
efflux was almost completely inhibited by MK-571: no change
in cellular fluorescence intensity was observed after 60 min
incubation in the presence of MK-571 (Figure S10). One
could imagine circumstances where moderate efflux of a
fluorophore could prove beneficial, such as improving contrast
when labeling intracellular structures. Phosphonofluorescein
efflux can be readily inhibited with the addition of MK-571 for
situations where efflux would be undesirable. Importantly, 3-
phosphonofluorescein 4 shows very low cellular toxicity
comparable that of calcein-AM,60 a reagent used as a viability
marker, when assayed using an MTT reduction assay (Figure
S11).61
Figure 3. Cellular retention of fluoresceins. (a) Comparison of the
relative brightness of fluorescein AMs in HEK cells. (b) normalized
intensity and (c) fluorescence images of 4-AM Open and FCl-AM
Closed loaded onto HEK cells at 500 nM in HBSS for 20 min. Cells
were sequentially washed with fresh HBSS and changes in
fluorescence intensity were measured by means of fluorescence
microscopy. All dyes were loaded at 500 nM in HBSS for 20 min at 37
°C. Error bars in (a) and (b) are SEM for n = 4 coverslips. Scale bar
is 50 μm. (d) Chemical structures of carboxyfluorescein AMs.
uration, which employs voltage-sensitive photoinduced elec-
tron transfer (PeT) from a lipophilic, electron-rich, aniline-
containing phenylene vinylene molecular wire into a
fluorophore.34 These VoltageFluors have primarily relied on
3-sulfonofluoresceins to prevent dye internalization and ensure
proper orientation within the plasma membrane.32,62
The voltage-sensing molecular wire domains of VFs are
typically installed via Heck coupling to fluoresceins, making
use of a 5- or 6-halogen. Since we also require a halogen handle
to install the phosphonate, we first protected 2,4- and 2,5-
dibromo benzaldehydes (7 and 8) with ethylene glycol and
used the corresponding acetals (9 and 10) to direct lithium-
halogen exchange with n-butyllithium exclusively to the ortho
position (Scheme 4). Slow addition of diethyl chlorophosphate
and subsequent hydrolysis in concentrated acid yielded 4- and
5-bromo phosphono-benzaldehdyes (13 and 14) which were
condensed with 4-chlororesocinol to the corresponding 2′,7′-
dichloro-3-phosphonofluoresceins (15 and 16). The bromine
Voltage Sensing with Phosphonofluoresceins. The
cellular uptake of 3-phosphonofluoresceins, like 4, can be
readily controlled by esterification of the 3-phosphonate, with
the free phosphonic acid showing excellent exclusion from
cells. Because of the tunable cellular permeability profile, high
water solubility, and lack of spirocyclization, we hypothesized
that 3-phosphonofluoresceins could be readily incorporated
into a voltage-sensitive fluorophore (VoltageFluor) config-
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Scheme 4. Synthesis of Phosphono-VoltageFluors
functional group enabled Pd-catalyzed Heck coupling to a
styrene molecular wire to produce para and iso phosVFs (17
and 18); however, only limited conversion occurred, and
significant amounts of dehalogenated and unreacted dye were
observed. We initially suspected this could be a result of the
presence of the phosphonic acid functional group, but Pd-
catalyzed cross coupling of phosphonate-containing precursor
13 and the same styrene resulted in a much greater 73% yield
(SI compound 19). The desired phosVFs were, however,
readily purified by reverse phase silica chromatography to give
the desired products in 25% yield.
Both para and iso phosVFs possess absorbance and emission
spectra nearly identical to those of pF.Cl (4), with the addition
of an absorbance band at around 370 nm, corresponding to the
aniline-containing molecular wire (Figure 4a, Figure S12).
Both phosVF dyes localize to cellular membranes of HEK293T
cells (Figure 4b, Figure S13) and are voltage sensitive (Figure
4c,d, Figure S14). Patch clamp electrophysiology coupled with
fluorescence microscopy reveal that para phosVF2.1Cl has a
voltage sensitivity of 26% ΔF/F per 100 mV (Figure 4c,d),
roughly the same sensitivity as the sulfonated analog, VF2.1Cl
(24%).31,63 The same trend was seen for iso phosVF2.1Cl and
iso VF2.1Cl with ΔF/F per 100 mV values of 11 and 9%
respectively (Figure S14). No significant differences were
observed in the relative brightness or signal-to-noise ratio when
both para probes were loaded at 250 nM (Figure S13),
suggesting that switching from a sulfonate to a phosphonate
has a negligible effect on the orientation or ability to load into
cellular membranes.
Figure 4. Cellular and in vitro characterization of phosphonated
VoltageFluors. (a) Normalized absorbance (solid line) and
fluorescence emission (dashed line) spectra of para phosVF2.1Cl
(17) in 0.1 M NaOH(aq). (b) HEK cells stained with 250 nM para
phosVF2.1Cl (17). Scale bar is 40 μm. (c) Plot of the fractional
change in fluorescence of para phosVF2.1Cl (17) vs time for 100 ms
hyper- and depolarizing steps ( 100 mV in 20 mV increments) for
single HEK cells under whole-cell voltage-clamp mode. (d) plot of
ΔF/F vs final membrane potential, revealing a voltage sensitivity of
approximately 26% per 100 mV. Error bars are SEM for n = 17
cells. If not visible, error bars are smaller than the marker.
CONCLUSIONS
■
In summary, we report the first synthesis of 3-phosphono-
fluoresceins, characterize the spectroscopic properties of this
new class of fluorophore, and use 3-phosphonofluorescein in
two orthogonal live-cell imaging applications. The new
synthetic route to 3-phosphonofluorescein provides access to
unhalogenated- and 2′,7′-dihalogen-3-phosphonofluoresceins.
2′,7′-Dichloro-3-phosphonofluorescein is more water-soluble
than its 3-carboxy analog. In addition, 3-phosphonofluor-
esceins, unlike 3-carboxyfluoresceins, do not spirocyclize, thus
rendering them cell-impermeant. Esterification of the phos-
phonic acid allows delivery of 3-phosphonofluoresceins to
living cells, where they show an almost 70-fold increase in
cellular brightness over carboxy fluorescein as a result of
improved accumulation and retention. Finally, we adapt the
synthesis of 3-phosphonofluoresceins to include 5- or 6-bromo
derivatives en route to voltage-sensitive 3-phosphonofluor-
esceins. These new phosVF dyes show excellent membrane
staining and have voltage sensitivity rivaling the best
fluorescein-based VF dyes.63 We imagine that 3-phosphono-
fluoresceins will be of utility in long-term imaging applications
that rely on a high degree of cellular retention and in voltage
imaging applications. We envision that 3-phosphono sub-
stitution will yield additional opportunities in the context of
xanthene dyes like rhodamines, and studies toward this end are
underway in our lab.
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Article
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c01139.
■
sı
Experimental section, supporting figures, spectra,
procedures, and analysis (PDF)
AUTHOR INFORMATION
Corresponding Author
Evan W. Miller − Departments of Chemistry, Molecular &
Cell Biology, and Helen Wills Neuroscience Institute,
University of California, Berkeley, California 94720, United
States; orcid.org/0000-0002-6556-7679;
Email: evanwmiller@berkeley.edu
■
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cytosolic calcium based on rhodamine and fluorescein chromophores.
J. Biol. Chem. 1989, 264 (14), 8171−8.
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Authors
Joshua L. Turnbull − Departments of Chemistry, University of
California, Berkeley, California 94720, United States
Brittany R. Benlian − Molecular & Cell Biology, University of
California, Berkeley, California 94720, United States
Ryan P. Golden − Departments of Chemistry, University of
California, Berkeley, California 94720, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c01139
Notes
The authors declare no competing financial interest.
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D. Virginia Orange: A Versatile, Red-Shifted Fluorescein Scaffold for
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ACKNOWLEDGMENTS
■
E.W.M. acknowledges generous support from the University of
California, Berkeley, Klingenstein-Simons Fellowship in the
Neuroscience, Camille Dreyfus Teacher-Scholar Fellowship,
and the National Institute for General Medical Sciences (R35
GM119855). B.R.B. was supported, in part, by a training grant
from NIGMS (T32GM666098).
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