Heavy Pnictogenium Cations as Transmembrane Anion Transporters in Vesicles and Erythrocytes

Article abstract of DOI:10.1016/j.chempr.2019.06.013

Our work on the complexation of fluoride anions using group 15 Lewis acids has led us to investigate the use of these main-group compounds as anion transporters. In this paper, we report on the anion-transport properties of tetraarylstibonium and tetraary

Full text of DOI:10.1016/j.chempr.2019.06.013

Article
Heavy Pnictogenium Cations as
Transmembrane Anion Transporters in
Vesicles and Erythrocytes
Gyeongjin Park, Dakota J.
Brock, Jean-Philippe Pellois,
Franc¸ois P. Gabbaı
¨
pellois@tamu.edu (J.-P.P.)
francois@tamu.edu (F.P.G.)
HIGHLIGHTS
Lewis acidic tetraarylstibonium
and tetraarylbismuthonium
cations as anion transporters
Transport of anions, including
fluoride, across artificial
membranes
Transporter-facilitated hemolysis
of erythrocytes in the presence of
fluoride anions
This work introduces a new approach to transmembrane anion transport based on
lipophilic and Lewis acidic tetraarylstibonium and tetraarylbismuthonium cations.
The stibonium cations are particularly appealing given that they readily transport
hydroxide, fluoride, and chloride anions in synthetic vesicles. When combined with
human erythrocytes, the most active stibonium cation uncovered in this study
quickly localizes in the cell membranes and induces hemolysis when fluoride is
present. This effect is assigned to the transporter-facilitated influx of toxic fluoride
anions.
Park et al., Chem 5, 1–13
August 8, 2019 ª 2019 Elsevier Inc.
https://doi.org/10.1016/j.chempr.2019.06.013

Please cite this article in press as: Park et al., Heavy Pnictogenium Cations as Transmembrane Anion Transporters in Vesicles and Erythrocytes,
Chem (2019), https://doi.org/10.1016/j.chempr.2019.06.013
Article
Heavy Pnictogenium Cations
as Transmembrane Anion Transporters
in Vesicles and Erythrocytes
1,3,
Gyeongjin Park,1 Dakota J. Brock,2 Jean-Philippe Pellois,1,2, and Franc¸ois P. Gabbaı
*
*
¨
SUMMARY
The Bigger Picture
The transport of anions through
human cell membranes usually
occurs through channel proteins,
which support numerous essential
processes, including synaptic
signal transmission, cell and
organelle acidification,
Our work on the complexation of fluoride anions using group 15 Lewis acids has
led us to investigate the use of these main-group compounds as anion trans-
porters. In this paper, we report on the anion-transport properties of tetraaryl-
stibonium and tetraarylbismuthonium cations of the general formula
[Ph3PnAr]+, where Pn = Sb or Bi and Ar = phenyl, naphthyl, anthryl, or pyrenyl.
Using egg yolk phosphatidylcholine (EYPC)-based large unilamellar vesicles, we
show that these main-group cations transport hydroxide, fluoride, and chloride
anions across phospholipid bilayers. A comparison of the properties of
[Ph3SbAnt]+ and [Ph3BiAnt]+ (Ant = 9-anthryl) illustrates the favorable role
played by the Lewis acidity of the central pnictogen element with respect to
the anion transport. Finally, we show that [Ph3SbAnt]+ accelerates the fluo-
ride-induced hemolysis of human red blood cells, an effect that we assign to
the transporter-facilitated influx of toxic fluoride anions.
transepithelial salt transport, cell
division, and apoptosis. With the
view to mimic and possibly one
day replace such channels for
therapeutic purposes, the design
of small molecules that facilitate
the transport of anions through
artificial phospholipid membranes
has become a topic of active
research. This article introduces
an approach to transmembrane
anion transport based on readily
accessible group 15 cations. The
results obtained in this study
demonstrate that lipophilic
tetraarylantimony and
INTRODUCTION
The toxicity of fluoride at high doses derives, in part, from its enzyme-inhibiting
properties.1,2 Logically, the adverse properties of these anions also originate from
its ability to penetrate cells, a step likely necessary before accessing vulnerable en-
zymes. Bacteria equipped with naturally occurring fluoride anion channels capable
of exporting the toxic anion display a greater resilience when exposed to high doses
of fluoride.2–5 These findings have served as an inspiration for the development of
synthetic derivatives that could be used to transport fluoride anions through cell
membranes. While Matile and co-workers obtained initial evidence that synthetic
ionophores based on naphthalene diimides are capable of transporting fluoride
ions,6,7 a team led by Gale has recently shown that strapped calix[4]pyrroles such
as A behave as selective fluoride anion transporters, a property correlated to the
favorable hydrogen bonds formed between the host and the anionic guest (Fig-
ure 1).8 There is also growing interest in the transport of hydroxide anions, a process
that could be used to defuse pH gradients across cellular membranes.9,10 Such pro-
cesses may be used to induce apoptosis, thus opening the door to applications in
cancer therapy.11–15
tetraarylbismuth cations are
particularly well suited for the
transmembrane transport of hard
anions, such as the fluoride and
hydroxide anions.
While the use of organic receptors dominates the domain of anion transport, a series
of recent reports indicate that Lewis acidic main-group compounds may constitute
promising platforms, as in the case of the pnictogen-, chalcogen-, and halogen-
bond-donor derivatives B,16 C,16 D,17 and E,18 which transport chloride ions through
artificial lipid bilayers (Figure 1). These precedents, as well as the knowledge we have
derived from our work on main-group Lewis acids as anion sensors,19–22 led us to
Chem 5, 1–13, August 8, 2019 ª 2019 Elsevier Inc.
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Chem (2019), https://doi.org/10.1016/j.chempr.2019.06.013
A
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Figure 1. Selected Examples of Relevant Transmembrane Anion Transporters
speculate that Lewis acidic pentavalent pnictogen derivatives may be potent fluo-
ride or hydroxide ion transporters. While contemplating this idea, it occurred to
us that pnictogenium cations could be appealing targets because of the known
cell-penetrating properties of their phosphorus congeners. Indeed, lipophilic phos-
phonium cations are known to quickly penetrate cell membranes, ultimately locating
in the mitochondrion as a result of a negative electrostatic gradient (Figure 2).23–25
Merging the concepts of cell penetration with those of Lewis acidity at the group 15
center, we have now decided to test whether pnictogenium cations would serve as a
new type of fluoride and hydroxide anion transporters (Figure 2).
RESULTS AND DISCUSSION
Pnictogenium Cations
While the use of tetraarylphosphonium cations was initially tempting, we contended
that the lack of Lewis acidity at the group 15 center might be problematic. Since
simple stibonium or bismuthonium cations are known to react with fluoride anions
to afford covalent l5-fluoro-stiboranes19,22,26–28 or -bismuthanes,29 respectively,
we decided to focus on the use of these heavy pnictogen cations as potential trans-
porters. The precedent that bismuth enjoys in medicine30 and the ease of prepara-
tion of tetraarylbismuthonium cations served as an additional incentive for studying
bismuth-based systems.31,32 Bearing in mind that lipophilicity might play a signifi-
cant role in the transport properties of these cations, we targeted cations [1]+–[8]+
(Figure 3) which could be readily prepared using existing protocols.22,31 While
[6]BF4 and [7]BF4 are new, we note that all other pnictogenium cations have been
previously described.19,22,33,34 All new salts have been fully characterized, and the
solid-state structure of [7]BF4 has been crystallographically determined (see Figures
S1–S10).
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1Department of Chemistry, Texas A&M
University, College Station, TX 77843, USA
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2Department of Biochemistry and Biophysics,
Texas A&M University, College Station, TX 77843,
USA
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*Correspondence: pellois@tamu.edu (J.-P.P.),
francois@tamu.edu (F.P.G.)
Figure 2. Cell-Penetrating and Anion-Binding Properties of Lipohilic Pnictogenium Cations
Left: Illustration of the ability of phosphonium cations to cross cell walls and target mitochondria.
Right: Graphical summary of the hypothesis tested in this study.
https://doi.org/10.1016/j.chempr.2019.06.013
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3K
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Figure 3. Structures of the Stibonium and Bismuthonium Cations Investigated as Anion
Transporters
Next, we measured the fluoride anion affinity of these cations in DMSO/H2O 98:2
(v/v) using KF as a fluoride source and UV-visible spectroscopy as a monitoring
method (Scheme 1; Figures S11–S18). All stibonium cations ([1]+–[4]+) display fluo-
ride binding constants in excess of 107 MÀ1, indicating essentially quantitative con-
version into the corresponding l5-fluoro-stiborane under these experimental condi-
tions. More progressive fluoride binding was observed with the bismuthonium
cations ([5]+–[8]+). In this case, fitting the resulting titration data to a 1:1 binding
isotherm afforded the binding constants Ka in Table 1. Because the binding con-
stants of [5]+ and [6]+ are lower than those of [7]+ and [8]+, these results suggest a
correlation between the hydrophobic character of the compounds and their fluoride
ion affinity. Another important conclusion from these titration experiments is the
higher Lewis acidity displayed by the antimony cations. While this conclusion may
appear surprising at first, we note that it is readily reproduced by fluoride anion af-
finity calculations since the computed fluoride anion affinity of [Ph4Bi]+ is 3.9 kcal/mol
lower than that of [Ph4Sb]+. We assign this difference to the more diffuse orbitals of
bismuth, a factor that would weaken the covalent component of the Bi–F bond. We
also note that relativistic effects in the sixth period lead to an anomalous increase of
the electronegativity (c = 2.01 for Bi versus 1.82 for Sb),35 thereby lowering the
Coulombic stabilization of the Bi–F bond. While the formation of fluorostiboranes
by the reaction of fluoride anions with stibonium cations is well understood, knowl-
edge about the corresponding bismuth species is more limited. To confirm that the
bismuthonium cations interact with fluoride, we conducted 19F NMR and 1H NMR
studies (Figures S20–S25). A resonance corresponding to the bismuth-bound fluo-
rine atom was observed upon addition of [6]+, [7]+, or [8]+ to solutions of tetra-n-bu-
tylammonium fluoride (TBAF) in d6-DMSO. These 19F NMR resonances appeared
between –81 and –77 ppm, a range that can be compared to the value reported
for pure Ph4BiF in CDCl3 (–84.6 ppm)29 or DMSO-d6 (–83.3 ppm, see Figure S19).
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Scheme 1. Binding Equilibrium Occurring between a Pnictogenium Cation and a Fluoride Anion
K represents the equilibrium constant.
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Table 1. Partition Coefficients, Fluoride Binding Constants, and Fluoride-Transport-Activity
Data Obtained for [1]+-[8]+
[Pn]+
[1]+
[2]+
[3]+
[4]+
[5]+e
[6]+
[7]+
[8]+
logKow
4.19
5.09
5.85
6.26
4.06
4.94
5.55
6.04
Ka (MÀ1 b
)
EC50 (mol %)c
6.91 (G0.70)
2.44 (G0.01)
0.41 (G0.05)
0.57 (G0.07)
–
nd
a
>107
1.32 (G0.18)
0.98 (G0.05)
0.88 (G0.13)
1.03 (G0.13)
–
>107
>107
>107
6.7 (G0.2) 3 103
1.7 (G0.1) 3 104
1.1 (G0.3) 3105
5.2 (G1.0) 3104
6.47 (G0.38)
1.24 (G0.03)
1.38 (G0.05)
0.99 (G0.06)
1.28 (G0.04)
1.50 (G0.08)
aCalculated octanol-water partition coefficient defined as logKow at 298 K.
bFluoride anion binding constant in DMSO/H2O 98:2 (v/v).
cEffective concentration needed to achieve 50% transport at t = 270 s (with valinomycin).
dHill coefficient derived from the fluoride efflux data (with valinomycin).
eThe low transport activity of this cation did not allow for Hill analysis at acceptable transporter concen-
trations.
Fluoride Anion Transport
To test the transport activities of [1]+–[8]+, we designed an assay in which a DMSO
solution (7 mL) of the pnictogenium cation (10 mM) was added to a solution contain-
ing fluoride-loaded large unilamellar vesicles (LUVs) prepared with egg yolk phos-
phatidylcholine (EYPC) suspended in a buffered aqueous solution (5 mL, [EYPC] =
0.7 mM) (Figure 4; experiment 1).8,17 Using a fluoride-selective electrode, we first
verified that fluoride efflux was negligible in the absence of any transporter (Fig-
ure S26). With this baseline established, we tested the effect of DMSO alone and
found that it induced a modicum of transport, as illustrated in Figure 5. Marked fluo-
ride transport was detected in the presence of stibonium cations ([1]+, [2]+, [3]+, or
[4]+), and the most hydrophobic derivatives [3]+ and [4]+ showed a higher activity.
In the case of the bismuthonium cations, the most hydrophilic cation [5]+, as well
as [6]+, showed no significant transport activity, suggesting that the higher Lewis
acidity of the antimony cations is a favorable factor (Figure 5A). The importance of
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Figure 4. Schematic Representation of Selected Anion-Transport Experiments Showing an
Idealized EYPC Unilamellar Vesicle
The vesicles were loaded with a 300 mM alkali metalfluoride solution (MF) buffered at pH 7.2 (10 mM
HEPES), and the external medium consisted of a 300 mM potassium gluconate solution also
buffered at pH 7.2 (10 mM HEPES). A fluoride selective electrode was used for measuring fluoride
efflux. EYPC concentration: 0.7 mM.
4
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A
B
Figure 5. Transporter-Facilitated Fluoride Efflux in the Absence of Valinomycin
Fluoride efflux from EYPC vesicles triggered by the addition of a DMSO solution (7 mL) containing
(A) only DMSO and [1]+–[4]+ and (B) only DMSO and [5]+–[8]+. Transporter concentration: 2 mol %
with respect to the lipid concentration. The other experimental conditions are those described for
experiment 1 in Figure 2.
hydrophobicity was also noted in the case of bismuth-based cations since the
anthracene- and pyrene-based systems [7]+ and [8]+ induced significant fluoride
efflux (Figure 5B). We also investigated the possible role played by the alkali cation
and found that using NaF, RbF, or CsF instead of KF led to the same fluoride efflux.
This result leads us to conclude that the fluoride transport activity of these pnictoge-
nium cation does not depend on the nature of the alkali cation, allowing us to rule
out metal-fluoride symport (Figure S27).
The transport activity observed in the absence of a cation transporter could be inter-
preted on the basis of two possibly complementary mechanisms. The first one would
be a simple FÀ/OHÀ antiport mediated by the pnictogenium cation, which would
lead to a drastic basification of the vesicle interior. Another relevant mechanism
that does not involve accumulation of hydroxide ions within the vesicle would rely
on the non-specific efflux of K+ and/or FÀ as a result of pnictogenium-induced mem-
brane destabilization. Independently of which mechanism is at play, we reasoned
that cation transport may be limiting anion efflux. For this reason, we became eager
to test the effect of valinomycin, a well-known cation transporter (Figure 4; experi-
ment 2).36,37 Under these conditions, we observed drastically improved fluoride
anion transport, especially in the case of the most hydrophobic pnictogenium cat-
ions [3]+, [4]+, [7]+, and [8]+ (Figures 6A, 6B, and S28). The resulting data were
modeled by the Hill equation (Figures S29–S35), leading to the parameters
compiled in Table 1.38–40 The first conclusion that can be derived from the fitted pa-
rameters is that all cations act as mobile carriers for the fluoride anion. Indeed, the
Hill coefficients (n < 2) calculated for [2]+–[4]+ were close to unity, suggesting that
each cation transports one fluoride anion.41 This conclusion is consistent with the
formation of l5-fluoro-stiboranes or -bismuthanes as the critical species involved
in fluoride shuttling.29 The larger Hill coefficient observed for some of the cations
could indicate the involvement of cooperative effects within the membrane where
two molecules of the cationic transporter could chelate or exchange the fluoride
anion.20,42 In addition, comparison of the data obtained for the antimony and
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A
B
C
Figure 6. Transporter-Facilitated Fluoride Efflux in the Presence of Valinomycin
(A and B) Fluoride efflux from EYPC vesicles (0.7 mM) in the presence of valinomycin (0.1 mol % with respect to the lipid concentration). Efflux was
triggered by the addition of a DMSO solution (7 mL) containing (A) only DMSO, [3]+, and [4]+ and (B) only DMSO, [7]+, and [8]+. Transporter
concentration: 2 mol % with respect to the lipid concentration. The other experimental conditions are those described for experiment 2 in Figure 2.
(C) Temperature-dependent transport experiments carried out with [3]+ and [7]+ and DPPC-based vesicles.
bismuth compounds points to the defining influence played by the elevated Lewis
acidity and fluoride anion affinity of the stibonium cations which display significantly
lower EC50 values. These data indicate that the tight complexation of fluoride
anions by these antimony-based systems does not impede anion catch-and-release
on either side of the membrane (Figures 6A and S28). The EC50 value of
0.41(G0.05) mol % (with respect to lipid concentration) measured for [3]+ can be
compared to the value of 0.9 mol % obtained by the Gale group for the Calix[4]pyr-
role A using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles
rather than EYPC vesicles. While the difference in the nature of the phospholipids
does not allow for a more detailed comparison, the low EC50 value obtained for
[3]+ validates our approach and shows that readily accessible main-group cations
can be potent fluoride transporters. It could be argued that the transport observed
in these experiments is the result of HF efflux coupled with transporter-facilitated hy-
droxide efflux. However, given that the fluoride concentration is 6 orders of magni-
tude higher that the hydroxide concentration, we see this scenario as highly unlikely.
To solidify our understanding of the role played by the lipophilicity of the pnictogenium
cations, we computed the n-octanol/water partition coefficient Kow, defined as Kow
=
[R4Bi+]octanol/[R4Bi+]water (see Figure S46).43–45 Within the antimony and bismuth series,
these partition coefficients increased gradually as the size of the polycyclic aromatic
substituent increased (Table 1). Interestingly, the data in Table 1 reveal that the most
lipophilic pnictogenium cations [4]+ and [8]+ are not the best transporters. We interpret
these results as indicating that the high lipophilicity of [4]+ and [8]+ may force them to
locate in the hydrophobic part of the membrane and thereby decrease access to
hydrated fluoride anions. It is also possible that the more lipophilic pyrene ring may
lower transmembrane mobility, also affecting fluoride anion transport. Such effects,
which have been discussed previously for hydrogen-bond-donor anion transporters,
indicate the subtlety that one should exert when optimizing the properties of these de-
rivatives.46 It is also possible that the more lipophilic cations can aggregate in the mem-
brane or even partly precipitate from solution, thus lowering anion transport.47–49
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To verify our interpretation that these cations behave as mobile fluoride ion carriers, we
tested the transport properties of [3]+ and [7]+ by using DPPC-based vesicles (DPPC =
1,2-dipalmitoyl-sn-glycero-3-phosphtidylcholine) (Figure 6C). This phospholipid is
known to form temperature sensitive bilayers that undergo a phase transition between
a gel and a liquid at 41ꢀC.48,49 It has been previously suggested that channel-type trans-
porters are less affected by this phase transition.50 However, in the case of [3]+ and [7]+,
we observed a drastic decrease in transport when the temperature of the assay was low-
ered from 45ꢀC to 25ꢀC, indicating that transport is significantly affected by the fluidity of
the bilayer. This observation asserts our interpretation that [3]+ and [7]+ behave as a mo-
bile fluoride ion carrier rather than as a channel-type transporter.51 Additionally, to rule
out a pnictogenium cation-induced destabilization of the membrane as a cause of
fluoride efflux, we carried out an assay based on carboxyfluorescein, a hydrophilic fluo-
rescent dye that does not cross phospholipid membranes and that experiences self-
quenchingwhen confinedtoavesicle. Thisdye isusedasamarkerofmembraneintegrity
since its release into the medium, as a result of membrane destabilization, is accompa-
nied by a marked fluorescence increase. Gratifyingly, no such effects were observed
when [3]+ or [7]+ was used as a transporter at a concentration close to its EC50 value (Fig-
ure S38). The stability of the membrane in the presence of the pnictogenium cation was
also assessedbydynamiclightscattering(DLS)studiesunderthe conditionsemployedin
the electrode assay. These studies showed that the scattering intensity and the average
size of the vesicles remained unchanged even 30 min after the addition of cation [3]+ or
[7]+ (Figure S39), providing further evidence for the stability of the membrane.
Activity toward Other Anions
As noted above, the observation that fluoride efflux occurred in the absence of
valinomycin suggests the possible involvement of a OHÀ/FÀ antiport mechanism.
Such a scenario is certainly supported in the case of the stibonium cations, which
displayed a high affinity for hydroxide anions.19,52 To add credence to this interpre-
tation, we tested the hydroxide transport properties of [1]+ by using the pH-sensitive
dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), which was loaded inside EYPC
vesicles (Figure 7A). This cation was selected because it shows good transport prop-
erties with respect to fluoride and does not absorb at 410 or 460 nm, the two wave-
lengths used to excite HPTS in ratiometric pH fluorescence measurements. Trans-
port was initiated by the addition of TBAOH (5 mM) to the vesicle solution (TBA =
tetra-n-butyl ammonium), which resulted in a base pulse of +1.36 pH unit of the buff-
ered solution (10 mM HEPES, starting pH 7.0).10 Hydroxide transport inside the
vesicle, facilitated by the membrane permeable TBA+ cation, was manifested by a
rapid change in the HPTS fluorescence profile and the normalized fractional fluores-
cence intensity If (Figure 7B). An initial slope analysis of the If versus time data
collected between 5 and 20 s showed that higher concentrations of [1]+ correlated
with an increased hydroxide influx (Figures S40–S45). This hydroxide transport
experiment supports the proposal that OHÀ/FÀ antiport is at least partly responsible
for the fluoride efflux observed in the electrode-monitored assay in the absence of a
cation transporter. Finally, we also tested the ability of [3]+ and [7]+ to transport chlo-
ride anions by using conditions analogous to those in experiment 2 (Figure 4) but
with KCl instead of KF (Figures S36 and S37). Monitoring the anion transport with
a chloride selective electrode showed that these two cations were also active in
the transport of chloride anions, as indicated by EC50 values of 0.61 (G0.04) mol
% (with respect to lipid concentration) obtained for [3]+ and 3.77 (G0.23) mol %
(with respect to lipid concentration) obtained for [7]+. We note that these EC50
values were higher than those obtained for fluoride anions, indicating that these
transporters remain selective for fluoride anions by a factor of 1.5 in the case of
[3]+ and 3.0 in the case of [7]+.8
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A
B
Figure 7. Hydroxide Transport Experiments with [1]+ as a Transporter
(A) Schematic representation of the hydroxide anion transport assay involving EYPC vesicles (EYPC
concentration: 0.1 mM). The vesicles were loaded with an HPTS (1 mM)/potassium gluconate
(100 mM) solution buffered at pH 7.0 (10 mM HEPES), and the external medium contained only
potassium gluconate and the buffer at the same concentrations and pH value. Transport was
initiated by the addition of TBAOH (5 mM). Hydroxide influx was monitored by measuring the
fluorescence intensity of the HPTS molecule (lem = 510 nm) at two excitation wavelengths
simultaneously (lex = 410 and 460 nm).
(B) The graph shows the normalized fluorescence intensity as a function of time. Transporter [1]+
was added as a DMSO solution 50 s after the base. [1]+ concentrations: 0 (pure DMSO), 0.5, 1, 2, 4,
and 6 mol % with respect to the lipid concentration.
Application to Fluoride Anion Transport in Biological Systems
With the anion-transport ability of tetraarylstibonium and tetraarylbismuthonium
established in model systems, we became eager to also assess the properties of
these new transporters in biological systems. In search of a model system, we
were drawn by a series of reports showing that fluoride induces hemolysis in mu-
rine erythrocytes as a result of oxidative stress.53–55 We hypothesized that these
toxic effects, which could be accelerated in the presence of a fluoride anion trans-
porter, could serve as a marker of activity for the cationic transporters described in
this study. To test this possibility, we suspended freshly obtained human erythro-
cytes in PBS buffer (6.8% by volume) and first tested their behavior in the presence
of 100 mM concentrations of [3]+ and [7]+. The experiment was monitored by mi-
croscopy, which revealed a different behavior for these two cations. Indeed,
whereas the erythrocytes remained visually unchanged upon addition of the anti-
mony cation [3]+, the bismuth cation [7]+ induced the rapid appearance of cell
ghosts, indicating lysis of the erythrocytes (Figure S47). This process was so fast
that no intact cells remained after 3 min. By contrast, the appearance of ghosts
was not observed with [3]+, even after 25 min (Figure 8A). The toxicity of [7]+,
which probably arises from the known oxidative properties of tetraarylbismutho-
nium cations,56,57 shows that such bismuth-based cations are not well suited for
application in biological systems. We therefore decided to focus our efforts on
[3]+, which appeared to be more innocuous. As argued above, [3]+ should be lipo-
philic, an attribute that should drive its localization in the cell membrane. We
tested this possibility by using fluorescence cell imaging and anticipated that
the anthracene fluorophore of [3]+ would provide a detectable signal. Because
of the weakly emitting nature of [3]+, we decided to carry out these experiments
by using a relatively high concentration of 100 mM. Images collected soon after
the addition showed a clear enhancement of fluorescence of the cell membranes,
suggesting that the transporter indeed localizes in the cell. Images taken 25 min
8
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Chem (2019), https://doi.org/10.1016/j.chempr.2019.06.013
A
C
B
Figure 8. Effect of [3]+ on Human Erythrocytes in the Absence and Presence of Fluoride Anions
(A and B) Bright-field and fluorescence images of a sample of human erythrocyte (0.2% by volume)
25 min after the addition of [3]OTf (100 mM). The insets show a magnified view of the cell marked by
an asterisk. The dark contrast of the cells in the bright-field image shows that the cells were intact,
and the fluorescence image shows localization of [3]+ in the membrane. The DAPI filter cube (from
Chroma Technology, lex/lem = 300–388 nm, 425–488 nm) was used to obtain the image in (B). Scale
bars, 10 mm.
(C) A hemolysis assay carried out with human erythrocytes shows the cooperative effects occurring
when [3]+ and fluoride were combined. The cells were suspended in PBS buffer (HyClone). [3]+ was
administered as the OTfÀ salt, and fluoride was administered as NaF. The rate of hemolysis is
denoted as rnX, where n = 1 or 2 and X = Sb, F, or SbF, as noted on the graph (n = 1 corresponds to
the rates observed in the 2–4 h window, and n = 2 corresponds to the rates observed in the 4–8 h
window; X = Sb stands for [3]+, X = F stands for FÀ, and X = SbF stands for [3]+ and FÀ combined).
Each experiment was carried out in triplicate, and the data points represent mean percentages. The
triplicated data showed high reproducibility such that some of the error bars were too small to
discern.
after the addition showed an even clearer contrast with a halo defining the cell
membrane of most cells (Figure 8B). These features, particularly the observation
of a halo, are characteristic of red blood cells (RBCs) stained by lipophilic fluores-
cent dyes.58,59 We confirmed the absence of autofluorescence by imaging RBCs
(0.2% by volume) in the absence of [3]+ (Figure S48).
Next, we decided to test whether transporter [3]+ would increase the sensitivity of
the RBCs of erythrocyte toward fluoride as a result of facilitated anion transport. We
investigated this possibility by carrying out a hemolysis assay, the results of which
are presented in Figure 8C. We first tested the independent toxicity of the fluoride
and [3]+ over the course of 8 h. When a 100 mM concentration of fluoride was used,
hemolysis did not appear to progress to any appreciable extent for the first 4 h of
the experiment. After this point, however, hemolysis started to increase and
reached 16.6% after 8 h. The long induction period may reflect the progressive
influx and accumulation of fluoride inside the cell. The addition of transporter
[3]+ led to a drastically different hemolysis profile. When the transporter was
used in a 5 mM concentration, the point taken at 2 and 4 h showed an increasing
extent of hemolysis. This trend became even clearer at 6 and 8 h, when hemolysis
reached 31.2% and 48%, respectively. Analysis of the rate of hemolysis showed that
the effect observed was cooperative rather than additive. For the 2–4 h window, the
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hemolysis rate measured in the presence of [3]+ and fluoride (r1
= 3.4%/h) was
SbF
Sb
F
9.7 times higher than the sum of the rates (r1 + r1 = 0.4%/h) obtained when
[3]+ (r1 = À0.1%/h) and fluoride were used alone (r1 = 0.49%/h). It follows that
[3]+ greatly shortened the toxicity induction period, which we propose resulted
from facilitated fluoride transport into the cell. Analysis of the data in the 4–8 h win-
dow of the experiment provided corroborating results. Indeed, the rate of hemoly-
sis in the presence of [3]+ and fluoride (r2SbF = 9.2%/h) was 2.8 times higher than the
Sb
F
sum of the rates (r2 + r2 = 3.3%/h) obtained when [3]+ (r2 = À0.1%/h) and
Sb
F
Sb
F
fluoride (r2 = 3.4%/h) were used alone. To a lesser degree, accelerated hemolysis
was also observed when [3]+ was used at a 1 mM concentration, as illustrated in
Figure S49.
Conclusions
This paper introduces a new approach to transmembrane anion transport based on
lipophilic and ‘‘fluoridophilic’’ pnictogenium cations. Evolved from the knowledge
that phosphonium cations possess biological membrane translocating properties
while their heavier analogs are Lewis acidic at the group 15 center, this approach
and its preliminary implementation illustrate how the structure of the ligands and
the nature of the Lewis acidic element govern the transport properties of these
main-group compounds. The stibonium cations are particularly appealing given
that they readily transport hydroxide, fluoride, and chloride anions in synthetic ves-
icles. Finally, we also investigated the behavior of [3]+ and [7]+ toward human eryth-
rocytes. While the bismuthonium cation [7]+ proved to be very toxic, the stibonium
cation [3]+ quickly localized in the cell membranes but did not induce notable he-
molysis when used in micromolar concentrations. However, when [3]+ and fluoride
were co-administered, the cells underwent accelerated hemolysis. We propose that
this phenomenon results from the transporter-facilitated influx of toxic fluoride
anions.
EXPERIMENTAL PROCEDURES
General Procedure Adopted for the Synthesis of [5]BF4-[8]BF4
This procedure was adapted from the literature.31 BF3,OEt2 (0.5 mmol) was trans-
ferred to a cooled (0ꢀC) CH2Cl2 solution (10 mL) containing triphenylbismuth difluor-
ide (0.3 mmol) and the arylboronic acid (0.4 mmol). The resulting solution was stirred
for 2 h at room temperature. An aqueous solution of NaBF4 (2.0 mmol) was then
added. The resulting mixture was stirred vigorously for 30 min. The aqueous phase
was separated and extracted with CH2Cl2. The combined organic extracts were
dried over MgSO4 and evaporated under reduced pressure to afford a solid residue,
which was taken up in CH2Cl2 (2 mL) and precipitated with Et2O (20 mL). After filtra-
tion, the solid product was washed with two portions of Et2O (20 mL, for [5]BF4 and
[6]BF4) or THF (10 mL, for [7]BF4 or [8]BF4).
Vesicle Preparation
The vesicles were prepared according to a previously established method.60 A thin
film of the lipid was prepared by evaporation of a solution of EYPC (30 mg) dissolved
in a 1:1 mixture of MeOH/CHCl3 (2 mL). This film was dried under vacuum overnight.
A buffered KF solution (1 mL, 10 mM HEPES, 300 mM KF [pH 7.2]) was then added,
resulting in a suspension that was then subjected to nine freeze-thaw cycles (liquid
N2, 40ꢀC water bath) and extruded 27 times through a 200 nm polycarbonate mem-
brane. To remove any extravesicular component, the vesicle suspension was passed
through a size exclusion column (Sephadex G-50) using a buffer solution (10 mM
HEPES, 300 mM KGlc [pH 7.2]) as an eluent. The DPPC vesicles were prepared in
a similar fashion, except that the water bath used during the freeze-thaw cycles
10
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was elevated to 50ꢀC. The same temperature was also used during the vesicle
extrusion.
Fluoride Efflux in the Presence of Valinomycin
The following assay was adapted from a previous report.8 A solution with a final
phospholipid concentration of 0.7 mM was obtained by combining an aliquot of
the KF-loaded EYPC vesicles with an aqueous solution (5 mL, pH = 7.2, [HEPES] =
10 mM) containing KGlc (300 mM). The fluoride-selective electrode was immersed
in this solution. After the signal of the voltage had stabilized (ꢁ60 s), the measure-
ment was initiated. At t = 0 s, valinomycin dissolved in DMSO (0.7 mM) was added
to the assay such that the final valinomycin concentration was 0.1 mol %. At t = 30 s,
the bismuthonium or stibonium salt ([1]-[3]OTf, [4]Br, and [5]BF4-[8]BF4) was added
to the assay. At t = 300 s, 50 mL of a Triton X-100 solution (10:1:0.1 H2O:DMSO:Triton
X-100 (v/v/v)) was added to lyse the vesicles, triggering complete release of
the fluoride cargo. The value corresponding to 100% fluoride efflux was recorded
at t = 420 s, 2 min after the vesicles were lysed. The DPPC-based assay was carried
out according to the same protocol and the same concentrations as those employed
for the EYPC-based assay. The only difference is that the transport experiments were
carried out at 25ꢀC and 45ꢀC.
Erythrocyte-Based Assays
Whole blood was purchased from the Gulf Coast Regional Blood Center (Houston,
TX). Erythrocytes were isolated from whole blood by centrifugation for 10 m at
1,250 3 g. The erythrocyte pellet was resuspended in PBS (HyClone) and
this washing procedure was repeated three times to remove plasma and the buffy
coat. To observe membrane localization of [3]+ by fluorescence microscopy, RBCs
were diluted to 2% volume in PBS and treated with 100 mM [3]+ for 5 or 25 min
at 37ꢀC. The solution was then rapidly diluted to 0.2% by volume in PBS
and added to a glass-bottom 96-well plate for imaging. Fluorescence microscopy
was performed with an inverted microscope (Olympus IX-81) with a 3100 objective
as well as a heated stage (37ꢀC), and images were taken with a Rolera-XR back-
illuminated electron-multiplying CCD camera (Qimaging). For fluorescence
imaging, a DAPI (lex/lem = 300–388 nm, 425–488 nm) filter cube was used
(Chroma Technology). The bright-field and fluorescence images of cells were
obtained with SlideBook 4.2.0.7 software (Intelligent Imaging innovations). For
hemolysis assays, RBCs were diluted to 6.8% volume in PBS and treated
with 100 mM NaF and/or 1 or 5 mM [3]+ as indicated, and ion transport was
allowed to continue for 1–8 h at 37ꢀC. At each time point, intact RBCs and
ghosts were sedimented by centrifugation for
2 min at 1,250 3 g. The
supernatant containing the heme was then transferred to an optical bottom
96-well plate for measurement of free heme by measuring the absorbance (l =
488 nm) using a GloMax-Multi+ detection system plate reader (Promega). Tripli-
cate experiments were performed, measured, and subjected to normalization. To
normalize hemolysis results, a positive control was conducted in tandem by treat-
ing RBCs with 1% Triton X-100.
DATA AND CODE AVAILABILITY
Crystallographic data for [7]BF4 have been deposited in the Cambridge Crystallo-
graphic Data Centre and are available under accession number CCDC: 1863621.
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.chempr.
2019.06.013.
Chem 5, 1–13, August 8, 2019
11

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ACKNOWLEDGMENTS
This work was supported by the National Science Foundation (CHE-1566474, grant
to F.P.G.), the Welch Foundation (A-1423, grant to F.P.G.), the National Institute of
General Medical Sciences (R01GM110137, grant to J.-P.P.), and Texas A&M Univer-
sity (Arthur E. Martell Chair of Chemistry, F.P.G.; FY19 Strategic Transformative
Research Program, grant to F.P.G. and J.-P.P.). We thank Dr. Iou-Sheng Ke for his
initial synthesis and characterization of [7]BF4.
AUTHOR CONTRIBUTIONS
G.P., D.J.B., J.-P.P., and F.P.G. conceived the study. G.P. carried out all experiments
involving the synthesis of all compounds, their characterization, and the use of the
transporters in synthetic vesicles. D.J.B. carried out all RBC-based assays.
DECLARATION OF INTERESTS
The authors declare no competing financial interests.
Received: August 23, 2018
Revised: October 2, 2018
Accepted: June 19, 2019
Published: July 15, 2019
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