Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
10.1002/anie.202006392
Angewandte Chemie International Edition
RESEARCH ARTICLE
OH···Cl- distance 2.29(6) Å (Figure 3). This water molecule
connects the chloride anion and the second receptor molecule by
acting as a hydrogen bond acceptor to the imidazole NHs, with
distances 2.13(7) and 2.25(8) Å. The assembly is further
stabilized with a hydrogen-bonded water dimer (Figure 3).
absence of cationophores (Figure 4e), confirming that K+/Cl-
symport did not significantly contribute to the Cl- transport
observed in the Cl-/NO3 exchange assay.
-
BisBzImPy derivatives 1-5 were then investigated using 8-
hydroxypyrene-1,3,6-trisulfonic acid (HPTS) assays (Figure 4f-
i).[23] The HPTS-KCl assay (Figure 4f) measures the activity of
anion transporters in facilitating H+/Cl- cotransport leading to the
dissipation of pH gradients. Consistent with the results of the ISE
assay, the unsubstituted BisBzImPy 1 emerged as the transporter
with the lowest activity in HPTS-KCl (HCl symport, Figure 4j)
assay with an EC50 = 4.8 mol%. The addition of electron-
withdrawing groups to the benzimidazole rings (2-5) increased the
ability of the compounds to facilitate H+/Cl- symport, with EC50
values of these compounds ranging from 0.23 mol% to 0.061
mol%, (Figure 4j, Table 1). The activity decreased in the order of
5 > 3 > 2 > 4. It should be noted that the HPTS assay is more
sensitive than the ISE assay because of the low Cl- efflux required
(5 mM compared with 489 mM in the ISE assay). This has led to
identification of 5 as the most active transporter despite its low
deliverability limiting its activity at high concentrations as
observed in the ISE assay. To further investigate the intrinsic
ability of transporters 1-5 to transport only chloride, the same
assay was employed at 1 mol% transporter (mol% rtl, final lipid
concentration 0.1 mM) and using the protonophore carbonyl
cyanide m-chlorophenyl hydrazone (CCCP) as an efficient proton
transporter. If H+ transport is the rate determining step of HCl
symport, CCCP will improve transport and accelerate the pH
dissipation (Figure 4k, S54-58).[23] Anionophores 1-5
demonstrated no CCCP dependence indicating that H+ transport
by these compounds was not the rate-limiting process in this
assay.
Figure 3: Side (a) and top (b) views of crystal structure of 2.Cl- complex.
Counterions (TEA+) and some solvent molecules have been omitted for clarity.
Thermal ellipsoids are at the 50% probability level. See also Figure S22 for
complete structures.
Transporters 1-5 were investigated for their chloride transport
properties via a Cl-/NO3- exchange assay using a chloride ion
selective electrode (ISE) (Figure 4a-e, Table 1). Briefly, 1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC)
unilamellar liposomes with a diameter of 200 nm were prepared
and loaded with 489 mM KCl, buffered at pH 7.2 and suspended
in 489 mM KNO3 solution buffered at pH 7.2. The chloride efflux
We next examined the abilities of transporters 1-5 to transport
only protons using a H+ transport assay by replacing potassium
chloride with potassium gluconate (KGlu) (Figure 4h) and using
valinomycin to facilitate K+ transport and dissipate the membrane
potential generated by H+ transport.[23] Gluconate is a relatively
-
due to Cl-/NO3 exchange was monitored using chloride ion
selective electrode upon addition of
a DMSO solution of
transporters 1-5 to the prepared liposomes.[23] At the same
concentration, compounds 1-5 showed better transport properties
when transporter loading volume increased from 10 μL to 40 μL
(Figures S77-80), presumably due to better deliverability in a
higher volume of DMSO. Chloride efflux at different
concentrations of the tested compounds (expressed as mol% with
respect to lipid concentration (rtl), final lipid concentration 1 mM)
was measured to obtain Hill plots and to calculate an EC50 value
(defined as the concentration required to achieve 50% the
chloride efflux at 270 s) and a Hill coefficient (n). The Hill
coefficient indicates the stoichiometry of the formed complex
during the anion transport across the lipid bilayer, while EC50 is
used as a measure of anion transporter potency.
large hydrophilic anion that can be considered untransportable[23]
.
Apart from the unsubstituted BisBzImPy 1 (EC50 = 2.2 mol%),
tested compounds 2-5 showed a remarkable protonophoric
activity with EC50 spanning from 0.031 to 0.0018 mol%, rtl, (Table
1). These values are lower than the EC50 for H+/Cl- symport,
confirming faster H+ transport than Cl- transport. The activity of
transporters 2-5 as protonophores decreased in the order of 3>
2> 5 > 4 (Table 1). To further investigate the potential interaction
between fatty acids and the tested anion transporters 1-5, oleic
acid (1 mol%) or BSA treated liposomes (to remove fatty acids)
were used (Figure 4k). It has been reported that some
transporters could transport H+ indirectly by facilitating the
transmembrane flip-flop of deprotonated fatty acids.[23-24] The
transport activity in KCl and KGlu assays were not affected by
addition or removal of fatty acids, which indicate that H+ transport
by 1-5 is fatty acid-independent and possibly occurs via
deprotonation of the NH groups. A selectivity assay was used to
demonstrate preferential transport of more lipophilic anions
Compounds 1 and 5 were excluded from Hill plot analysis due
to low potency of compound 1 and the limited solubility of
transporter 5. Tested anionophores 2-4, showed comparable Cl-
/NO3- exchange activity (EC50 = 0.42-0.58 mol%). Transporter 2
was the most active with EC50 = 0.42 mol%, followed by
transporters
3 and 4 (EC50 = 0.49 and 0.58 mol% (rtl),
respectively) (Figure 4d, Table 1). A cationophore coupling assay,
using valinomycin (Vln) or monensin (Mon) was used to
investigate the mechanism of transport facilitated by the
anionophores. Such assays are used to determine the activities
of Cl- uniport (coupling with valinomycin) and H+/Cl- symport
(coupling with monensin) (Figure 4e).[23] The results of the assays
showed that compounds 1-5 were capable of both Cl- uniport and
H+/Cl- symport (Figure 4e). Very low Cl- efflux was observed in the
-
including Br-, I-, NO3 , and ClO4- over Cl- for all anionophores 1-5
(Figures S69-S73). Identical transport rates were found when the
K+ in the HPTS assay was replaced with Na+ (Figures S74-75)
consistent with the lack of metal ion transport. A liposomal
leakage experiment performed using self-quenching calcein
confirmed the absence of non-specific membrane leakage (Figure
S76).[24]
3
This article is protected by copyright. All rights reserved.