Tetra-arylborate lipophilic anions as targeting groups

Article abstract of DOI:10.1039/d0cc07924c

Tetraphenylborate (TPB) anions traverse membranes but are excluded from mitochondria by the membrane potential (Δψ). TPB-conjugates also distributed across membranes in response to Δψ, but surprisingly, they rapidly entered cells. They accumulated within

Full text of DOI:10.1039/d0cc07924c

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Tetra-arylborate lipophilic anions as targeting
groups†
Cite this: Chem. Commun., 2021,
7, 3147
5
a
a
b
b
Kishore K. Gaddale Devanna, Justyna M. Gawel, Tracy A. Prime, Filip Cvetko,
b
a
c
b
Received 7th December 2020,
Accepted 16th February 2021
Cristiane Beninca, Stuart T. Caldwell,
Andrew M. James, Evgeny V. Pavlov, Michael P. Murphy* and
Alexander Negoda, Andrew Harrison,
´
b
d
be
a
Richard C. Hartley
*
DOI: 10.1039/d0cc07924c
rsc.li/chemcomm
Tetraphenylborate (TPB) anions traverse membranes but are excluded potential energy well on the other side of the membrane,
from mitochondria by the membrane potential (Dw). TPB-conjugates traversing the activation energy barrier of the membrane core
6
,7,9
also distributed across membranes in response to Dw, but surprisingly, (Fig. 1).
they rapidly entered cells. They accumulated within lysosomes bilayers
Hence, TPB rapidly permeates phospholipid
and its distribution is determined by the Dc,
6
,7,10,11
12
following endocystosis. This pH-independent targeting of lysosomes resulting in exclusion from the mitochondrial matrix.
makes possible new classes of probe and bioactive molecules.
1
5
TPB anions have been widely used in analytical chemistry,
16
17
18,19
catalysis and solid electrolytes, and as substrates for oxidative
2
0,21
The ability to direct molecules to an appropriate location within and Suzuki-type cross-coupling.
Despite their similar struc-
the cell facilitates development of bioactive and probe com- ture to TPP, and their complementary response to Dc, the use of
pounds. For example, molecules are targeted to the mitochondrial TPB anions to deliver compounds to different sub-cellular
matrix by conjugation to the lipophilic triphenylphosphonium compartments has not been explored. This may be because the
1–4
cation. The large hydrophobic surface area of the triphenylpho- Dc across the plasma membrane, which is negative inside,
sphonium group enables rapid crossing of biological membranes would be expected to disfavour intracellular delivery. There has
by lowering the activation energy for movement through the been only one exploration of lipophilic anion distribution
4,5
membrane core. Furthermore, the positive charge drives exten- within cells, by conjugation of the monocarborane (1-carba-
1
3,14
sive accumulation within the mitochondrial matrix due to the closo-dodecaborate) lipophilic anion to a porphyrin.
large membrane potential (Dc negative inside).
Surpri-
2,4,5
14
singly, the monocarborane conjugate was taken up by cells,
To deliver molecules to other cell compartments in a similar although the mechanism was not investigated. Thus, we inves-
way, we explored conjugation to lipophilic anions. Just as the tigated whether TPB anions could be used to deliver com-
archetypal lipophilic cation is tetraphenylphosphonium (TPP) pounds, despite their negative charge, within the cell.
the corresponding lipophilic anion is tetraphenylborate (TPB).
We synthesized a universal TPB-conjugate precursor 5 that
These molecules have identical radii (4.2 Å) and, apart from could be derivatized through amide coupling to carboxylic acids.
6
,7
charge, are similar. Both are water soluble, facilitating bio-
logical uses, although TPB has a more negative free energy of
8
hydration. Lipophilic ions bind to a potential energy well on
the membrane surface, before flipping, to the corresponding
a
School of Chemistry, University of Glasgow, Glasgow, G12 8QQ, UK.
E-mail: Richard.Hartley@glasgow.ac.uk
b
MRC Mitochondrial Biology Unit, Hills Road, University of Cambridge, CB2 0XY,
UK. E-mail: mpm@mrc-mbu.cam.ac.uk
c
Department of Physiology and Biophysics, Dalhousie University, Halifax,
Nova Scotia, Canada
d
New York University, College of Dentistry, Department of Molecular Pathobiology,
3
45 East 24th Street, New York, NY 10010, USA
e
Department of Medicine, University of Cambridge, Cambridge, UK
†
Electronic supplementary information (ESI) available: Author contributions,
movies, synthetic procedures, NMR spectra and supplementary figure. See DOI:
0.1039/d0cc07924c
1
Fig. 1 Membrane permeation by TPB lipophilic anions (adapted from ref. 9).
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Fig. 2 Transport of TPB-conjugates across a Black Lipid Membrane (BLM).
Compounds were added to the both sides of a BLM to create a concen-
tration gradient. Insets show expansions of the x-axis intersection that
indicates reversal potential (Erev). (A) TPB gradient = 10 mM (cis)/1 mM (trans),
Scheme 1 Synthesis of TPB-conjugates. Conditions: (a) (i) TFA-CH
2
Cl
2
,
,
0
2 2
1C – RT, 2 h (ii) ClCO Me, 0 1C - RT, 3.5 h. (b) (i) [B(Pin)] , Pd(dppf)Cl
2
KOAc, DMSO, 70 1C, 18 h (ii) KF, MeCN–MeOH (1 : 1), RT, 1 min (iii) L-(+)-
tartaric acid, THF, RT, 2 min (iv) MeCN 4 min (c) (i) 5 equiv. PhMgCl, THF,
Erev B50 mV. (B) TPB gradient = 60 mM (cis)/10 mM (trans), Erev B31 mV. (C)
0
1C, 30 min then reflux 16 h, (ii) Na
RT. (d) KOH, MeOH–H O (2 : 1) reflux, 4 h. (e) (i) RCO
ii) ion exchange.
2
CO
3
(aq), RT, 1 h (iii) Bu
4 2 2
NBr, CH Cl ,
TPBM = 5 mM (cis)/0.5 mM (trans), Erev B32.5 mV. (D) Current recorded at 10
mV Æ 100 nM TPBM. Inset shows magnification of the region at voltage
application. (E and F) Charge accumulated on BLM with different concen-
trations of TPBM and TPBE as a function of applied voltage. (G) Data from
independent experiments at voltage jump +50 mV. n = 4–6 Æ SEM.
2
2
H, coupling agent
(
The key synthetic step was the addition of three identical phenyl
22
substituents to an aryltrifluoroborate (Scheme 1). As tetraaryl-
borates with electron-rich substituents are prone to oxidation and (Fig. 2c). The greater membrane disruption by TPBM compared to
protodeborylation, we incorporated an electron-withdrawing TPB is caused by its enhanced lipophilicity. Therefore, only low
sulfonamide. To assess intracellular distribution and functional concentrations can be used and it was not possible to assess the
properties we made a minimal TPB derivative with the sulfona- membrane permeation of TPBM and TPBE directly. Instead, to
mide piperazine replaced with a morpholine (TPBM), to avoid estimate the amount of compound crossing the bilayer in
23
24,25
N-protonation targeting acidic compartments;
gated to the chromanol group of a-tocopherol as a generic cargo induced by steps from 0 mV to defined voltages before/after
TPBE); and two fluorescent probes, TPBCoumarin and TPBBO- addition of the compounds (Fig. 2d).
DIPY, to allow imaging. In the absence of compounds the transient (capacitive) currents
a TPB conju- response to a voltage we measured transient ionic currents
(
To see if TPB-conjugates could rapidly distribute across are determined by the membrane’s dielectric properties. In the
membranes in response to voltage we used a phospholipid presence of a TPB-conjugates the transient currents are the sum of
27
black lipid membrane (BLM) system (Fig. 2). In agreement with the capacitive current and that caused by anion redistribution.
2
6
previous observations, we detected increasing currents due to Charge transfer by the lipophilic anions crossing the bilayer,
TPB crossing a BLM as a function of voltage (Fig. 2a). In the derived by subtracting the current in the absence of compound,
presence of a TPB gradient (1 mM : 10 mM) across a BLM the depends on the compound concentration and the applied voltage
reversal potential was close to the theoretical Nernst equilibrium (Fig. 2e and f). Combined data for a +50 mV voltage jump are
potential of 60 mV (Fig. 2a), demonstrating that the observed shown in Fig. 2g. TPBM at 100 nM induced the largest charge flux,
current is caused by TPB transfer across the bilayer. At higher while higher concentrations caused non-specific conductance. The
TPB concentrations, but the same gradient, the current TPBE conductance was less (Fig. 2e and f). These results indicate
increased and the reversal potential shifted towards 0 mV, that TPB-conjugates cross a BLM in a voltage-dependent manner.
due to non-specific conductance caused by membrane disruption
We next assessed whether TPB-conjugates crossed biological
(Fig. 2b). TPBM disrupted the BLM at lower concentrations than membranes in response to a Dc using sub-mitochondrial
TPB, leading to a larger non-specific conductance, hence the particles (SMPs), which are inverted mitochondrial inner
reversal potential for TPBM was considerably lower than 60 mV membrane vesicles (Fig. 3a). Proton pumping by the respiratory
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Fig. 4 Cell distribution of TPBCoumarin. (A), C2C12 cells were incubated
with 100 nM TPBCoumarin (green). Distribution imaged 5 min after
addition. (B) MitoTracker (red) was compared with TPBCoumarin (green).
(
C) LysoTracker (magenta) compared with TPBCoumarin (green). Colocali-
zation is shown in white. (D) Cells were incubated with 20 mM Pitstop2 for
0 min before addition of TPBCoumarin and compared with control. The
3
bar chart shows mean Æ SEM of 3 independent experiments. ** p o0.01,
by Student’s t-test. Scale bar = 20 mm.
Importantly, these attributes of TPB-conjugates are greatly
enhanced over lipophilic cations due to the different inter-
action of lipophilic anions with biological membranes. This is
primarily because of the large dipole potential from positive within
6,7,9,29
the phospholipid bilayer core to negative at the surface
which lowers the activation energy barrier for lipophilic anion
transport, relative to cations, hence the membrane conductivity of
5
6,7,9–11
TPB is B10 -fold greater than that for TPP.
Similarly, the
binding constant for TPB to the potential energy well near the
3
4
6,7,9,29
membrane surface is B10 –10 fold greater than for TPP.
Addition of a hydrophobic linker/cargo increases this membrane
adsorption further still.
Fig. 3 Uptake of TPB derivatives by submitochondrial particles (SMPs). (A),
Upon energization with NADH proton pumping by the respiratory chain
generates a Dc, negative inside. (B–D), An ion-selective electrode (ISE) was
We next assessed the uptake and distribution of TPB-
calibrated (5 Â 1 mM additions) then SMPs (0.2 mg protein per ml) were conjugates using a fluorescent probe, TPBCoumarin, with C2C12
added followed by NADH (1 mM) to generate Dc and subsequently FCCP cells by confocal laser scanning microscopy (Fig. 4a and Movie 1,
(1 mM) to dissipate Dc.
ESI†). Despite its negative charge, TPBCoumarin was rapidly taken
up by cells showing punctate staining, as was TPBBODIPY (Fig. S1
and Movie 2, ESI†). Parallel staining with MitoTracker (Fig. 4b)
showed TPBCoumarin was not colocalizing with mitochondria
chain generates a positive-inside Dc, which should drive uptake while LysoTracker (Fig. 4c) showed partial colocalization with
1
2
of lipophilic anions (Fig. 3a).
lysosomes. This suggests the punctate staining upon uptake of
Using an ion-selective electrode (ISE) to measure TPB concen- TPBCoumarin is due to uptake by endocytosis with initial localiza-
2
8
30
tration showed its rapid uptake into SMPs upon induction of a tion within endosomes which then fuse with lysosomes. Sup-
Dc by NADH, which was reversed by dissipating the Dc with porting this, uptake was greatly decreased by Pitstop 2 (Fig. 4d),
31,32
FCCP (Fig. 3b). Similarly, TPBM (Fig. 3c) and TPBE (Fig. 3d) also which inhibits clathrin-mediated endocytosis.
This shows
showed uptake into SMPs in response to Dc. The greater there is rapid initial uptake by endocytosis followed by redistribu-
hydrophobicity of TPBM and TPBE enhanced their membrane tion to lysosomes. Critically, there was no co-localization with
adsorption compared to TPB. Therefore, TPB-conjugates rapidly mitochondria, consistent with their exclusion from the negatively-
cross biological membranes in response to a Dc and adsorb charged matrix. Cell toxicity of TPBE was also negligible below
strongly to membranes.
10 mM (Fig. S2, ESI†).
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Despite their negative charge, and the negative-inside Dc
across the plasma membrane, TPB display a novel intracellular
distribution. Most lysosome targeting is achieved through ion-
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6
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