Fructose controlled ionophoric activity of a cholate-boronic acid

Article abstract of DOI:10.1039/c4ob00165f

Wulff-type boronic acids have been shown to act as ionophores at pH 8.2 by transporting Na⁺ through phospholipid bilayers. A cholate-boronic acid conjugate was synthesised and shown to be an ionophore, although the hydroxyl-lined face of the cholate moiety did not enhance ion transport. Mechanistic studies suggested a carrier mechanism for Na⁺ transport. The addition of fructose (>5 mM) strongly inhibited ionophoric activity of the cholate-boronic acid conjugate, mirrored by a strong decrease in the ability of this compound to partition into an organic phase. Modelling of the partitioning and ion transport data, using a fructose/boronic acid binding constant measured at pH 8.2, showed a good correlation with the extent of fructose/boronic acid complexation and suggested high polarity fructose/boronic acid complexes are poor ionophores. The sensitivity of ion transport to fructose implies that boronic acid-based antibiotic ionophores with activity modulated by polysaccharides in the surrounding environment may be accessible. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00165f

Organic &
Biomolecular Chemistry
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Fructose controlled ionophoric activity of a
cholate–boronic acid†
Cite this: Org. Biomol. Chem., 2014,
12, 2576
James R. D. Brown, Inmaculada C. Pintre and Simon J. Webb*
Wulff-type boronic acids have been shown to act as ionophores at pH 8.2 by transporting Na+ through
phospholipid bilayers. A cholate–boronic acid conjugate was synthesised and shown to be an ionophore,
although the hydroxyl-lined face of the cholate moiety did not enhance ion transport. Mechanistic
studies suggested a carrier mechanism for Na+ transport. The addition of fructose (>5 mM) strongly inhib-
ited ionophoric activity of the cholate–boronic acid conjugate, mirrored by a strong decrease in the ability
of this compound to partition into an organic phase. Modelling of the partitioning and ion transport data,
using a fructose/boronic acid binding constant measured at pH 8.2, showed a good correlation with the
extent of fructose/boronic acid complexation and suggested high polarity fructose/boronic acid com-
plexes are poor ionophores. The sensitivity of ion transport to fructose implies that boronic acid-based
antibiotic ionophores with activity modulated by polysaccharides in the surrounding environment may be
accessible.
Received 21st January 2014,
Accepted 27th February 2014
DOI: 10.1039/c4ob00165f
www.rsc.org/obc
Introduction
The recognition of polyols, in particular saccharides, by
boronic acids is an example of a dynamic covalent chemistry
that has provided a wealth of applications in supramolecular
chemistry, with recent examples in sensing,1 drug delivery,2
separation3 and hydrogel formation.4 The interaction of lipo-
philic or membrane-bound boronic acids with saccharides or
glycolipids has risen to particular prominence in recent
years,5,2b driven in part by the development of 2-(hydroxy-
methyl)phenylboronic acids able to bind a wider range of
sugars at close to physiological conditions.6 Lipophilic boronic
acids have also been known for some time to also transport
Fig. 1 Scheme showing the transport of boronic acid–diol complexes
through bilayers either as (a) trigonal planar or (b) tetrahedral boron
species. (c) Main 1 : 1 boronate complex formed with fructose.12
saccharides, particularly fructose, across supported mem- tetrahedral boronate anions, which can be covalently linked to
branes,7 bulk organic phases8 and phospholipid bilayers.9
the boronic acid, an added lipophilic tetraalkylammonium
Amphiphilic boronic acids are suggested to transport sac- salt, or it can be a counterion symported with the sugar from
charides by forming complexes that are sufficiently lipophilic the sending phase.7,11 In the latter case a crown ether is often
to diffuse across the membrane. Two mechanisms have been added to form a lipophilic complex able to pass through the
proposed for boronic acid-mediated diol transport across apolar membrane phase.10a Despite the implication that
membranes (Fig. 1), with transport either occurring through cation transport is concomitant with saccharide transport
trigonal planar boronate esters or tetrahedral boronate anions; transport,7,9b,11 boronic acid mediated M+ transport across
the pH of the sending phase and boronic acid pKa determines phospholipid bilayers has not been explicitly measured and
which mechanism is followed.10 A cation is associated with compared to the rate of concomitant saccharide transport.
The ability of lipophilic boronic acids embedded in phos-
pholipid bilayers to complex sugars suggests new ways of con-
Manchester Institute of Biotechnology (MIB) and School of Chemistry, University of
trolling ion flux across phospholipid bilayers, with ion flow
Manchester, 131 Princess St., Manchester M1 7DN, UK.
across the membrane controlled by the presence of biological
E-mail: S.Webb@manchester.ac.uk
catechols13 or saccharides.14 Saccharides are ideal for control-
†Electronic supplementary information (ESI) available: NMR spectra of 3. K+
transport by 3. U-tube picrate transport data. Effect of fructose on Na+ transport
by 3 or 4. See DOI: 10.1039/c4ob00165f
ling ion transport as these highly polar molecules cannot cross
the phospholipid bilayer, they will not discharge a pH gradient
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Cholic acid 4 was first coupled to mono-protected 1-(benzyl-
oxycarbonylamino)-3,6-dioxa-8-aminooctane,
the
product
hydrogenolysed, then reductively aminated with 2-formyl-
phenylboronic acid to give compound 3 in 59% yield from the
acid.
Ion transport assays
To screen the ability of these compounds to transport cations
through bilayers we chose the phospholipid vesicle based
8-hydroxypyrenetrisulfonate (HPTS pKa = 7.3) assay,19 using an
interior pH of 6.5 and an exterior pH of 8.5. The exterior pH
was selected to be above the pKa of aminomethylboronic acids
(pKa ∼ 6.5)7 and corresponding saccharide complexes (pKa <
6.5),20 a strategy designed to favour M+/OH− co-transport.
EYPC/cholesterol vesicles were used as these bilayers have the
right balance between fluidity and impermeability. Fluidity
allows assembly in the membrane and diffusion through the
membrane, but pure EYPC bilayers gave unsatisfactory levels
of background leakage.
The choice of buffer was crucial as boronic acid complexa-
tion of diols is strongly pH dependent, with the ideal pH
between the pKa of the diol and the boronic acid.21 Buffer type
and concentration can also strongly affect the binding of
boronic acids to saccharides, with stronger binding at low
buffer concentrations.22 To obtain precise pH control, the stan-
dard procedure for the HPTS assay was modified, with HPTS-
loaded vesicles directly added to buffer at pH 8.5 rather than
addition of an external aliquot of NaOH (the base pulse). The
ionophore was then added after 180 s to initiate transport,
which also eliminates the jump in fluorescence observed if the
base pulse is applied after ionophore addition.
Fig. 2 Boronic acids and cholic acid.
and unlike lipophilic polyols they will not disrupt bilayer mem-
branes. Although the affinities of sugars for most boronic
acids is generally rather weak, some sugars, such as fructose,
exhibit binding constants with phenylboronic acids in buffer
in the order of 100 M−1
.
Previously we developed cholate-based ionophores that can
be turned on or off by complexation to metal ions or pro-
teins.15 Adding boronic acid mediated recognition/transport
with mono- or bis-cholate scaffolds was expected to provide
some interesting ionophores, especially as oligocholates are
known to transport metal ions and dyes through bilayers,
through either channel or carrier mechanisms.16,17 The high
activity of cholates stems from their facial amphiphilicity,
which provides hydrophobic convex surfaces that can be pre-
sented to the bilayer and concave hydrophilic surfaces that can
interact with alkali metal ions.
Linking cholates with boronic acids was hoped to produce
saccharide-switchable ion transport across phospholipid
bilayers. Fructose forms boronic acid complexes with a variety
of geometries and stoichiometries,12 including 1 : 2 fructose to
boronic acid,12,18 and we wondered if fructose could assemble
boronic acid–cholate conjugates into dimers that might form
channels17 or “hairpin” carriers with a sequestered hydrophilic
cavity. The compounds we investigated for cation transport
ranged from phenylboronic acid 1 and Wulff-type 2-(N-methyl-
aminomethyl)phenylboronic acid 2, to a more amphiphilic
Wulff-type boronic acid conjugate 3 that has a single facially
amphiphilic cholate tail (Fig. 2).
Initially cholate–boronic acid 3 was assayed for its ability to
allow sodium ions to cross a bilayer. Addition of cholate–
boronic acid 3 (10 μM) to give a membrane loading of 5% mol/
mol gave an immediate increase in fluorescence that suggested
Na+/H+ antiport or Na+/OH− symport across the bilayer of
these EYPC/chol vesicles. Cholic acid 4 (pKa ∼ 4.6)23 did not
conduct sodium ions across the membrane and gave little
transport above the background methanol control (Fig. 3).
Results and discussion
Synthesis of 3
Boronate-cholate derivative 3 was synthesised in two steps
from cholic acid 4. The short triethylene glycol spacer was
added between the cholate body and the boronic acid to give
flexibility around the complexation site and to project it away
Fig. 3 Transport of Na+ across EYPC/chol bilayers by boronic acid 2 ( ),
boronate-cholate 3 ( ) and cholate 4 ( ). The methanol control has
from the cholate, which will become embedded in the bilayer. been subtracted from the data. First order curve fits are to guide the eye.
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To confirm that the boronic acid is the active moiety in the ability of these Wulff-type boronic acids to act as
mediating ion transport, phenylboronic acid 1 and 2-(N- ionophores.
methylaminomethyl)phenylboronic acid 2 were also assessed
If the complexation of the boronic acid group to fructose
for their ability to transport Na+ through an EYPC/chol bilayer. blocks the activity of these ionophores, then the rate of Na+
Phenylboronic acid (10 µM, 5% mol/mol) did not transport transport should depend on the fructose concentration. Ion
sodium ions above the background rate, but the 2-(N-methyl- transport by 3 was monitored in the presence of 2.5 mM,
aminomethyl) derivative 2 (10 µM, 5% mol/mol) gave good 5 mM, 10 mM and 100 mM fructose, which revealed a steady
transport of Na+ at a rate above that observed for cholate decrease in activity as the concentration of fructose increased.
boronic acid 3. This observation suggests that the cholate A plot of the extent of transport after 1200 seconds against the
hydroxyls play little role in assisting ion transport and the concentration of fructose showed an inverse dependence, a
sodium ion is co-transported in close proximity to an anionic relationship that mirrors the fraction of free ionophore 3
boronate headgroup. The pKa of 2 is ∼6.5,7 and at pH 8.5 the expected at different concentrations of fructose (Fig. 4b).
major species in solution will be the tetrahedral boronate
Mechanistic investigations
anion, but phenylboronic acid 1 has a higher pKa of 8.8,21a,22
suggesting that at pH 8.5 the major boron species in solution Smith and co-workers have shown that boronic acids transport
will be uncharged; this trigonal planar boronic acid would be saccharides across bilayers via a carrier mechanism, which
unable to antiport Na+/H+ or symport Na+/OH− across the implies a similar mechanism for Na+ transport by 2 and 3. A
membrane.
classical test for an ion carrier mechanism is the ability to
transport ions through a bulk organic phase in a U-tube experi-
ment.19 The boronic acids 2 and 3 were assessed for their
ability to transport sodium picrate across a chloroform phase,
using 0.01% wt/vol sodium picrate in sodium phosphate
buffer (2.5 mL, 20 mM phosphate, 100 mM NaCl, pH 8.5) in
the sending phase and sodium phosphate buffer (2.5 mL,
20 mM phosphate, 100 mM NaCl, pH 6.5) in the receiving
phase. In addition, cholic acid 4 and dibenzo-18-crown-6 were
also assessed for their ability to transport sodium picrate.
Boronic acid 3 (1 mM) transported sodium picrate through the
chloroform layer at a rate comparable to dibenzo-18-crown-6
(respectively 1% and 3% picrate transport after 6 h, ESI
Fig. S5†).26 As expected, cholate 4, lacking the boronic acid,
was unable to transport sodium picrate through the organic
phase (∼0.07% after 6 h). Surprisingly, boronic acid 2 was also
relatively ineffective (0.2% after 6 h), which was ascribed to its
fairly high solubility in the aqueous phase.
Effect of fructose on ion transport
Based upon literature binding constants (K from 100 to 500
M−1) in methanol–buffer mixtures at near neutral pHs,22,24,25
we hoped that 100 mM fructose would be sufficient to give
extensive complexation of the boronic acid headgroups in
these ionophores. The effect of this concentration of fructose
on ion transport by ionophore 3 was assessed using the HPTS
assay. During formation and purification of the EYPC/chol
vesicles, all pH 6.5 buffer solutions also contained 100 mM
fructose, maintaining the osmotic balance across the vesicle
membrane. As previously, an aliquot of this vesicle solution
was transferred to iso-osmotic buffer with 100 mM fructose at
pH 8.5 before ionophore 3 was added at 180 s. Under these
conditions, little Na+ transport was observed, with discharge of
the pH gradient only marginally above the methanol control
(Fig. 4a). As expected the low level of transport exhibited by
cholate control 4 was found to be little affected by the addition
of fructose (ESI Fig. S3†). A similar switching off of ion trans-
port was observed for boronate 2 in the presence of 100 mM
fructose, showing that complexation to fructose diminished
Given that lipophilic boronate complexes are known to
transport fructose through organic phases,7–9 the relationship
between fructose transport and Na+ transport was also
assessed. The ability of ionophore 3 to transport fructose/Na+
through the organic CHCl3 phase was assessed using a U-tube
and a combination of 1H-NMR analysis (fructose transport)
with UV-visible spectroscopy (Na+ transport). A CDCl3 (99.8
atom % D) solution of 3 (1 mM) was transferred to a glass
U-tube. A receiving phase of sodium phosphate buffer (D2O
containing 0.75% sodium 3-(trimethylsilyl)-2,2,3,3-d4-propio-
nate salt as an internal standard, 20 mM phosphate, 100 mM
NaCl, pH 6.5) was added to one side of the U-tube, and to the
other side was added a source phase of fructose (1 M) and
0.01% wt/vol sodium picrate in sodium phosphate buffer (D2O
containing 0.75% internal standard, 20 mM phosphate,
100 mM NaCl, pH 8.5). Aliquots were taken at intervals from
1
the receiving phase and analysed by H NMR spectroscopy for
Fig. 4 (a) Transport of Na+ across EYPC/chol bilayers by 2 ( ), 2 with
100 mM fructose ( ), 3 ( ) and 3 with 100 mM fructose ( ). First order
curve fits are to guide the eye. (b) Plot of Na+ transport by 3 after 1200 s
at different concentrations of fructose. Curve fitted using eqn (2) with K
the presence of fructose, but no fructose transport was
detected over 10 h. Similarly, UV-visible measurements of the
receiving phase showed a concomitant lack of picrate in the
receiving phase, confirming that little Na+ transport was
= 400 M−1
.
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occurring. Both observations suggest that the fructose complex greater than the concentration of boronate, this gives the
of 3 is too polar to efficiently partition into the apolar phase. relationship below:
The lack of fructose transport by 3 is similar to that observed
KD þ K′ K½fructoseꢀ
D
by Karpa et al. for a cholesterol–phenylboronic acid conjugate,
which they ascribed to slow diffusion across the aqueous–
organic interface for very lipophilic boronic acids.27
KDðobsÞ ¼
ð1Þ
1 þ K½fructoseꢀ
By approximating the values of KD and K′ from the distri-
D
To measure changes in lipophilicity caused by complexa-
tion to fructose, the distribution constant (KD) for cholate
boronic acid 3 and cholate 4 in the presence of fructose was
measured. Solutions of 3 or 4 in 1-octanol (10 mM) were pre-
pared and an equal volume of pH 8.5 buffer was added. The
aqueous–organic mixture was vigorously mixed for 180 s
before standing for 1 h. To calculate the concentration of 3 or
4 in the organic layer, the UV-visible absorbances (260 nm) of
aliquots from the organic layer were measured. The same pro-
cedure was repeated for 0.01, 0.1 and 0.5 M fructose in pH 8.5
phosphate buffer. A plot of the buffer/octanol distribution con-
stant (KD) at pH 8.5 for ionophore 3 and cholic acid 4 as a
function of changing fructose concentration revealed a strong
decrease in the lipophilicity of 3 in the presence of fructose,
decreasing from 2.0 in the absence of fructose to 1.2 at concen-
trations of fructose above 0.1 M. In contrast the pH 8.5 buffer/
octanol distribution constant for cholic acid 4 was relatively
unaffected by the presence of fructose, and was between 2.2
and 2.6 at all concentrations of fructose tested (Fig. 5a). These
observations are consistent with ionophore 3 binding to fruc-
tose to produce a complex that is less able to partition into the
membrane and mediate transport.
To allow us to model these changes in KD, the binding
affinity (K) of compound 2 for fructose was determined under
conditions similar to those used for the HPTS assays (pH 8.5).
By using pyrocatechol violet in a competitive displacement
assay28 and following the method developed by Wang,22 the
affinity of 2 for fructose in phosphate buffer (20 mM NanHmPO4,
100 mM NaCl, pH 8.5) was determined as K ∼ 230 M−1. The
partition data for fructose + 3 were then fitted to a simple
model, where the measured value of the distribution constant
bution coefficients of 3 with and without 1 M fructose, com-
bined with the approximate binding constant for 2 to fructose
in pH 8.5 buffer, a good fit was found to the distribution con-
stant data using eqn (1) (Fig. 5a).
This analogy was extended by fitting the HPTS responses
for 3 after 1200 s (ESI Fig. S4†) to eqn (2). In this relationship,
1 is the normalised response in the absence of fructose, and
0.17 is the estimated normalised response at saturating fruc-
tose concentrations.
1 þ 0:17K½fructoseꢀ
Response ¼
ð2Þ
1 þ K½fructoseꢀ
This equation gave a reasonable fit (Fig. 4b) with values of
K between 300 and 400 M−1, similar to the measured affinity of
2 for fructose and consistent with complexation of fructose
inhibiting of ion transport. This agreement is remarkable
given that ion transport recorded by an HPTS assay is a combi-
nation of factors, including the rate of ionophore distribution
across the population of vesicles.29a
The similarity between the decline in ionophoric activity of
3 with increasing fructose and the decrease in KD with increas-
ing fructose suggests that ionophoric activity is modulated by
the ability of the boronate to partition into the bilayer. Support
for this suggestion comes from models of carrier-mediated ion
transport across bulk organic phases.30 By assuming that
transport across a bilayer will be broadly analogous to trans-
port through a bulk organic phase and that low saturation con-
ditions exist at both bilayer–aqueous interfaces, diffusion of
the Na+/boronate complex through the buffer–bilayer interface
would be the rate limiting step. The rate of transport across
the bilayer would then be proportional to the ability of the
ionophore to partition into the membrane.
KD(obs) will be an average of KD and K′ weighted according to
D
the fraction of free boronate and fructose-boronate complex
respectively (Fig. 5b). With the concentration of fructose much
Conclusions
2-(Methylaminomethyl)phenylboronic acids have been shown
to transport Na+ across phospholipid bilayers. Boronic acid–
cholate conjugate 3 transported Na+ through carrier rather
than channel mechanisms despite cholate derivatives being
well known to form channels in phospholipid bilayers.16,17
The conjugate 3 was less effective than the smaller 2-(methyl-
aminomethyl)phenylboronic acid 2 at transporting Na+ across
bilayers, perhaps due to slower diffusion through the mem-
brane or slower redistribution across the vesicle population,29
but 3 was more effective at transporting across a bulk organic
phase. We have also shown that Na+ transport by 2-(methyl-
aminomethyl)phenylboronic acids 2 and 3 is not coupled to
saccharide transport, and that fructose complexation instead
switches off ion flow. Mechanistic studies suggest that
Fig. 5 (a) Changes in the distribution constant KD for ionophore 3 (red,
), and cholate 4 (blue, ) in the presence of 0 M, 0.01 M, 0.1 M, and 0.5
M fructose. Curve fit added for 3 using eqn (1) and K = 230 M−1, with
linear curve fit for 4 to guide the eye. (b) Model mechanism for the par-
tition of 3 into the bilayer in the presence of fructose.
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complexation to fructose shuts down ionophoric activity due indicated by (br). Due to quadrupolar relaxation, the aryl
to the lower lipophilicity of the Na+/saccharide/boronate com- carbon atoms attached directly to boron are not always
plexes inhibiting partitioning into the bilayer.
observed by 13C NMR.24 Electrospray mass spectra were
The studies presented here may open a pathway towards measured on Micromass Prospec and Micromass Platform
saccharide-sensitive ionophores, provided that high selectivity spectrometers; samples were prepared using 50 : 50 : 0.1 aceto-
for targeted saccharides under physiological conditions6 can nitrile–water–formic acid solution. High resolution mass
be engineered into the boronic acid moiety. Several design spectra (HRMS) were made on a Thermo Finnegan MAT95 XP
rules can be elucidated for the construction of such saccharide instrument. Infrared spectra were recorded on a Bruker Alpha-
sensitive boronic acid ionophores. The boronic acid must be P spectrometer and analysed using OPUS 6.5 software package.
designed so that the pKa lies below the pH of the sending Elemental analysis was performed using a Thermo Scientific
(exterior) solution. It may not be necessary to have a crown FLASH 2000 Series CHNS/O Analyzer. UV-Visible absorption
ether or other Na+ complexing site appended to the boronic measurements used Sigma Spectrophotometer Silica (Quartz)
acid for sodium ion transport,11 as the hydroxyl-lined cholate cuvettes (10 mm pathlength) and were recorded on a Jasco
on 3 did not provide greater activity than simple boronic acid V660 spectrophotometer with Jasco EHC-716 Peltier tempera-
2. Other simple saccharides should produce a different effect ture control. Fluorescence spectroscopy was carried out on a
on ionophoric activity, depending upon binding constant and Perkin-Elmer LS55 fluorimeter, with an attached Julabo F25-
complex lipophilicity. Covalently linking together cholates into HE water circulator for temperature control. All pH measure-
bis(cholate) dimers17 with boronic acids at both termini did ments were recorded on a HANNA pH 212 microprocessor pH
not give any improvement in Na+ transport activity, and these meter using a Hamilton MINITRODE pH electrode that was
compounds also followed a carrier mechanism with similar calibrated using Fisher Chemicals standard buffer solutions
activity.31 Transport of other cation/anion combinations are yet (pH 4.0 – phthalate, 7.0 – phosphate, and 10.0 – borate).
to be investigated, although preliminary studies indicate K+ is Unless otherwise stated all binding constants and pKa values
transported in much the same way as Na+ (ESI Fig. S6†).
Given recently reported biological applications of a new
generation of boronates able to complex to polysaccharides
under physiological conditions,2b,5e,32 work is ongoing to gene-
were determined at 20 °C.
Synthesis of compound 3
N-[8′-(Benzyloxycarbonylamino)-3′,6′-dioxaaminooctyl]-
rate new types of boronic acid-based antibiotics that are 3α,7α,12α-trihydroxy-5β-cholan-24-amide. To
a solution of
responsive to the concentration of targeted polysaccharides in cholic acid (1 g, 2.4 mmol) in dry dimethylformamide (20 mL)
the surrounding environment.
was added O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HBTU, 0.91 g, 2.4 mmol, 1 eq.) and N,N-
diisopropylethylamine (DIPEA, 0.42 mL, 2.4 mmol, 1 eq.). The
reaction mixture was stirred for 1 hour, over which time the
solution took on a yellow coloration, indicating the formation
Experimental section
Benzeneboronic acid 1 and cholic acid 4 were purchased from of the active benzotriazolyl ester. A solution of 1-(benzyloxy-
Sigma-Aldrich and used as received. Phospholipids were carbonylamino)-3,6-dioxa-8-aminooctane (0.68 g, 2.4 mmol,
obtained from Avanti Polar Lipids. Unless otherwise stated, all 1 eq.) in dry dimethylformamide (5 mL) was then added to the
other reagents and solvents were obtained from commercial solution, which was stirred overnight. The solution was diluted
suppliers (Sigma-Aldrich, Fluka, Acros and Alfa Aesar). Anhy- with dichloromethane (50 mL) and washed successively with
drous dichloromethane was obtained by distillation from dilute hydrochloric acid (1 M, 25 mL), saturated sodium hydro-
calcium hydride, and anhydrous tetrahydrofuran was obtained gen carbonate solution (25 mL) and brine (25 mL). The
by distillation from sodium. Thin layer chromatography (TLC) organic layer was dried over magnesium sulphate, filtered, and
was carried out using Merck aluminum-backed F254 silica gel the filtrate evaporated in vacuo. Flash chromatography (SiO2,
plates and visualised with either UV light (256 nm or 365 nm), eluent: ethyl acetate–methanol 9 : 1 v/v) of the residue provided
alkaline aqueous KMnO4, ninhydrin, 2,4-dinitrophenylhydra- the title compound as a white foam (1.06 g, 66%). 1H-NMR
zine, or cerium molybdate. Preparative column (flash) (400 MHz, CD3OD, 25 °C): δH ppm 0.58 (3H, s, C18/19 CH3),
chromatography was carried out using commercially available 0.79 (3H, s, C18/19 CH3), 0.91 (3H, d, J = 4.7 Hz, C21H3),
normal-phase silica gel. 1-(Benzyloxy carbonylamino)-3,6- 1.15–2.22 (24H, steroid CH/CH2), 3.33 (5H, t, J = 5.1 Hz, 2 ×
dioxa-8-aminooctane and 2-(methylamino-methyl)phenyl- NCH2 + C3H), 3.43–3.53 (8H, m, CH2OCH2CH2OCH2), 3.73
boronic acid
2
were synthesised according to literature (1H, m, C7H), 3.86 (4H, m, C12H + OH), 5.02 (2H, s, PhCH2),
procedures.33,34
5.68 (1H, br t, J = 5.2 Hz, amide NH), 6.65 (1H, br, carbamate
NMR spectra were measured on Bruker DPX300, AV400, or NH), 7.27 (5H, m, CHAr). 13C-NMR (100 MHz, CD3OD, 25 °C):
AMX500 instruments and were assigned using COSY, HMBC, δC ppm 12.5, 17.4, 22.5, 23.3, 26.3, 27.6, 28.1, 30.4, 31.7, 33.1,
HMQC and DEPT spectra where appropriate. Coupling con- 34.7, 34.8, 35.4, 35.5, 39.2, 39.4, 39.5, 40.8, 41.6, 46.4, 46.5,
stants J are given in hertz (Hz); multiplicities are given as 66.7, 68.4, 69.9, 70.0, 70.1, 70.2, 71.8, 73.1, 128.1, 128.2, 128.5,
singlet (s), doublet (d), triplet (t), quartet (q), quintet (qn), 136.6, 156.7, 174.5. MS (ES+): m/z 673.5 [M + H]+, 695.6
sextet (sxt), septet (spt), or multiplet (m); broad signals are [M + Na]+, 1346.3 [2M + H]+.
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(8′-Amino-3′,6′-dioxaaminooctyl)-3α,7α,12α-trihydroxy- Procedure for HPTS fluorescent assay of metal ion transport
5β-cholan-24-amide. To a solution of N-[8′-(benzyloxycarbonyl- for boronocholates with and without fructose present
amino)-3′,6′-dioxaaminooctyl]-3α,7α,12α-trihydroxy-5β-cholan-
24-amide (0.2 g, 0.3 mmol) in methanol (10 mL) was added
A lipid thin film was first prepared by the addition of spectro-
scopic grade chloroform to egg yolk phosphatidylcholine
acetic acid (1.7 μL, 0.03 mmol) and 10% palladium on acti-
(EYPC, 40 mg, 52 μmol) and cholesterol (5 mg, 13 μmol) in a
vated charcoal (0.02 g). The reaction vessel was degassed three
round bottomed flask (5 mL). Chloroform was slowly evapor-
ated in vacuo at room temperature to leave a lipid thin film on
the interior of the flask. HPTS phosphate buffer (1.2 mL,
100 μM HPTS, 20 mM MnHmPO4, 100 mM MCl, pH 6.5 where
times and stirred under hydrogen overnight. The solution was
filtered over Celite and evaporated in vacuo before the addition
of dichloromethane (10 mL); the organic layer was washed suc-
cessively with saturated sodium hydrogen carbonate solution
M+ = Na+ or K+ as appropriate) was added, followed by detach-
(25 mL) and brine (25 mL). The organic layer was dried over
ment of the lipid film from the interior of the flask by vortex
magnesium sulphate, filtered, and the filtrate evaporated
agitation, and extruded through a polycarbonate membrane
in vacuo to yield the title compound as a pale yellow oil (0.16 g,
(200 nm pore size, 19×). To remove unencapsulated HPTS, an
1
99%). H-NMR (400 MHz, CD3OD, 25 °C): δH ppm 0.71 (3H, s,
aliquot of the suspension (1 mL) was diluted with phosphate
C18/19 CH3), 0.92 (3H, s, C18/19 CH3), 1.03 (3H, d, J = 6.5 Hz,
buffer (1.5 mL, pH 6.5) and purified by gel permeation
C21H3), 1.28–2.34 (24H, steroid CH/CH2), 3.15 (2H, t, J =
chromatography (GPC) on a PD10 desalting column (eluted
with 3.5 mL phosphate buffer). This provided a purified
suspension of HPTS-encapsulated vesicles (15.5 mM total lipid
concentration).
5.3 Hz, CH2NH2), 3.38 (2H, t, J = 4.7, NHCH2) 3.58 (2H, t, J =
5.9, NHCH2CH2), 3.69 (4H, s, OCH2CH2O), 3.73 (2H, t, J =
5.1 Hz, CH2CH2NH2), 3.82 (1H, m, C7H), 3.97 (1H, m, C12H).
13C-NMR (100 MHz, CD3OD, 25 °C): δC ppm 13.1, 17.9, 23.3,
An aliquot (25 μL) of the vesicle suspension was transferred
24.4, 28.0, 28.8, 29.7, 31.0, 31.3, 33.4, 34.2, 36.0, 36.6, 37.1,
to
a fluorescence cuvette containing phosphate buffer
40.3, 40.6, 40.8, 41.1, 43.2, 43.3, 47.6, 48.1, 68.0, 69.2, 70.8,
71.4, 71.5, 73.0, 74.2, 177.2. MS (ES+): m/z 539.5 [M + H]+.
HRMS for C30H54N2O6: expected 539.4015, found 539.4017.
(8′-((2-Boronobenzyl)amino)-3′,6′-dioxaaminooctyl)-3α,7α,12α-
trihydroxy-5β-cholan-24-amide (3). To a solution of (8′-amino-
3′,6′-dioxaaminooctyl)-3α,7α,12α-trihydroxy-5β-cholan-24-amide
(0.1 g, 0.19 mmol) in anhydrous methanol (5 mL) was added
2-formylphenylboronic acid (28 mg, 0.19 mmol) and stirred for
45 minutes. The reaction mixture was cooled to 0 °C and
sodium borohydride (36 mg, 0.95 mmol) was added slowly
portionwise, the resulting solution was stirred for 45 minutes
at 0 °C and for a further 3 hours at room temperature. The
solution was diluted with dichloromethane (20 mL) and
washed successively with dilute HCl (1 M, 5 mL), saturated
sodium hydrogen carbonate solution (5 mL) and brine (5 mL).
The organic layer was dried over magnesium sulphate, filtered
and the filtrate evaporated in vacuo to yield the title compound
(1.975 mL, 20 mM MnHnPO4, 100 mM MCl, pH 8.5 where M+ =
Na+ or K+ as appropriate) with stirring (0.19 mM total lipid
concentration) and fluorescence measurements were immedi-
ately started (ex. 405 nm and 460 nm, em. 510 nm, time inter-
val 8 s) as a function of time. After 180 s the relevant
ionophore was added (20 μL, 0.01 mM total ionophore concen-
tration, 5 mol%) and vesicles were lysed after 1200 s by the
addition of Triton X-100 (25 μL, 25% v/v in distilled water).
The change in the normalized ratio I460/I405 as a function of
time gave the rate of M+/OH− antiport across the phospholipid
bilayer. The above procedure was repeated at fructose concen-
trations of 0.25 mM, 0.5 mM, 10 mM, 0.1 M, 0.5 M, achieved
by ensuring each buffer composition contained the relevant
concentration of fructose.
U-tube metal picrate transport experiments
1
A chloroform solution of the potentially transporting species
(1 mM, 5 mL) was transferred to a glass U-tube (internal dia-
meter 10 mm) and incubated at 25 °C. To one side of the
U-tube was added a receiving phase of phosphate buffer
(2.5 mL, 20 mM NanHmPO4, 100 mM NaCl, pH 6.5), and to the
other side was added a source phase of 0.01% sodium picrate
in phosphate buffer (2.5 mL, 20 mM NanHmPO4, 100 mM
NaCl, pH 8.5). Addition of the source phase marked the start
of the experiment; aliquots (1 mL) were taken at intervals from
the receiving phase and analysed for the presence of picrate by
UV spectroscopy (356 nm); after each measurement the sample
was immediately replaced back in the receiving phase of the
U-tube. The chloroform was stirred (300 rpm) for the entire
experiment to aid diffusion through to the receiving phase.
3 as a white foam (116 mg, 91%). H-NMR (400 MHz, CD3OD,
25 °C): δH ppm 0.70 (3H, s, C18/19 CH3), 0.92 (3H, s, C18/19
CH3), 1.01 (3H, d, J = 6.7 Hz, C21H3), 1.05–2.35 (24 H steroid
CH/CH2), 3.09 (2H, t, J = 5.7 Hz, CH2NH), 3.29 (2H, t, J =
5.7 Hz, NHamideCH2), 3.36 (1H, m, C3H), 3.53 (2H, t, J = 5.1 Hz,
NHamideCH2CH2), 3.67 (4H, m, OCH2CH2O), 3.81 (3H, t, J =
5.4 Hz, CH2CH2NH + C7H), 3.95 (1H, m, C12H), 4.07 (2H, s,
PhCH2), 7.12–7.24 (3H, m, CHAr), 7.39–7.45 (1H, m, CHAr).
13C-NMR (100 MHz, CD3OD, 25 °C): δC ppm 13.1, 17.8, 23.2,
24.3, 27.9, 28.8, 29.6, 31.2, 33.3, 34.1, 35.9, 36.5, 37.0, 40.2,
40.5, 41.0, 43.0, 43.2, 48.1, 48.2, 49.1, 49.3, 54.9, 68.0, 69.0,
70.7, 71.2, 71.4, 72.9, 74.0, 79.5, 126.8, 127.7, 128.4, 128.9,
129.2, 131.5, 176.9. MS (ES+): m/z 637.5 (100%) [M − 2H2O +
H]+, 673.6 [M + H]+. HRMS for C37H57BN2O6: expected
637.4388,
found
637.4372.
Elemental
analysis
for
U-tube fructose transport experiments
C37H62BN2O9·CH2Cl2: expected C: 58.92%, H: 8.33%, N:
3.62%; found C: 58.46%, H: 8.19%, N: 3.41%. νmax/cm−1 3357 A chloroform solution of the potentially transporting species
(OH), 2930, 2869, 1649, 1214, 1076, 745.
(1 mM, 5 mL) was transferred to a glass U-tube (internal
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Organic & Biomolecular Chemistry
diameter 10 mm) incubated at 25 °C. To one side of the U-tube constant Keq2 was determined by plotting 1/P as a function
was added a receiving phase of phosphate buffer (2.5 mL D2O of Q, where P is defined as:
containing 0.75% % 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid
1
½Ioꢀ
as an internal standard, 20 mM NanHmPO4, 100 mM NaCl, pH
6.5), and to the other side was added a source phase of fruc-
tose (1 M) and 0.01% sodium picrate in phosphate buffer
(2.5 mL D2O containing 0.75% 3-(trimethylsilyl)propionic-
2,2,3,3-d4 acid as an internal standard, 20 mM NanHmPO4,
100 mM NaCl, pH 8.5). Addition of the source phase marked
the start of the experiment; aliquots (0.8 mL) were taken at
intervals from the receiving phase and analysed for the pres-
ence of D-fructose by 1H NMR spectroscopy, after each
measurement the sample was immediately replaced back to
the receiving phase of the U-tube. The chloroform was stirred
(300 rpm) for the entire experiment to aid diffusion through to
the receiving phase.
P ¼ ½Loꢀ ꢁ
ꢁ
ð3Þ
QKeq1 Q þ 1
where Lo is the total amount of boronic acid, Io is the total
amount of PV, Keq1 is the binding constant of the PV-2
complex, and Q is the ratio of the concentration of free PV to
complexed PV,
½Iꢀ
A ¼
ð4Þ
½ILꢀ
where I is the concentration of free PV in solution, and IL is
the concentration of complexed PV in solution. The binding
constant for 2 with a diol can be calculated by dividing Keq1 by
the gradient of a plot of 1/P vs. Q.
Distribution coefficient determinations
Acknowledgements
Solutions of relevant ionophores were prepared in 1-octanol
(10 mM, 0.5 mL) and sodium phosphate buffer (20 mM
NanHmPO4, 100 mM NaCl, pH 8.5) was added (0.5 mL). The
aqueous–organic mixture was vigorously mixed using a vortex
mixer for 180 s before standing for 1 h to allow separation of
the organic and aqueous layers. Aliquots (100 µL) of the
1-octanol layer were transferred to a UV-visible cuvette contain-
ing HPLC-grade methanol (1.9 mL) and mixed before measure-
ment (260 nm). The procedure was repeated for different
fructose concentrations in sodium phosphate buffer (20 mM
NanHmPO4, 100 mM NaCl, pH 8.5, [fructose] = 0.01, 0.1, 0.5 mol
L−1). To provide a maximum absorbance reading solutions of
relevant ionophores were prepared in 1-octanol (5 mM, 100 μL)
and transferred to a UV-visible cuvette containing HPLC-grade
methanol (1.9 mL, 0.25 mM total ionophore concentration) and
mixed before measurement (260 nm).
J.R.D.B. and S.J.W. thank the EPSRC for funding (EP/F055315/1).
I.C.P. would like to thank the EU Seventh Framework Pro-
gramme for a Marie Curie Intra-European Fellowship (Grant
PIEF-GA-2012-328025 MagNanoVes).
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