Synthesis and ion transport activity of oligoesters containing an environment-sensitive fluorophore

Article abstract of DOI:10.1039/c1ob06047c

A series of oligoesters based on a rigid triphenyl-diyne core is described. The molecules were readily synthesized from key intermediates, and retained good solubility properties. One of the compounds displayed modest ion transport activity in vesicles, w

Full text of DOI:10.1039/c1ob06047c

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PAPER
Synthesis and ion transport activity of oligoesters containing an
environment-sensitive fluorophore†
Joanne M. Moszynski and Thomas M. Fyles*
Received 29th June 2011, Accepted 5th August 2011
DOI: 10.1039/c1ob06047c
A series of oligoesters based on a rigid triphenyl-diyne core is described. The molecules were readily
synthesized from key intermediates, and retained good solubility properties. One of the compounds
displayed modest ion transport activity in vesicles, was capable of forming highly conducting single
channels in planar bilayers and exhibited an irregular non-linear current–voltage response. All the
reported molecules had minimal aqueous fluorescence while being highly fluorescent in less-polar
media including lipid vesicles; their partitioning into the membrane could be monitored by a significant
blue-shift and increase in fluorescence intensity, as well as a decreased extent of quenching in vesicles
over that in water. The combined data indicated that the compounds are highly aggregated in aqueous
solution, which limits their membrane partitioning and ion transport activity, in agreement with
mechanisms proposed for other ‘simple’ oligoester channels.
chains,22–24 as well as a modified diphenylacetylene or ‘Dip’
Introduction
moiety.25 The Dip molecules exhibit environment-sensitive flu-
orescence due to the diphenylacetylene chromophore,26–28 this
allowed direct detection of partitioning dynamics which in turn
lent support to a proposed mechanism based on rate-limiting
aqueous phase aggregation.25 The Dip oligoesters were also found
to be highly transport-active, with rates enhanced by up to 5-fold
over the saturated analogs, due to more efficient partitioning of
the rigid Dip moiety and/or increased stabilization of the channels
formed within the membrane.18,29
Synthetic ion channel research has been extensively pursued
for the past several decades, and entire classes of transport-
active molecules have been made over this time.1–6 A number
of these compounds exhibit interesting properties in addition
to ion transport; they have been used as antibiotic agents7–10
and sensors for analytes such as sugars.11 Synthetic systems are
also becoming increasingly sophisticated; ion transport activity
can be regulated by voltage,12–14 light15 and ligand-binding16 and
additional examples continue to be reported in the literature.
One ongoing focus has been the design and synthesis of
structurally ‘simple’ compounds which are believed to act as
aggregate channels in the membrane.1 While these structures are
easily accessible by relatively facile syntheses and can be highly
transport-active,17–19 in most cases the in-membrane structures of
these species remain unknown. Accordingly, increasing attention is
being paid to characterizing the fate of these compounds as they
interact with lipid bilayers. Fluorescently-labelled synthetic ion
channels offer one such possibility of monitoring the bilayer parti-
tioning and dynamics of putative channel-forming compounds,20,21
which could lead to a better understanding of how these ‘simple’
channels act.
While the Dip fluorescence is a valuable tool, its intensity is
fairly low; a more efficient fluorophore would be more useful as
a ‘light up’ membrane probe,30 as well as simplifying detection of
low concentrations of compound. An extended rigid fluorophore
derived from diphenylacetylene would therefore be promising in
this regard. In addition, as the Dip studies indicated a positive
correlation existed between compound rigidity and ion transport
activity, the potential to enhance this further by a longer rigid
aromatic moiety was another motivation. A number of structures
were considered but a majority were rejected based on the known
poor solubilities of oligo phenylacetylenes.31 We reasoned that this
was in part due to the high symmetry of these compounds and as
we were interested in end-differentiated molecules, we focussed
our attention on lower symmetry fluorophores.
Recent efforts from our own lab have involved a series of
oligoester ion channels containing both fully-saturated alkyl
This paper reports the design, synthesis and functional char-
acterization of a series of potential channel formers built upon
an extended, tri-aromatic ‘Trip’ scaffold. An example of such a
target structure; HO2C-Trip-Hex-G(12)-OH‡ is shown below, in
Department of Chemistry, University of Victoria, PO Box 3065, Victoria,
BC, Canada. E-mail: tmf@uvic.ca; Fax: +1 250 721 7147; Tel: +1 250
7217192
† Electronic supplementary information (ESI) available: synthetic exper-
imental details and characterization (1H and 13C NMR, HPLC, MS) of
new compounds; transport and fluorescence assay details; bilayer clamp
summary data, as discussed in text. See DOI: 10.1039/c1ob06047c
‡ The trivial naming of the compounds follows from the linear structures
with the carboxyl and hydroxyl termini explicitly specified; named subunits
are linked as esters. The G(12) unit indicates a diester of 3-hydroxy glutaric
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comparison to the previously-developed Dip oligomer HO2C-Dip-
Hex-Hex-G(12)-OH. The Trip-containing molecules retain similar
structural features as the Dip oligomers such as a membrane-
spanning length of approximately 3.5 nm from carboxyl to
hydroxyl termini, and an overall dissymmetry to potentially access
voltage-gated ion transport behaviours.
Results & discussion
Synthesis
The synthesis of the Trip scaffold-containing key intermediate
6 began with the facile Sonogashira32,33 coupling of propargyl
alcohol to the known34 4-bromo-4¢-iodobiphenyl compound 1 to
produce 2 in 89% yield (Scheme 1). Under the utilized conditions,
only the iodo substituent underwent the coupling reaction. The
next step required a higher-temperature Sonogashira reaction
with TMS acetylene to effect cross coupling of the bromo
substituent of 2, to produce 3 in a fairly good yield of 71%.
After a simple TMS deprotection with K2CO3, the terminal alkyne
4 underwent another cross-coupling reaction with the prenyl-
protected aromatic iodide 5, to furnish the protected Trip scaffold
6. Compound 5 was made simply by the ester coupling of prenyl
alcohol to 4-iodophenylacetic acid.†
Once in hand, the Trip scaffold was extended into dimeric or
trimeric oligoesters by utilizing the ester coupling and deprotection
reactions previously developed for the Dip compounds.25 The alkyl
‘tails’ appended to the hydroxyl terminus of 6 under the standard
conditions were either known from previous work (compounds
9 and 11),23 or easily synthesized from commercially-available
starting materials (compound 7).†In all three cases, the ester
coupling reactions between the protected alkyl tails and the Trip
scaffold 6 were fairly good yielding; 71% for the reaction yielding
8, 73% for 10, and ~60% for each of the 2 steps leading to
14, via 12 and 13. The final step in all three syntheses was the
simultaneous removal of the prenyl and tBDMS protecting groups
present on the carboxyl and hydroxyl termini, respectively. This
was accomplished in one step using TMS triflate,25,35 leading to
the final products HO2C-Trip-G(E3)-OH, HO2C-Trip-G(12)-OH
and HO2C-Trip-Hex-G(12)-OH in overall acceptable yields.
The final compounds were easily purified by HPLC to produce
high purity samples for further analysis, and they retained
favourable properties throughout the syntheses, with the final,
deprotected oligomers being soluble in solvents such as methanol
and chloroform at millimolar concentrations. The fact that the
key intermediate 6 could be easily diversified into the desired
final compounds using known and highly-optimized reactions
Scheme 1 Synthesis of Trip-containing molecules. Abbreviations stan-
dard except PRE is prenyl (–CH2CHC(CH3)2).
in a maximum of 9 steps from commercially-available starting
materials is also significant.
Ion transport activity
Lipid vesicle assays
acid with a 12-carbon alkyl ester. The a,w-hydroxy acids of 6 and 10
carbons are indicated as Hex and Dec, respectively, while the ester of 4-
carboxymethyl-4¢-(hydroxymethyl)diphenylacetylene is referred to as Dip.
Only the final structures are named in this way.
The vesicle-based HPTS assay was used to assess ion transport
activity.36 In this experiment, the pH-sensitive dye (HPTS) is en-
trapped in vesicles to which a putative channel-forming molecule
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is introduced. An aliquot of NaOH is then added to initiate a
pH gradient. By monitoring the differential emission from the
acid and conjugate-base forms of the dye as ions are transported
across the membrane and the pH gradient is collapsed, the extent
of transport by the channel forming-molecule can be obtained
by procedures reported previously.22 Apparent rate constants can
then be obtained by plotting the extent of transport as a function
of concentration of the assayed compound. The results of this
assay for the polyether-tailed HO2C-Trip-G(E3)-OH are shown
in Fig. 1A and B; the compound is active at low concentrations,
as above-baseline activity was observed down to 3 mM of added
transporter. At concentrations above 20 mM, however, the rate
appears to level off, with only minor rate increases from 20 mM
to the maximum tested concentration of approximately 150 mM
being observed (Fig. 1B). Fitting the concentration-rate profile to
a saturation model gave a Vmax of 0.0155 s-1 and a concentration
at half maximal activity of 9.8 mM (r2 = 0.94).
test for surfactant-like activity, the carboxyfluorescein (CF) assay
was therefore carried out on HO2C-Trip-G(E3)-OH. This assay
detects large defects in the membrane of diameters greater than
˚
10 A, the approximate size of CF, by measuring the extent of
fluorescence enhancement as the self-quenched encapsulated dye
is released and diluted in the exterior aqueous medium.2,36 As
shown in Fig. 1C and D, extensive CF efflux equal to that of
Triton X100 was observed for HO2C-Trip-G(E3)-OH at relatively
low concentrations (~80 mM, Fig. 1D). Above 80 mM, further
increases in compound concentration up to 260 mM actually
reduced the extent of CF efflux, although this is most likely due
to poor solubility of the Trip compound in aqueous solution at
these high concentrations. This ‘saturation’ of CF efflux occurred
at a similar concentration range as did the HPTS ion-transport
activity, suggesting that the saturation is simply a solubility issue.
Note however that CF release requires a significantly higher
concentration of compound at the half-maximum (50 mM) that
was required in the HPTS assay.
Planar bilayer assay
The vesicle-based transport results suggest that HO2C-Trip-
G(E3)-OH behaves as a membrane disruptor or surfactant in
a manner that is closely akin to Triton X100. This was an
intriguing observation in light of the long history of single-
channel observations of Triton itself, and of other structurally-
derived ion channels.37,38,19,36 The original observation of Triton
single channels reported regular, long-lived (up to a few minutes),
predominantly ‘square-top’ type conductance behaviour with
conductances ranging from 100 to 400 pS, although shorter
lifetime openings and multiple-step openings were also observed;39
other common surfactants failed to produce single channel events.
Triton channels were found to be cation-selective, and had voltage-
dependent conductance under certain conditions. Despite reports
from other groups that support the existence of Triton single-
channels,40,41 they remain elusive due to their poor reproducibility
and the unconventional methods used to introduce Triton to the
bilayer membrane.18,29
Examples of bilayer activity observed for HO2C-Trip-G(E3)-
OH are presented in Fig. 2; other Trip-containing molecules were
completely inactive by this transport assay as well. HO2C-Trip-
G(E3)-OH exhibits a variety of behaviours ranging from short-
lived, highly-conducting spikes through irregular yet longer-lived
openings to almost idealized square-tops of fairly low conductance
(~30 pS for the trace in Fig. 2A). As shown in Fig. 2B, multiple
behaviours were usually observed in a single trace over a short
period of time, indicating a variety of active structures. Openings
varied greatly in both lifetime and conductance, and occurred in
approximately equivalent proportions in each tested electrolyte
(KCl, CsCl, NMe4Cl). The bilayer results thus reinforce the
similarity to Triton, and that HO2C-Trip-G(E3)-OH can act in
a dual role as both a surfactant and a discrete ion channel at low
concentrations.
Fig. 1 Vesicle-based transport activity. HPTS (A, B) and CF (C, D) data
for HO2C-Trip-G(E3)-OH. Raw data from both assays is shown in panels
A and C. The data were then analysed as discussed in text and SI to
provide transport rate constants (HPTS assay, B) or extent of CF efflux (CF
assay, D).
Other Trip-containing molecules were completely inactive, with
rates no higher than that of the THF or MeOH controls. The
activity of HO2C-Trip-G(E3)-OH is modest in comparison to the
previously-reported Dip oligomers; the maximum rate achieved
is only 40% of that exhibited by HO2C-Dip-Hex-Hex-(G12)-OH,
the most active in the series. The supposition that rigidity leads to
improved transport activity is therefore in question. The relatively
low activity of the only compound uncovered among the first few
examples prepared limited our enthusiasm for preparing the full
range of sequence isomers.
Saturation behaviour, as shown by HO2C-Trip-G(E3)-OH, has
been associated with membrane-disruption by surfactants.36 Struc-
turally, the amphiphilic HO2C-Trip-G(E3)-OH resembles a classic
surfactant such as Triton X100, which solubilises membranes at
or below its critical micelle concentration (cmc) of 200 mM. To
The observed behaviours were further analysed by a procedure
recently developed in our lab,42 the results of which are shown
in the bottom panel of Fig. 2. Each type of conductance activity
is assigned a corresponding colour (green for square-top, yellow
for flickers, blue for multiple opening, red for spikes, purple for
erratic), which is plotted on an activity grid. The horizontal axis
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Fig. 3 Conductance versus potential for HO2C-Trip-G(E3)-OH (1 M
NMe4Cl, diPhyPC) determined within the lifetime of a single opening
event. The three segments occurred in three separate experiments over a
span of 10 min.
Fig. 2 Voltage clamp activity of HO2C-Trip-G(E3)-OH. Conditions: A:
diPhyPC, 1 M CsCl, MeOH solution added to a bulk concentration of
0.5 mM, + 150 mV, 5 min following addition. B: diPhyPC, 1 M KCl,
concentration as A, +100 mV, 1 h following addition. C: cumulative activity
grids, see text.
shows three sections of the same experiment separated by about
10 min in which the conductance of these openings is clearly a
function of applied potential. Equally clearly there are dynamic
processes that alter the pore during the time of the opening such
that the magnitude and sign of the voltage dependence varies.
The Hille diameters in these cases range from 0.4 to 1.2 nm.
The asymmetry of the conductance behaviour implies overall
asymmetry of the conducting pore. The same must be true of the
channels formed by Triton X100, which are sometimes reported
to exhibit a voltage-dependent response, although this was not
conclusively observed by all authors.42
represents the event lifetime (on a log scale from 10 ms to 100
s), while conductance (in half log unit steps from 3 to 3000 pS)
is on the vertical axis. Every experimental record was analysed
and the activities were summed to produce the grids shown. The
color intensity reflects the frequency of a particular conductance-
duration within a type of conductance behavior. The dominant
activity is of the multiple-opening type (40% of all records contain
this activity). Other prevalent activities are spikes and erratic
behaviours (25% each). Only 10% of the observations were of
the square-top type shown in Fig. 2A and flicker activity was only
observed on a single occasion. Although statistically unfounded,
the summary grids further confirm the strong similarity of HO2C-
Trip-G(E3)-OH and Triton.42
Fluorescence studies
Fluorescence in solution
Previous studies on the Dip oligoesters exploited their
environment-sensitive excimer fluorescence to track the partition-
ing of the molecules from an aqueous solution into the vesicle
membrane. In water the compounds emit at 380 nm from an
excimer state; the introduction of lipid vesicles shifts this to
the ‘monomer’ emission at 320 nm. Over time, the recovery of
the 380 nm emission suggests the formation of in-membrane
aggregates.25
Consistently high conductances are produced by HO2C-Trip-
G(E3)-OH. In 75% of collected traces, conductances of over 100
pS were predominant, regardless of the type of activity observed,
and in over 20% of total experiments, the predominant behaviour
showed conductances greater than 1 nS. Occasionally, truly giant
pores corresponding to Hille diameters43 greater than 10 A were
˚
observed. These frequent large openings are due to channel
formation, and not membrane rupture, as direct detergent action
on the bilayer was rarely observed during the bilayer experiments.
These types of very large pores are rare in the synthetic ion channel
literature; some Triton openings are of high conductance,42,44 but
these do not occur with the Dip oligoesters,25 suggesting that the
increased rigidity of the Trip scaffold may be able to better stabilize
these larger channels.
Dissymmetric oligoesters potentially could give rise to voltage-
dependent conductance, but this has not been observed for any
previous compound of this class.45,25 As noted above, large long-
lived openings, sometimes of the square-top type, are formed by
HO2C-Trip-G(E3)-OH and it was therefore possible to alter the
applied potential during the period of a single opening. A non-
rectifying channel would show a constant conductance; Fig. 3
The low fluorescence intensity of the Dip moiety and its small
(~15 nm in MeOH) Stokes shift prompted the development
of the Trip fluorophore; it was therefore satisfying to observe
that in organic solutions such as THF or methanol, the Trip
compounds exhibit fluorescence intensity enhanced by at least
an order of magnitude over that seen for the Dip oligoesters
(Fig. 4). Significant fluorescence signal can still be observed at
sub-micromolar concentrations, below the detectable limit for the
Dip molecules. The absorption, excitation and emission spectra of
the Trip-containing compounds are all significantly red-shifted
in comparison to the Dip moiety as well. Quenching studies
such as those reported for the Dip molecules were also carried
out, and the Trip fluorophore can be quenched by CuSO4 in
methanolic solution, yielding derived Stern–Volmer constants of
approximately 230 M-1 (Table 1). The Trip molecules are also
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Table 1 Comparison of representative photophysical parameters of the
assess due to their extremely low aqueous fluorescence. However,
even at these very low intensities, the fluorescence response was
clearly not linear with concentration, and the maximum intensity
was reached at concentrations as low as 3 mM; they are evidently
far less soluble than the less-rigid precursors.
Dip and Trip fluorophores in methanolic solution
Property
Dipa
Tripb
e (M-1cm-1)
20 000
289
305
90 000
313
325
Absorption (lmax, nm)
Excitation (lmax, nm)
Emission (lmax, nm)
CuSO4 quenched/KSV
Besides aggregation-induced self-quenching, another possible
explanation for these molecules’ very low fluorescence in aqueous
solution is that water actually quenches the Trip fluorophore
by some specific solvent effect47 as has been postulated for a
number of known membrane probes.51–53 To determine whether
this was indeed a factor, a Stern–Volmer type experiment in
which a solution of compound 6 in MeOH was titrated against
increasing concentrations of aqueous buffer was carried out.
Selected scans are shown in Fig. 5A, while 5B illustrates a plot of
the fluorescence intensity as a function of water concentration. It
is evident from the data that any water quenching does not follow
a linear relationship. Rather, the observed behaviours are much
more consistent with an aggregation process as the fluorescence
intensity remains unchanged up to 40% water (22 M), and then
suddenly decreases by half at 45% water (24.8 M). Therefore, it
appears that above some critical concentration of water (24.5 M,
as determined from a logistic fit of the data) the Trip-containing
compound collapses into an aggregate, resulting in a dramatic
fluorescence decrease. The nature of the aggregate is not known
but it must have significant stability to form in the presence of a
significant concentration of methanol.
320
365
Yes/1000 M-1
Yes/230 M-1
a 16 mM HO2C-Dip-Hex-Hex-(G12)-OH25 b 16 mM HO2C-Trip-G(E3)-OH
Fig. 4 A: Excitation (dashed lines) or emission (solid lines) spectra of
14 mM HO2C-Trip-G(E3)-OH in CH3OH (black line) or aqueous buffer
(10 mM Na3PO4, 100 mM NaCl, pH = 6.4) (grey line). B: Emission
wavelength maximum as a function of solvent polarity in a variety of
tested solvents. C: photograph under UV light of solutions shown in A.
visibly fluorescent at low micromolar concentrations in MeOH
under UV illumination (Fig. 4C).
While the Trip fluorophore exhibited an intense emission in
organic solution, this was much reduced in water, as clearly
demonstrated in Fig. 4A and C. The fluorescence observed
in aqueous solution is also red-shifted in comparison to that
present in MeOH, shifting by 15 nm for HO2C-Trip-G(E3)-OH,
and nearly 40 nm for the longer trimer HO2C-Trip-Hex-G(12)-
OH. These characteristics are immediately suggestive of excimer
emission, analogous to that observed for the Dip isomers; however,
the extremely low intensity of this emission makes it of little
practical utility. More interestingly, the Trip compounds exhibit
solvatochromism that can be roughly correlated (r2 = 0.94) to the
polarity of the solvent, based on the ET scale46 (Fig. 4B).
The low fluorescence intensity of the Trip molecules in aqueous
solution was not completely unexpected as they were discovered
to visibly aggregate at fairly low concentrations; aggregation-
induced self-quenching47 is therefore a reasonable possibility. The
extent of aqueous aggregation is commonly assessed by the pyrene
assay.48,49 This was carried out for both the fully-saturated and Dip
oligoesters, yielding similar apparent cmc values of approximately
20 mM. The Trip molecules cannot be assayed in this manner
due to their fluorescence overlapping that of pyrene, however, an
apparentcmcof approximately5 mMwas obtained for HO2C-Trip-
G(E3)-OH from the point at which a concentration-intensity plot
deviated from linearity (data in the SI).50 The extent of aggregation
of the more hydrophobic Trip compounds was yet more difficult to
Fig. 5 A: Fluorescence emission spectra (Ex~325 nm) of 14 mM 6 in
aqueous buffer/MeOH mixtures; from top to bottom, solid lines = [buffer]
of 22, 24.8, 27.5, 30.3 and 41.3 M. Dashed line = 100% MeOH. B:
fluorescence intensity as a function of water concentration, fit to a logistic
function, r2 > 0.99.
A linear Stern–Volmer relationship was observed when CuSO4
was used as quencher. In analogy to the Dip molecules, the
quenching observed for the Trip fluorophore in aqueous solution
was significantly more efficient than in MeOH, with KSV values in
water approximately four-fold higher (1000 M-1, see the ESI for
spectra†). The fact that quenching by copper ion was observed
in both media for the Trip fluorophore indicated that analogous
experiments as carried out for the Dip compounds, in particular
quenching in the presence of vesicles, could be conducted on these
molecules as well.
Membrane localization
When a suspension of lipid vesicles was introduced into an
aqueous solution of HO2C-Trip-G(E3)-OH, the fluorescence
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intensity rapidly increased by approximately 2-fold, as shown
in Fig. 6A. This rapid increase in intensity is analogous to that
seen for the Dip compounds when interacting with vesicles, and
was previously used to monitor bilayer partitioning.25 As the
fluorescence intensity of the Trip molecules is much higher in
less-polar media, this suggests that bilayer partitioning is also
being observed by the experiment in Fig. 6A. Significantly, the
response displayed by the Trip isomers, which levels off after a few
minutes, is much faster than that observed for the Dip oligomers,
which continued to exhibit intensity changes for periods of time
up to 1 h. Further indication that the Trip molecule is associated
with the bilayer comes from the fact that the quenching seen with
externally-added aqueous copper is decreased by over 50% when
the compound is incubated with vesicles (see ESI†).
reported by You and Gokel; these authors suggest that the ion
transport activity observed for these compounds is only a fraction
of what could be possible.21
Polarity in vesicles
The titration results in Fig. 6B reveal a shift of the emission
maxima from that initially seen in water (385 nm) to 375 nm, and
the growth of a new band at 356 nm. A blue-shifting of emission
maximum upon vesicle introduction is a common occurrence for
many polarity-sensitive fluorophores, and has been used to probe
the polarity of the bilayer environment,47,56 and the localization of
synthetic ion channels.
The membrane localization behaviours of the Trip molecules
were determined for the polyether-tailed HO2C-Trip-G(E3)-OH
as well as the longer trimer HO2C-Trip-Hex-G(12)-OH, as both
compounds exhibited a linear ET-wavelength dependence (Fig. 4
and 7).
Fig. 6 A: Emission over time of an aqueous solution of 16 mM
HO2C-Trip-G(E3)-OH to which 0.5 mM lipid (as vesicles) has been
added at the time indicated. B: Static emission spectra of 16 mM
HO2C-Trip-G(E3)-OH at varying lipid concentrations. From bottom to
top, lipid concentrations = 0, 0.25, 0.5, 1, 2.5, 5 mM. Inset: Plot of CPS
versus lipid concentration, saturation not reached, Kp not determined. The
line is to guide the eye. Ex = 325 nm.
Attempts to quantify the extent of partitioning are shown in
Fig. 6B; in this experiment, a constant concentration of HO2C-
Trip-G(E3)-OH was titrated against increasing concentrations of
lipid vesicles (0–5 mM). This procedure has been used by others
to obtain a partitioning constant (Kp)51,54,55 however, in order for
this to occur, the maximal response upon complete partitioning
must be known. A Kp could not be obtained for HO2C-Trip-
G(E3)-OH (inset to Fig. 6B), as saturation was not reached at the
assayed lipid concentrations, and further increases were precluded
by the high turbidity of the suspension above these concentrations.
Despite this, the results are significant as they give at least a rough
indication of the affinity of this compound for the membrane. It is
clearly modest in comparison to that of known membrane probes
such as DPH (diphenylhexatriene), which exhibits lipid EC50
values (indicating the lipid concentration needed to incorporate
50% of the probe molecule) of 43 mM.54 HO2C-Trip-G(E3)-OH
is not fully partitioned even at 5 mM of lipid. Therefore, despite
its transport activity, the efficiency of partitioning for the Trip
molecule is poor.
Fig. 7 Solvent effects for HO2C-Trip-Hex-G(12)-OH. Fluorescence emis-
sion spectra (Ex~325 nm) of 17 mM compound in selected solvents (solid
lines), and pre-loaded into vesicles at ca. 3.5 mM (dashed line). INSET:
lMax as a function of solvent polarity. Black circles = tested solvents, open
circles = fit of vesicle wavelengths onto linear relationship.
While HO2C-Trip-G(E3)-OH was simply incubated with vesi-
cles for a short period of time, the more hydrophobic trimer was
pre-incorporated into the lipid, as it does not freely partition from
solution. Once the two maxima were compared to the linear
emission-polarity relationships, they corresponded to derived
in-vesicle ET values of 39 and 50 for HO2C-Trip-Hex-G(12)-
OH (Fig. 7). This compound showed two equivalent maxima,
possibly indicating that the Trip moiety is acting as two separate
fluorophores that probe different membrane environments. This is
plausible as the length of the Trip scaffold from carboxyl terminus
˚
to hydroxyl terminus is approximately 20 A, so it can clearly extend
through different regions of the bilayer.
This poor partitioning has consequences when considering the
vesicle-based activity assays discussed above (Fig. 1). In these
assays, the concentration of lipid as vesicles is kept constant at
0.5 mM, a concentration at which the compound is incompletely
partitioned. Similar incomplete partitioning has been recently
observed for a series of fluorescently-labelled peptidic ion channels
The ET derived polarity for HO2C-Trip-G(E3)-OH is very high
(ET = 57), comparable to or more polar than MeOH (ET = 55.4).
A number of conformations could account for this high value;
for example this could indicate that the compound is protruding
into the phosphocholine head group region. Another possibility
open to HO2C-Trip-G(E3)-OH is that the high polarity ET value
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is reporting an aqueous aggregate, as the fluorescence intensity of
this molecule in aqueous solution is relatively high in comparison
to the other, more hydrophobic analogs, and the blue-shift from
aqueous to in-vesicle of 10 nm is not as significant as that seen
for HO2C-Trip-Hex-G(12)-OH (up to 49 nm). The increasing
intensity of the band at 356 nm, which only appears in the presence
of vesicles and corresponds to a low-polarity ET value of 39,
must be tracking the actual membrane-inserted species (Fig. 6B).
Significantly, the presence and intensity of this band appears to be
inversely correlated with transport activity.
either a Macherey–Nagel “Nucleosil” RP C18 analytical (4 mm ¥
250 mm) or a Grace Davison “Alltima” RP C18 semi-prep (10 mm
¥ 150 mm) column. Solvents used (ACN, CH3OH; HPLC-grade)
were filtered through a Millipore sub-micrometre filter before use.
HPLC elution was monitored at various UV wavelengths (typically
254, 280 and 220 nm) and fluorometrically. Fluorescence spectra
◦
were run on a PTI QM-2 instrument at T = 20 C in either 10 ¥
10 mm quartz cells equipped with a micro stir rod, or 1 ¥ 10 mm
quartz cells (pyrene, CF assay). The syntheses followed directly
from previously-reported procedures.25
Sonogashira coupling:32 To a round bottomed flask equipped
with a septum, 1.2–1.5 equivalents (in relation to the alkyne
starting material) of the halogen-containing reactant and 6–10%
CuI were dissolved in dry solvent (typically DMF or THF), which
was then deoxygenated under vacuum. The alkyne reactant, 3–5%
Pd(PPh3)4, and 2–5 equivalents of NEt3 were then sequentially
added. The reaction was then stirred in the dark under N2 at
temperatures ranging from rt to 70 ◦C. Reactions were monitored
by TLC (silica gel, EtOAc/hexanes as eluent, visualized by UV,
p-anisaldehyde and/or vanillin stain). Once complete, reactions
were cooled if necessary, diluted with EtOAc and washed with
saturated EDTA (2–3 times), H2O (once), saturated NaCl (once),
dried over sodium sulfate, and concentrated under vacuum. Unless
noted otherwise, the crude products were purified by column
chromatography on silica gel, typically using EtOAc/hexanes as
eluent.
Conclusions
A series of triphenyldiynes based on a highly fluorescent Trip
chromophore was synthesized and characterized. While their
ion transport activity in vesicle assays was modest, the active
compound, HO2C-Trip-G(E3)-OH, exhibited a ‘dual role’ as both
a membrane-disrupting agent as well as a discrete ion channel,
similar to the reported activity of other ‘Triton-like’ molecules.
Based on the functional similarities between HO2C-Trip-G(E3)-
OH and the Triton channels, it is tempting to extrapolate to
a common mechanism based on the additional environmental
information provided by the compounds prepared. The following
structural constraints appear to be established: the Trip unit of
HO2C-Trip-G(E3)-OH is in a relatively polar but membrane-
associated environment, possibly as a surface-exposed structure,
while the Trip unit of HO2C-Trip-Hex-G(12)-OH resides in a
significantly less polar environment. The inactive compounds are
thus inserted into the membrane but are unable to form productive
channels. The partitioning of HO2C-Trip-G(E3)-OH is relatively
poor, yet the channels form easily and reliably. The channels
once formed are highly conductive in most cases, and can show
asymmetry. Taken together, these results suggest the insertion of
the Trip occurs from one side of the bilayer. Since it is unlikely
that the glycol portion directs entry, and it is also unlikely that the
carboxylate leads the insertion, a U-shaped insertion seems most
probable and most consistent with the data. The aggregation of
these U-insertions in one leaflet, followed by flip-flop to the other
would build a structure of the right size. It could also lead to the
observed, but irregular asymmetry of the conductance-potential
relationship as different numbers of molecules would be engaged at
different times. Triton is more flexible than HO2C-Trip-G(E3)-OH,
but the same sequence of events could lead to similar conducting
structures.
Ester coupling:25 To a solution of 1.3–2 equivalents of either
the alcohol or acid building block in relation to 1 equivalent of
the other (excess reagent choice determined by ease of synthesis
or availability) in dry solvent (typically THF) were added 1.3–2
equivalents of DIC, HOBt and 2.6–4 equivalents of DIPEA. The
reaction was sealed under an atmosphere of N2, and stirred in
the dark at temperatures varying between rt and 50 ◦C. Reaction
completion was monitored by TLC. Once complete, the reaction
was cooled (if necessary), filtered to remove DIU, and diluted
with DCM or EtOAc. The organic phase was extracted with
H2O (twice), saturated NaHCO3 (twice), rinsed with sat. NaCl
(once), dried with anhydrous sodium sulfate, and concentrated
under vacuum. Unless noted otherwise, the crude product was
purified by column chromatography on silica gel, typically using
EtOAc/hexanes as eluent.
THP removal: pTsOH (5–25%) was added to a solution of
compound in ~10–30% CH3OH: DCM, which was stirred at rt
for 1–3 h, as monitored by TLC. Once complete, the reaction was
diluted with DCM, washed with H2O (once), saturated NaHCO3
(twice), and saturated NaCl (once), then dried over sodium sulfate
and concentrated under vacuum. If necessary, further purification
was carried out as noted.
Experimental
General procedures
Prenyl deprotection: Adapted:35 0.025–0.1 equivalents TMSOTf
were added to the compound dissolved in DCM, which was stirred
at rt. Once complete (as monitored by TLC, generally < 1 h), the
reaction mixture was diluted further into DCM, washed with H2O,
saturated NaHCO3 and saturated NaCl, then dried over sodium
sulfate and concentrated under vacuum. Further purification was
carried out as noted. Full spectroscopic and characterization data
for all new compounds is available in the ESI.
Most chemicals and solvents were used as received from known
suppliers, except THF which was dried and distilled before use.
NMR spectra werecollected on at300MHzor 500MHz(protons).
UV spectra were run on a Cary 5 UV-VIS spectrometer in a 10 ¥
10 mm quartz cell. ESI Mass spectra were recorded on a Waters
MicroMass Q-TOF instrument running in negative ion mode;
Trip-containing compounds aggregate in solution so spectra
frequently contain intermolecular fragment-transfer (M+Trip)
and fragment (M-Trip) ions in addition to the molecular ion.
HPLC was performed using an HP Series 1100 instrument, with
Transport assays: the bilayer clamp,57,58 HPTS,22 CF,59 pyrene,
quenching and partitioning assays, as well as vesicle preparation25
have all been reported previously, details of the minor
7474 | Org. Biomol. Chem., 2011, 9, 7468–7475
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The Royal Society of Chemistry 2011
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modifications utilized are available in the ESI. Bilayer clamp
experiments conducted: diPhyPC, 1 M electrolyte (KCl, CsCl,
NMe4Cl), 5 mL of a 1 mM MeOH solution introduced to 5 mL
of electrolyte (0.5 mM bulk aqueous concentration), potentials
between 150 and -150 mV, 2–5 h of observations in replicate for
each electrolyte. Full trace summary information is available in
the ESI.†
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