Transmembrane signal transduction by cofactor transport

Article abstract of DOI:10.1039/d1sc03910e

Information processing and cell signalling in biological systems relies on passing chemical signals across lipid bilayer membranes, but examples of synthetic systems that can achieve this process are rare. A synthetic transducer has been developed that tr

Full text of DOI:10.1039/d1sc03910e

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Transmembrane signal transduction by cofactor
transport†
Cite this: DOI: 10.1039/d1sc03910e
b
a
Istvan Kocsis,a Yudi Ding,a Nicholas H. Williams
and Christopher A. Hunter
*
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Information processing and cell signalling in biological systems relies on passing chemical signals across
lipid bilayer membranes, but examples of synthetic systems that can achieve this process are rare. A
synthetic transducer has been developed that triggers catalytic hydrolysis of an ester substrate inside
lipid vesicles in response to addition of metal ions to the external vesicle solution. The output signal
generated in the internal compartment of the vesicles is produced by binding of a metal ion cofactor to
a head group on the transducer to form a catalytically competent complex. The mechanism of signal
transduction is based on transport of the metal ion cofactor across the bilayer by the transducer, and the
system can be reversibly switched between on and off states by adding cadmium(II) and ethylene
diamine tetracarboxylic acid input signals respectively. The transducer is also equipped with a hydrazide
moiety, which allows modulation of activity through covalent conjugation with aldehydes. Conjugation
with a sugar derivative abolished activity, because the resulting hydrazone is too polar to cross the
bilayer, whereas conjugation with a pyridine derivative increased activity. Coupling transport with
catalysis provides a straightforward mechanism for generating complex systems using simple components.
Received 16th July 2021
Accepted 17th August 2021
DOI: 10.1039/d1sc03910e
rsc.li/chemical-science
switches has been used in conjunction with liposomes to
induce passive18 or active19 actions within the system. Multi-
Introduction
functional membrane molecules have been used to design
complex synthetic systems capable of self-reproduction20 or
recognition-based transmembrane signalling.21
Cascade reaction pathways are a characteristic of biological
systems through which complex functions are accomplished.
Cellular communication, for instance, takes place via inter-
connected signalling and amplication cascades.1 In order to
achieve this, biological cells synergistically couple several
processes both simultaneously and subsequently using mainly
protein mediators.2 However, designing articial systems that
can mimic the complexity of such processes is not trivial.
Synthetic liposomes have proven to be useful models of some
properties of natural cells, allowing the study of both biological
and synthetic components at the lipid bilayer interface.3 The
hydrophobic environment of a bilayer acts as a localization
point for hydrophobic molecules,4 providing a quasi-two-
dimensional matrix and raising the apparent concentration of
membrane-bound molecules in comparison to the total bulk
solution.5,6 This property has been exploited in catalysis7 and
molecular imprinting.8–10 The compartmentalization afforded
by liposomes has been used to study signal transduction,11–13
cascade reactions14–16 and transport phenomena.17 More
recently, the concept of synthetic molecular motors and
We have recently reported a new class of membrane mole-
cules that act as signal transducers across lipid bilayers.22–24 The
membrane-anchored molecules are equipped with two different
switchable head groups which allow them to transmit chemical
signals across the lipid bilayer membranes of vesicles by
a membrane translocation mechanism. Coupling of an external
stimulus to a change in the polarity of one of the head groups
was used to control the location of the transducer molecules in
the lipid bilayer. Complexation of metal ions on the inside of
the vesicles by the second head group lead to formation of
a catalyst, which hydrolysed an ester substrate to generate the
amplied output signal.
Here, we describe a different signal transduction mecha-
nism, which is based on transport of an externally added metal
ion cofactor across a lipid bilayer membrane to generate
a catalyst for substrate hydrolysis on the inside of vesicles.
Scheme 1 shows the structure of the transducer molecule 5. The
steroid core acts as a membrane anchor to embed the
compound in the lipid bilayer of vesicles, and the pyridine-
oxime head group is a switchable catalyst, which hydrolyses
esters when bound to a transition metal ion. The acyl hydrazide
head group was designed as a point for covalent attachment of
different aldehydes by hydrazone formation in order to regulate
the properties of the catalytic head group.25–28 Hydrazones can
aDepartment of Chemistry, University of Cambridge, Lenseld Road, Cambridge CB2
1EW, UK. E-mail: herchelsmith.orgchem@ch.cam.ac.uk
bDepartment of Chemistry, University of Sheffield, Sheffield S3 7HF, UK
† Electronic supplementary information (ESI) available: Details of the synthesis
and spectroscopic characterisation of new compounds, characterisation of
hydrazone formation, ester hydrolysis experiments and cadmium transport
experiments. See DOI: 10.1039/d1sc03910e
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Scheme 2 Chemical structures of the ester substrate Ac-HPTS and
aldehyde derivatives 6–9.
uorescence was observed over time (see Fig. S6†). Repeating
this experiment in the presence of Cd2+ resulted in a steady
increase in uorescence intensity, which indicates hydrolysis of
the ester to give the uorescent product HPTS. This result shows
that complexation of Cd2+ by the pyridine-oxime head group of
5 leads to a catalytically active complex. A pH of 6.6 ensures that
the pyridine-oxime head group is protonated and inactive, but
the metal complex is deprotonated for nucleophilic catalysis.
We have previously used to Zn2+ pyridine-oxime complex to
catalyse this reaction, but we found a signicantly higher
activity for the Cd2+ complex (see Fig. S6†).
Scheme 1 Synthesis of 5.
The behaviour of 5 embedded in the bilayer of DOPC vesicles
was then investigated. Vesicles were prepared containing 5 in
the lipid membrane and Ac-HPTS encapsulated in the inner
compartment. When external Cd2+ was added to the vesicles,
a signicant increase in uorescence was observed, which
indicates hydrolysis of the substrate is taking place on the
be formed both in organic and aqueous solutions and are stable
at neutral pH.29–32 The presence of the hydrazide therefore
provides a handle for straightforward modication of the
properties of the system and for in situ functionalisation in
vesicles.33 However, we have found that compound 5 is able to
transduce chemical signals across a lipid bilayer without any
modication.
Results and discussion
Synthesis
Compound 5 was synthesized starting from lithocholic acid as
shown in Scheme 1. An 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) mediated coupling with t-butyl carbazate,
followed by condensation with chloroacetyl chloride gave 2.
Substitution with pyridine-oxime derivative 3 and subsequent
deprotection of the hydrazide gave 5. Pyridine-oxime 3 and the
ester substrate (Ac-HPTS in Scheme 2) were synthesized as
previously described.22 The aldehyde derivatives 6, 7 and 8,
which were used to conjugate the hydrazide moiety are
commercially available, and the sugar derivative 9 was prepared
using the route shown in Scheme 2.
Fig. 1 External addition of Cd2+ to DOPC vesicles containing 2.5 mol%
5 embedded in the lipid membrane and 250 mM Ac-HPTS encapsu-
lated in the inner compartment at pH 6.6 with 50 mM MES buffer and
100 mM NaCl. At (a) CdSO4 (0–2500 mM) was added, and at (b) Triton-
X 100 and NaOH were added to attain complete substrate hydrolysis.
The fluorescence emission intensity due to the hydrolysis product
Transmembrane signalling
Initial assessment of catalytic properties of 5 was carried out in
aqueous solution with no vesicles present. When 5 was added to
a
buffered solution containing Ac-HPTS, no change in HPTS is shown (lex ¼ 415 nm, lem ¼ 510 nm).
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inside of the vesicles. Fig. 1 shows that addition of increasing
amounts of external Cd2+ to the vesicle suspension increases the
rate of internal substrate hydrolysis. These results indicate that
addition of Cd2+ to the outside of the vesicles results in
formation of a catalytically active pyridine-oxime complex,
which is able to hydrolyse the substrate on the inside of the
vesicles. In other words, 5 must transport Cd2+ across the
bilayer.
To test this hypothesis, an HPTS transport assay was used.30
Vesicles were prepared at pH 6.6 with CdSO4 on both the inside
and the outside of the vesicles and with HPTS encapsulated on
the inside. Addition of H2SO4 to the external solution was used
to generate a pH gradient across the bilayer, and changes in the
internal pH of the vesicles was monitored using the uores-
cence emission of HPTS (the ratio of the emission intensities at
510 nm and 460 nm provides a direct measure of local pH).
Fig. 2 shows the results obtained in the presence and absence of
5 in the bilayer membrane. In the control experiment (addition
of just DMSO), there was a small jump in the uorescence
emission on addition of external H2SO4, but subsequent
changes were small. In the presence of 5, the change in internal
pH was much larger, and no additional change in pH was
observed on lysing the vesicles with detergent, which shows that
the internal pH had equilibrated to match the external pH.
These results are characteristic of ion transport across the
bilayer. The possible transport pathways that would allow
equilibration of pH across the lipid bilayer are Cd2+/H+ antiport,
Cd2+/OHꢀ symport, OHꢀ/SO42ꢀ antiport and H+/SO42ꢀ symport.
However, the high dehydration energy associated with sulfate
Fig. 3 Transmembrane signal transduction by cofactor transport. In
the resting OFF state, the pyridine-oxime head group (red) is inactive.
The input signal is a metal ion cofactor (blue circle) added to the
external vesicle solution. Binding to the pyridine-oxime head group of
the membrane-embedded transducer in the outer leaflet leads to
transport of the metal ion across the bilayer to the inner compartment.
The metal complex of the pyridine-oxime head group (green) formed
in the inner leaflet catalyses hydrolysis of an ester substrate (grey balls)
encapsulated on the inside of the vesicles to generate a fluorescent
product (yellow balls) as the output signal of the ON state.
lipid bilayer, due to the steroid core, which provides a hydro-
phobic membrane anchor. The pyridine-oxime head group can
access both the outer and inner aqueous solutions. When
cadmium ions are present in the external solution, binding to
the pyridine-oxime head group leads to transport of the metal
ion across the bilayer. The active complex could be the singly
charged 1 : 1 complex, the neutral 2 : 1 complex, or transport
could proceed via a relay process where one transducer in the
outer leaet passes a metal ion to another transducer in the
inner leaet, but these mechanisms are difficult to distinguish.
Regardless of the precise mechanism, the result is that exter-
nally added cadmium ions appear in the inner compartment of
the vesicles. Formation of the catalytically active Cd2+ complex
of the pyridine-oxime head group on inner surface of the vesicle
bilayer leads to hydrolysis of the substrate inside the vesicles.
transport (DG ¼ +1080 kJ molꢀ1
)
means that the SO4
2ꢀ
31
mediated pathways are very unlikely. This experiment therefore
suggests that 5 transports Cd2+, which is consistent with the
results of the catalysis experiment shown in Fig. 1.
Fig. 3 illustrates the signal transduction mechanism that
accounts for the results in Fig. 1 and 2. Transducer 5 sits in the
Hydrazone modulation of activity
The second head group on 5 is an acyl hydrazide, which
provides a site for conjugation of aldehydes and an opportunity
for modulation of catalytic activity by formation of hydrazone
head groups of different polarity. Starting from compound 5,
hydrazones were formed either prior to incorporation into
vesicles or by in situ functionalisation of vesicles containing 5.
The reactions were quantitative, and Fig. 4 shows HPLC traces
of vesicle suspensions containing the hydrazone conjugates of 5
Fig. 2 Comparison of the effect 5 (black) and of DMSO (red) on
cadmium transport across vesicle bilayer membranes. DOPC vesicles
containing 1 mM HPTS were prepared at pH 6.6 and suspended in
10 mM MES buffer with 100 mM CdSO4 and either 5 or DMSO. At t ¼ obtained using the four different aldehydes shown in Scheme 2.
1 min, H2SO4 was added to lower the external pH to 6.1, and the local
pH inside the vesicles was monitored using the ratio of the fluores-
The hydrazones are stable in the aqueous conditions used for
these experiments, and no traces of the starting hydrazide 5
cence emission at 510 nm due to the phenol (lex ¼ 405 nm) and
were observed in any case. The in situ conjugation reactions
phenolate (lex ¼ 460 nm) forms of HPTS. At t ¼ 6 min, the vesicles
were much faster with aldehydes 6, 7 and 8 (complete conver-
sion in less than half an hour) compared with 9, presumably
were lysed by addition of Triton-X 100 to obtain the HPTS emission at
a pH of 6.1.
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catalysis by the aldehyde itself. When the Cd2+ transport
experiment shown in Fig. 2 was repeated using the hydrazone
conjugate formed by 5 and 6, the rate of transport was the same
as that observed for 5 alone (see Fig. S8†). These experiments
suggest that the pyridyl hydrazone increases the rate of the
catalytic reaction and does not increase the rate of transport by
chelating metal ions.
We conclude that the modulation of activity observed in
Fig. 5 is due to the effect of the hydrazone head groups on the
accessibility of the pyridine-oxime complex to the inner vesicle
solution (Fig. 6). The hydrophilic sugar moiety in 9 holds the
hydrazide head group in the aqueous solution, preventing the
transducer from crossing the bilayer and forcing the catalytic
head group into the membrane. As result, the metal ion
complex is not formed in the inner compartment, and there is
no substrate hydrolysis. The benzoic acid and n-heptyl head
groups appear to have little effect of the distribution of the
transducer in the membrane or on the accessibility of the
catalytic head group to the inner compartment. The enhanced
activity associated with the pyridyl head group is presumably
due to a change in the way in which the transducer sits at the
membrane interface that increases the activity of the catalyst in
the inner leaet.
Fig. 4 HPLC-UV traces (absorption at 280 nm) of DOPC vesicles
containing 5 or the hydrazone conjugates formed with aldehydes 6–9.
due to the polarity of the sugar group, which limits access of 9 to
the membrane anchored hydrazide.
Fig. 5 shows signal transduction experiments carried out
with the hydrazone conjugates. Vesicles were prepared con-
taining the hydrazone embedded in the lipid membrane and Ac-
HPTS encapsulated in the inner compartment. Aer 30
minutes, Cd2+ was added to the external solution of the vesicle
suspension. As observed previously for 5, in the absence of Cd2+
,
there was no signicant change in uorescence with time, but
addition of Cd2+ lead to catalysis of substrate hydrolysis. Fig. 5
shows that the nature of the aldehyde moiety has a dramatic
effect on activity. Conjugation of 5 with the sugar aldehyde 9
completely abolished signal transduction. The benzoic acid and
n-heptyl hydrazones show similar activity to the parent hydra-
zide, and the pyridyl hydrazone shows enhanced activity.
Additional control experiments were carried out to investi-
gate the possible role of the pyridyl hydrazone in increasing the
activity of the system. Compound 4 has a protected hydrazide
and cannot form hydrazones. When compound 4 was used in
place of 5, the rate of the catalysed reaction was unaffected by
the addition of aldehyde 6 (see Fig. S7†). Thus the enhanced
activity observed with 6 is not due to metal ion transport or
Reversible ON/OFF switching
The coupling of cofactor transport with catalysis represents
a novel mechanism for transmembrane signal transduction.
Catalytic generation of the hydrolysis product on the inside of
the vesicles is switched on by the presence of metal ions added
to the outside of the vesicles. It should therefore be possible to
make this system reversible by raising and lowering the external
metal ion concentration. Fig. 7 shows experiments on the most
active system, the hydrazone derivative formed by conjugation
of 5 and 6. Catalysis on the inside of the vesicles is switched on
by adding Cd2+ to the external vesicle solution and then
switched off again by external addition of EDTA. The transducer
transports Cd2+ into the internal compartment of the vesicles,
Fig.
5 Effect of aldehyde conjugation on transmembrane signal
transduction. At t ¼ 30 min, 1 mM CdSO4 was added to DOPC vesicles
containing 250 mM Ac-HPTS encapsulated in the inner compartment
at pH 6.6 with 50 mM MES buffer and 100 mM NaCl and with 2.5 mol%
of different transducers embedded in the lipid bilayer: the parent Fig. 6 Modulation of catalytic activity by hydrazone formation. The
hydrazide 5 (blue) or the hydrazone conjugates formed with aldehydes sugar head group locates preferentially in the aqueous phase, forcing
6 (red), 7 (yellow), 8 (green) or 9 (black). The fluorescence emission due the pyridine-oxime head group inside the lipid bilayer, and blocking
to the hydrolysis product HPTS is shown (lex ¼ 415 nm, lem ¼ 510 nm). any interaction with the metal ion cofactor.
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We have further shown that the hydrolytic activity of vesicle
systems containing 5 and Ac-HPTS can be controlled by turning
the system on and off by loading/unloading of liposomes with
Cd2+.
The multifunctional nature of the compounds presented
here open new avenues for the design of complex biochemical
tools. The catalytic function can be exploited to trigger drug
release from vesicles34 while at the same time making use of the
hydrazone formation for implementation in post injection
repurposing of drugs.35 Similarly, the dual transport/catalysis
property can be exploited for the design of nanoreactors,16,36
where compartmentalization via liposomes can lead to easily
controllable sequence reactions as well as simple purication
methods for the products.
Data availability
Fig. 7 Reversible ON–OFF switching of signal transduction using the
hydrazone conjugate formed by 5 and 6 (2.5 mol% in DOPC vesicles
containing 250 mM Ac-HPTS encapsulated in the inner compartment
at pH 6.6 with 50 mM MES buffer and 100 mM NaCl). In the resting
state, there is no hydrolysis of the ester substrate inside the vesicles.
Addition of Cd2+ (blue circle) to the external vesicle solution initiates
catalysis in the inner compartment of the vesicles, giving an ON state.
Subsequent addition of EDTA (orange triangle) to the external vesicle
All supporting data is provided in the ESI.†
Author contributions
IK, CAH and NHW devised the experiments, YD synthesised
solution removes the Cd2+ from the transducer head group turning the compound 9, IK carried out the experiments, and all authors
system OFF. Addition of more Cd2+ to the external vesicle solution
contributed to the manuscript.
turns the system back ON again. The fluorescence emission intensity
due to the hydrolysis product HPTS is shown (lex ¼ 415 nm, lem ¼ 510
nm).
Conflicts of interest
There are no conicts to declare.
where the activated catalytic head group hydrolyses the ester
substrate. When EDTA is added externally, the transducer
Acknowledgements
transports the Cd2+ back out of the vesicles and delivers it to the
EDTA, which cannot cross the bilayer. The removal of Cd2+
We thank the Engineering and Physical Sciences Research
Council (EP/R002584/1) for funding.
deactivates the catalytic head group and halts hydrolysis of the
substrate. Subsequent addition of more Cd2+ to the external
vesicle solution switches the catalysis on the inside of the
vesicles back on again. The rate of increase in uorescence
emission intensity appears slower in the second ON phase, but
this reects the consumption of substrate.
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