Template effects of vesicles in dynamic covalent chemistry

Article abstract of DOI:10.1039/d0sc03185b

Vesicle lipid bilayers have been employed as templates to modulate the product distribution in a dynamic covalent library of Michael adducts formed by mixing a Michael acceptor with thiols. In methanol solution, all possible Michael adducts were obtained in similar amounts. Addition of vesicles to the dynamic covalent library led to the formation of a single major product. The equilibrium constants for formation of the Michael adducts are similar for all of the thiols used in this experiment, and the effect of the vesicles on the composition of the library is attributed to the differential partitioning of the library members between the lipid bilayer and the aqueous solution. The results provide a quantitative approach for exploiting dynamic covalent chemistry within lipid bilayers. This journal is

Full text of DOI:10.1039/d0sc03185b

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Template effects of vesicles in dynamic covalent
chemistry†
Cite this: DOI: 10.1039/d0sc03185b
All publication charges for this article Carlo Bravin
have been paid for by the Royal Society
of Chemistry
and Christopher A. Hunter
*
Vesicle lipid bilayers have been employed as templates to modulate the product distribution in a dynamic
covalent library of Michael adducts formed by mixing a Michael acceptor with thiols. In methanol solution,
all possible Michael adducts were obtained in similar amounts. Addition of vesicles to the dynamic covalent
library led to the formation of a single major product. The equilibrium constants for formation of the Michael
adducts are similar for all of the thiols used in this experiment, and the effect of the vesicles on the
composition of the library is attributed to the differential partitioning of the library members between the
lipid bilayer and the aqueous solution. The results provide a quantitative approach for exploiting dynamic
covalent chemistry within lipid bilayers.
Received 8th June 2020
Accepted 22nd July 2020
DOI: 10.1039/d0sc03185b
rsc.li/chemical-science
Results and discussions
Introduction
Michael acceptor 1 was obtained through a two-step synthesis
involving formation of the cyanoacetamide of n-decylamine
followed by an aldol condensation with benzaldehyde
(Scheme 1, ESI Section S2†). The lipophilic chain of 1 is
a membrane anchor, ensuring that the Michael acceptor is
efficiently incorporated into vesicle membranes.
Dynamic covalent libraries (DCL) employ reversible covalent
bonding to interchange different chemical components of
1
–5
compounds in a mixture.
This synthetic tool has led to
6
–8
discoveries such as complex reaction networks, synthetic self-
replicators, drug delivery systems
9,10
11–14
and stimuli responsive
15–19
assemblies.
The composition of a DCL can be modulated by
Separate reaction of 1 with each of thiols 2a–2d (Scheme 1) in
methanol gave adducts 3a–3d which could be characterised by
a template, which selectively amplies a complementary
molecular target in the mixture. A variety of template effects
based on inorganic anions, organic molecules or external
20
physical stimuli have been reported. DCLs have been studied
2
1,22
23,24
in multiple solvents systems,
at nanosystem interfaces,
25,26
and in response to changes in solvent environment.
Here we
report the behaviour of a DCL in the multiphase environment
provided by a membrane lipid bilayer in an aqueous phase,
which opens the possibility for spatial and temporal control of
27–30
the properties of the mixture.
Vesicles have been used to develop an understanding of
molecular events at lipid bilayer interfaces in biological
31–35
systems,
synthetic membrane anchored receptors have been Fig. 1 A vesicle can act as a template to change the composition of
a dynamic covalent library.
used for reaction control and transmembrane signal trans-
36–41
duction,
and a number of dynamic processes have been
42–44
investigated in the presence of lipid bilayers.
Here, we show
that vesicles can be used as templates to modulate the product
distribution of a DCL by exploiting the differential partitioning
properties of the library components (Fig. 1).
Department of Chemistry, University of Cambridge, Lenseld Road, Cambridge CB2
1
EW, UK. E-mail: herchelsmith.orgchem@ch.cam.ac.uk
†
Electronic supplementary information (ESI) available: Experimental details and
characterization of all new compounds, HPLC analysis of the adducts formed,
UV-Vis kinetic experiments. See DOI: 10.1039/d0sc03185b
Scheme 1 Addition of thiols 2a–2d to 1 leads to formation of the
corresponding Michael adducts 3a–3d.
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HPLC-MS and HPLC-ELSD (ESI Section S4†). Mixtures of
Michael acceptor 1 and thiols 2a–2d were then used to form
a DCL containing all of Michael adducts. Methanol was chosen
for these control experiments to ensure good solubility of all
library components and avoid any bias due to precipitation. A
mixture containing each of the four thiols 2a–2d at a concen-
tration of 0.15 mM in methanol was prepared. Fig. 2a shows the
HPLC trace obtained one hour aer adding the thiol mixture to
a 0.15 mM solution of 1. A mixture of all four Michael adducts
was obtained along with some unreacted 1.
Fig. 2b shows the corresponding HPLC trace obtained one
hour aer adding the thiol mixture to an aqueous solution of
Fig. 3 Time course for equilibration of the dynamic covalent library. At
DOPC vesicles containing 0.15 mM 1 in HEPES buffer at pH 7.2 time zero, 1 (3 mM) was mixed with 2a–2e (3 mM each) in 1.0 mL of
(
ESI Section S3†). There is a dramatic shi in the product methanol. After 1 hour (arrow), an aliquot of this mixture (0.1 mL) was
added to 2.0 mL of a vesicle solution (1 mM DOPC) in HEPES buffer at
distribution with formation of a single major product 3a in the
presence of vesicles. Addition of the same thiol mixture to an
aqueous solution containing only HEPES buffer at pH 7.2 gave
a mixture of all four Michael adducts, which demonstrates that
the change in product distribution is due to the presence of the
vesicles and is not due to phase separation or precipitation of
some of the library components in water (ESI Section S5 and
Fig. S16†). Experiments were carried out to demonstrate that
the thiol–Michael addition reactions were occurring under
reversible conditions. In separate experiments, four different
libraries each composed of three of the thiols were equilibrated
with 1 in methanol for one hour, then the fourth thiol was
added, and the system was allowed to re-equilibrate. In each
case, the same product distribution was obtained (ESI Section
S4.3†), conrming the reversibility of the process.
pH 7.2. The fraction of each product was calculated from the integrals
of the HPLC traces corrected by the relevant response factors. [ ] ¼
3a, [ ] ¼ 3b, [ ] ¼ 3c, [ ] ¼ 3d, [ ] ¼ 1.
a change in the product distribution was observed (arrow in
Fig. 3). The system was fully re-equilibrated aer one hour
giving Micheal adduct 3a as the only major product. The
dynamic nature of this process was demonstrated by equili-
brating thiols 2b, 2c and 2d with vesicles containing 1, and then
adding thiol 2a aer one hour. Michael adduct 3b was initially
observed as the major product. On addition of 2a, rapid
Fig. 3 shows the evolution with time of the DCL containing 1
and all four thiols. The mixture was fully equilibrated aer one
hour in methanol. At this point, vesicles were added and
Fig. 2 HPLC traces of the product distribution obtained 1 hour after
mixing 1 (0.15 mM) with 2a–2e (0.15 mM each) (a) in methanol, and (b) Fig. 4 (a) UV-Vis absorption spectra recorded after addition of 1.0 eq.
after addition of this mixture to an aqueous solution of vesicles (DOPC, of 2a to vesicles (1 mM DOPC) containing 0.15 mM 1 in HEPES buffer at
1
mM) containing 0.15 mM 1 in HEPES buffer at pH 7.2. The corre- pH 7.2. The black line is the spectrum at t ¼ 0, and the dashed line is the
sponding product distributions calculated from the integrals of the spectrum of vesicles without 1. (b) Time course for the formation of 3a
HPLC traces corrected by the relevant response factors are also for addition of different amounts of 2a to the solution of vesicles con-
shown.
taining 1: [2a] ¼ 0.03 mM [ ], 0.06 mM [:], 0.08 mM [C], 0.15 mM [-].
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exchange occurred to give the same product distribution ob- presence of vesicles. As expected from the DCL results shown in
tained by directly mixing all four thiols with vesicles containing Fig. 2a, the equilibrium constants are practically identical in
1, with 3a as the major product (ESI Fig. S14†).
methanol (ESI Section S6.3†), indicating that there is no
In order to understand the origin of the templating effect of intrinsic difference in the reactivity of the thiols or the stability
vesicles in the DCL experiment, the reaction of each of the of the Michael adducts. In contrast in the presence of vesicles,
individual thiols with 1 was investigated. Michael acceptor 1 has the equilibrium constants span three orders of magnitude.
a UV-Vis absorption band at 300 nm which disappears in the
Fig. 5a shows the relationship between the measured values
45–47
less conjugated Michael adduct.
Even in the presence of of log K and the octanol–water partition coefficients of the
vesicles, which cause some background scattering, the UV-Vis corresponding thiols, c log P, calculated using MarvinSketch.
absorption band of 1 can be used to monitor reaction with The values of log K measured in vesicles correlate rather well
a thiol. Fig. 4a shows UV-Vis absorption spectra recorded aer with c log P, and the slope of the line of best t is close to one,
addition of thiol 2a to vesicles containing 1. The formation of which suggests that the stability of the Michael adduct in the
adduct 3a was quantied by monitoring the disappearance of presence of vesicles is directly related to the hydrophobicity of
the absorption band at 300 nm, and Fig. 4b shows how the time the thiol.
course of the reaction depends on the concentration of 2a.
An explanation for this result is shown in Fig. 5b. Reaction of
The rate of formation of 3a is similar in all of the experi- a thiol with 1 in vesicles can be considered as two coupled
ments, and equilibration is complete aer 30 minutes. equilibria: in the rst step, the thiol partitions between the
However, the amount of 3a formed depends strongly on the aqueous solution and the hydrophobic membrane with an
concentration of 2a. The end points of the kinetics experiments equilibrium constant P, which depends on the solubility of the
in Fig. 4b were used to calculate an equilibrium constant thiol; then the reaction with 1 takes place inside the lipid bilayer
5
ꢂ1
of (2.1 ꢀ 0.2) ꢁ 10 M for formation of the Michael adduct 3a with an equilibrium constant K
in the presence of vesicles. When the same experiment was thiol.
carried out in methanol solution an equilibrium constant of
b
, which is independent of the
2
ꢂ1
(
8.0 ꢀ 0.7) ꢁ 10 M was obtained.
Conclusions
Equilibrium constants (K) for reaction with 1 were measured
for each of the thiols, both in methanol solution and in the Vesicles have been used to template the product distribution in
a DCL composed of a Michael acceptor and a mixture of thiols.
In methanol solution, a mixture of all possible Michael adducts
was observed, but addition of vesicles led to re-equilibration of
the DCL to give a single major product. Equilibrium constants
were measured for Michael adduct formation, and the results
show that the origin of the template effect is differential parti-
tioning of the reactants between the aqueous solution and
hydrophobic membrane of the vesicles. These ndings provide
useful guidelines for the design of molecular components for
exploiting dynamic covalent chemistry within lipid bilayers.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
C.B. thanks Marie Skłodowska Curie Actions for Individual
Fellowship (H2020-MSCA-IF-2018 number 837706).
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