α-Helical peptide vesicles with chiral membranes as enantioselective nanoreactors

Article abstract of DOI:10.1039/c7cc05869a

We report peptide vesicles with chiral membranes as enantioselective nanoreactors. The peptide vesicles are able to selectively encapsulate only a single enantiomer from a racemic mixture solution through preferential diffusion across the membranes. Notab

Full text of DOI:10.1039/c7cc05869a

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
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α-Helical Peptide Vesicles with Chiral Membranes for
Enantioselective Nanoreactors
Received 00th January 20xx,
Accepted 00th January 20xx
Xi Chen, Yanqiu Wang, Huaxin Wang, Yongju Kim and Myongsoo Lee*
DOI: 10.1039/x0xx00000x
www.rsc.org/
We report peptide vesicles with chiral membranes as
enantioselective nanoreactors. The peptide vesicles are able to
selectively encapsulate only a single enantiomer from a racemic
mixture solution through preferential diffusion across the
membranes. Notably, the confined space of the vesicle acts as an
enantioselective nanoreactor for the encapsulated enantiomer
which undergoes a further chemical transformation to yield a
highly enantiopure product.
In living cells, many biological processes undergo within
compartments surrounded by a lipid membrane which is
selectively permeable to maintain homeostasis by regulating
the passage of some substances while preventing others from
entering the interior. The membrane encloses catalytically
active species in a confined space to protect them from the
surrounding environment and allows access and release of
chemical substances into and out of the compartments
through active and passive diffusions in which chemical
transformations undergo.1 Dendrimers and vesicles as
biological mimics have been constructed using the self-
assembly of amphiphilic molecules with the aim to obtain
confined space.2 For example, the dendrimers consisting of
hydrophobic, catalytic cores and hydrophilic exteriors can be
used to create confined space to undergo chemical reactions
in aqueous solution.3-6 Within the confined inner space
provided by dendritic architecture, reactions can undergo with
higher selectivity and greatly enhanced reaction rate.
Figure 1. (a) Chemical structures of
vesicle used as a nanoreactor. Enantioselective permeation and further chemical
transformation of D-enantiomer inside the vesicle containing reactant
1 and 2. (b) Schematic representation of
A
.
hydrophilic ones on each side similar to lipid bilayer
membranes. For selective chemical reactions to take place
inside nanoreactors, specific reactants should be able to enter
the interior of the vesicles by selective diffusion through the
membrane. The semi-permeability of the membranes plays a
profound role in selective access for substrates to the interior
of vesicles.7,9 However, most of the polymer vesicles are based
on non-chiral membranes which are far from the membrane
cavities with chiral environments.10
Another example is provided by amphiphilic block
copolymer vesicles which have been used to encapsulate many
different cargos, especially enzymes to generate catalytic
compartments to improve the efficiency and selectivity of the
catalytic process.7-9 Various amphiphilic block copolymers have
been employed to form vesicles with different sizes and
membrane thicknesses to be used as nanoreactors. The
membranes consist of a middle hydrophobic layer covered by
Given that folded helical peptides are intrinsic chiral
secondary structures, one of the ideal candidates for the
construction of chiral membranes is their self-assembly into an
extended membrane structure that allows chiral cavities
between the helical strands.11-15 For example, the
incorporation of oligoether side chains into short hydrophobic
peptides induces an α-helical conformation through
aggregation into vesicular membranes with chiral void space.16
However, the vesicles exhibit low chiral discrimination
capabilities that hamper vesicles to use as highly efficient
enantioselective nanoreactors, most probably, due to internal
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peptide
2 containing two pyrenes as side groups shows two
negative bands at 206 nm and 222 nm, demonstrating that the
peptide backbone adopts an α-helical structure.20 Even after
adding the helix promoting solvent 2,2,2-trifluoroethanol (TFE),
the CD intensity at λ= 222 nm did not change, indicating nearly
maximal helicity.21 We envisage that the laterally-grafted rod-
like α-helical peptides would be aligned parellel to each other
to form flat membranes with
a planar nematic-like
arrangement.22 Indeed, transmission electron microscopy
(TEM) images revealed the formation of spherical objects with
diameters ranging from ~ 70 nm to ~100 nm (Figure 2c). Cryo-
TEM investigations confirmed the presence of spherical
aggregates with a dark outer circle expected from the 2-D
projection of hollow vesicles with a uniform thickness of ~3 nm
(Figure 2c, inset). Dynamic light scattering (DLS) measurements
revealed that the aggregates have an average diameter of ~90
nm (Figure 2b), which is in reasonable agreement with the size
observed by TEM. In addition, the concave structures observed
in the SEM image indicate the characteristics of deformed
hollow spheres formed through the drying process, providing
further evidence of the hollow nature of the vesicular
structures (Figure 2d). The results demonstrate that the helical
conformation triggers the formation of the membrane
structures.
Figure 2. (a) CD spectra of
1
and
2
, each in 0.1 wt % aqueous solution, (b) DLS
trace of
image. (d) SEM image of
2 in 0.1 wt% aqueous solution. (c) TEM image of 2, inset; cryo-TEM
2.
chiral peptide layers buried by achiral polymeric surfaces.
To overcome this limitation, we considered that lateral
grafting of hydrophobic aromatic segments as self-assembling
units into a hydrophilic α-helical peptide would form highly
efficient enantioselective membranes because of the exposure
of the α-helical peptide layers with chiral void spaces to
external environment.17 The hydrophilic α-peptide surfaces
would allow remarkable accessibility of enantiomers to chiral
cavities. Here, we report peptide vesicles with enantioselective
membranes consisting of planar nematic-like arrangement of
short α-helical peptides at the surfaces. The vesicles are able
to encapsulate only a single enantiomer from a racemic
mixture solution through selective diffusion across the peptide
membrane. The selectively captured enantiomer inside the
To gain insight into the formation of the hollow spherical
structure, we performed TEM experiments with highly diluted
conditions of peptide
2 (Figure 3a-c). The images showed
coexistence of ~3 nm-sized micelles and laterally-associated
small fragments of flat membrane structures which begin to
deform into buds at 0.003 wt% and then transform into
vesicles at 0.1 wt %, most probably due to lowering surface
energy.23 Considering the wall thickness and the height of
calculated dimer of ~3 nm, the hydrophilic peptide helices are
aligned one-direction, parallel to the flat membrane surface to
form peptide bilayers with hydrophobic pyrene units inside
(Figure 3d). The fluorescence emission showed a strong
excimer peak at longer wavelength region together with
monomer emission at shorter wavelengths (Figure S4),
indicative of pyrene packings inside the membranes with
vesicles undergoes
a click reaction to yield a highly
enantiopure reaction product in a confined space while
preventing the opposite enantiomer from entering interior
(Figure 1). As a result, the confined space can represent a
highly efficient enantioselective nanoreactor for transporting
enantiomer which is separated from the external environment
by a peptide membrane.
The membrane structure of the vesicles consists of the
lateral association of rod-like α-helical peptides which allows
the vesicular walls to function as
a highly efficient
enantioselective membrane. The α-helix-forming peptide
consists of a AKAK(A)4KAKAK(A)4KAKA amino acid sequence
with high helix propensity (Figure 1a).18 To endow self-
assembling feature with the peptide chain, two pyrenes were
symmetrically grafted into the peptide chain through click
chemistry of lysine residues with pyrene.19 The building blocks
were synthesized by a solid state peptide synthesis method
with a combination of both natural amino acids and a pyrene-
grafted lysine. Peptide
1 with a lack of pyrene adopts a
Figure 3. TEM images of intermediate structures formed at different
random coil conformation, as confirmed by circular dichroism
(CD) measurements, irrespective of concentration changes.
However, the incorporation of pyrene into the peptide chains
drives the random coil conformation of the peptides to
transform into an α-helical one (Figure 2). The CD spectrum of
concentrations of
2
, (a) 0.001 wt %, (b) 0.003 wt % (c) 0.1 wt %. (d) Energy-
and schematic
minimized packing structure of the dimeric association of
2
representation of the self-assembly into a vesicle through flat bilayer membrane
as intermediate structures.
2 | Chem. Commun., 2017, 00, 1-4
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packing frustration in the self-assembled state.19,24 The results larger than that of G1, indicative of inverse proportionality
imply that the vesicular structure of peptide originates from between permeability and enantioselectivity. Notably, the
2
budding the flat membranes consisting of the planar nematic- larger dipeptide, G3, is internalizedinto the vesicle interiors
like alignment of rod-like α-helical peptides on the external with perfect enantioselectivity with the ee value of 100 % upto
surfaces and hydrophobic pyrene inside the membranes 4 hr permeation, thereafter gradually decreases. This result
(Figure 3d).
indicates that the peptide vesicles are able to encapsulate only
Considering that the vesicular walls consist of the planar a single enantiomer in racemates above a certain size of guest
arrangements of rod-like α-helical peptides on the membrane through enantioselective diffusion across the vesicular
surfaces with frustrated pyrene packing inside the membranes, membranes (Figure 4c). The preferential internalization is
the the vesicular membranes are intrinsically porous with attributed to the lateral arrangements of helical peptides
chiral void spaces between the helical peptide parallel to basal plane which generate chiral porous membrane
arrangements.16,25 Accordingly, we envisioned that the structures. The chiral void spaces of vesicular membranes
vesicular walls function as enantioselectively permeable would preferentially associate with one enantiomer over the
membranes.26 To corroborate the permeability of the vesicular other one due to the large difference in free energy required
walls, we added protected amino acids (0.02 mM) to a solution to fit into the voids. Indeed, isothermal titration calorimetry
of peptide
2 at room temperature. The internalization of a (ITC) measurements with G2 revealed that the binding affinity
series of amino acid derivatives into the vesicular interior was for L-enantiomer is higher (Ka = 14,867 + 1,273) than that of L-
monitered by tracing high-performance liquid chromatography enantiomer (Ka =7,503 + 160) (Figure S6). This result suggests
(HPLC) after separation of the free racemate from the solution that the preferential association with membrane voids
using a Sephadex column. The concentration measurements of facilitates selective diffusion across the membrane.
the internalized racemic guests showed that the smaller
The preferential diffusion led us to envision that the peptide
molecules diffuse into the vesicle interiors faster than the vesicles can be used as an enantioselective nanoreactor that
larger molecules (Figure 4a), indicative of the size-selective provides protected spaces by peptide membranes for the
permeability of the vesicular walls.
chemical reactions of transporting enantiomers from the
More importantly, the internalization of the guests across environment. To explore the capability of the peptide vesicles
the vesicular walls is enantioselective as the diffusion rates of as enantioselective nanoreactors, N3-substituted trp-lys
each enantiomer are prominently different (Figure S5). The (L)- dipeptide G4 was selected to carry out a click reaction with a
enantiomers of both monopetides, G1 and G2 are internalized alkyne-functionalized hydrophilic dendrimer,
faster than the (D)-enantiomers, while their dipeptide G4 shows perfect enantioselectivity upto 4 hr
enantioselectivities in enantiometric excess (ee%) decrease permeation (Figure 5a), similar to G3. It should be noted that
A
because
over diffusion time (Figure 4b). The enantioselectivity of G2 is
the D-form internalizes preferentially to the L-form, as
opposed to the other amino acids. To corroborate
enantioselective reactions inside the vesicular cavities, first we
encapsulated dendritic alkyne
A that remains inside the
vesicles without leakage. The encapsulation of reactant
A
inside the vesicles was confirmed by MALDI-TOF and HPLC
after separation of the untrapped one by gel permeation
chromatography (GPC) using a Sephadex LH-20 column. Then,
we added racemic G4 into the vesicle solution encapsulating
reactant
A inside. After 1 hr diffusion, MALDI-TOF analysis
showed addtional peaks corresponding to a reaction product P
(Figure S7), indicating that the click reaction of G4 readily
undergoes with the encapsulated dedritic alkyn inside the
vesicles (Figure 5b).
Chiral HPLC analysis showed that the intensity associated
with the reaction product of the D-enantiomer, P(D), gradually
increases without any noticeable traces of the opposite
enantiomer product, P(L), over 4 hr (Figure 5b, inset),
demonstrating that the vesicular membranes allow only the D-
enantiomer in external environment to internalize for click
reactions. Because the click reactions between alkyne and
azide groups undergo rapidly before entering the other
enantiomer (L-form) into the reaction space, the reaction is
able to yield highly enantiopure product upto 4 hr permeation.
After 4 hr, we found that the L-enantiomer begins to undergo
click reaction, leading enantiopurity of the reaction product to
decrease. This result indicates that the interior of the peptide
Figure 4. Molecular structures of racemic amino acids. (a) Permeation of racemic
amino acids into the inside of vesicle with diffusion time. (b) Enantiomeric excess
(ee%) of encapsulated L-enantiomers as
a function time. (c) Schematic
representation of enantioselective permeation. D; D-enantiomer and L; L-
enantiomer.
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A. (a) Permeation of each
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inside vesicles with diffusion time, inset; HPLC chromatograms of the reaction
products formed from an encapsulated enantiomer. (c) Schematic
representation of enantioselective encapsulation and further chemical
transformation inside the vesicle nanoreactor.
A; dendritic alkyne, D; D-
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In conclusion, we have demonstrated that
a pyrene-
substituted helical peptide self-assembles into vesicular
structures with enantioselective membranes. The peptide
vesicles are able to encapsulate only a single enantiomer from
a racemic mixture environment through selective diffusion
across the peptide membranes. The encapsulated enantiomer
23 Y. Kim and M. Lee, Chem. Eur. J. 2015, 21, 11836.
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C. Yu and V. W. Yam, Org. Lett., 2009, 11, 4302.
separating from the opposite enantiomer in the environment.
Such a unique vesicles will offer an opportunity to construct
versatile reaction spaces for chiral differentiation, facile drug
discovery, and enantioselective modifications of intracellular
biomolecules.
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There are no conflicts to declare.
This work was supported by the 1000 Program, and NSFC
(no. 51473062, no. 21574055, no. 21634005 and no.
21550110493).
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