Crystal Packing-Guided Construction of Hetero-Oligomeric Peptidic Ensembles as Synthetic 3-in-1 Transporters

Article abstract of DOI:10.1002/anie.202101489

Strategies to generate heteromeric peptidic ensembles via a social self-sorting process are limited. Herein, we report a crystal packing-inspired social self-sorting strategy broadly applicable to diverse types of H-bonded peptidic frameworks. Specificall

Full text of DOI:10.1002/anie.202101489

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International Edition: doi.org/10.1002/anie.202101489
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3-in-1 Transporters
Hot Paper
Crystal Packing-Guided Construction of Hetero-Oligomeric Peptidic
Ensembles as Synthetic 3-in-1 Transporters
Jie Shen, Ruijuan Ye, and Huaqiang Zeng*
Abstract: Strategies to generate heteromeric peptidic ensem-
bles via a social self-sorting process are limited. Herein, we
report a crystal packing-inspired social self-sorting strategy
broadly applicable to diverse types of H-bonded peptidic
frameworks. Specifically, a crystal structure of H-bonded alkyl
chain-appended monopeptides reveals an inter-chain separa-
tion distance of 4.8 ꢀ dictated by the H-bonded amide groups,
which is larger than 4.1 ꢀ separation distance desired by the
tightly packed straight alkyl chains. This incompatibility results
in loosely packed alkyl chains, prompting us to investigate and
validate the feasibility of applying bulky tert-butyl groups,
modified with an anion-binding group, to alternatively inter-
penetrate the straight alkyl chains, modified with a crown ether
group. Structurally, this social self-sorting approach generates
highly stable hetero-oligomeric ensembles, having alternated
anion- and cation-binding units vertically aligned to the same
side. Functionally, these hetero-oligomeric ensembles promote
transmembrane transport of cations, anions and more interest-
ingly zwitterionic species such as amino acids.
ions[12–37] or anions,[38–60] with much less on molecular species
such as water[61–71] and glucose.[72] Specific to synthetic amino
acid transporters, we are aware of only two artificial trans-
porter systems based on a pillar[5]arene derivative by Hou in
2013[73] or dynamic covalent bonds by Gale in 2015.[74]
In 2017, we demonstrated a modularly tunable molecular
strategy toward rapid evolution of highly K+-selective chan-
nels.[26] In this strategy, cation-binding and -transporting
crown either units were vertically aligned to the same side
using a monopeptide-based H-bonded scaffold, generating
cation-transporting channels such as 6V10 (Figure 1a). To
unambiguously confirm that these crown ethers were indeed
aligned to the same side, we have made extensive and
combinatorial attempts to grow the X-ray quality single
crystals over a long period of two years, eventually and very
recently giving rise to single crystals of 6V10 by dissolving it in
DMSO at 10 mM and allowing the crystals to slowly grow at
room temperature over three months. The crystal structure of
Introduction
Aside from serving as the main building blocks for protein
synthesis, amino acids are also involved in a myriad of
biochemical pathways and physiological processes, including
nucleotide biosynthesis, DNA methylation, cellular signaling,
activation of mechanistic target of rapamycin, cytoprotection
against cell death, cellular transformation, learning, memory,
etc.[1–10] In cells or cellular organelles, homeostatic balance in
amino acid composition is maintained by at least sixty
specialized amino acid transporters (AATs) in a sophisticated
manner.[8] Dysregulated AATs could lead to homeostatic
imbalance and metabolic reprogramming. These contribute
to many forms of human diseases and the pathogenesis of
cancer.[8–10] Synthetic amino acid transporters, which may be
used to complement or replace the malfunctioned AATs to
ensure regulated delivery of required amino acids, may offer
tangible therapeutic benefits.
Nevertheless, over the past four decades of intensive
research since the first report on the synthetic transporter in
1982,[11] study on the synthetic membrane transporters has
overwhelmingly focused on transport of inorganic cat-
[*] Dr. J. Shen, R. Ye, Prof. H. Zeng
Figure 1. a) Chemical and crystal structures of crown ether-containing
K+-transporting channel 6V10, revealing loosely packed side chains
among the decyl chains. b) Computationally calculated binding ener-
gies per crown ether group, H-bonding segment and straight decyl
chain at the level of wB97XD/6–311G** in the gas phase for 6V10.
Note that a tightly packed docane generates a binding energy of
ꢀ12.98 Kcalmolꢀ1 per molecule.
Department of Chemistry, College of Science, Hainan University
Haikou, Hainan 570228 (China)
E-mail: 2733910004@qq.com
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202101489.
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6V10 confirms our early premise, underlying the directional
Specifically, the computed methylene (CH2) groups of the
self-assembly of crown ether units that forms a transmem-
brane pathway for mediating transport of cations (Figure 1a).
This directional assembly obviously results from a directional
parallel arrangement involving the H-bond-forming amide
groups among adjacent mono-peptide scaffolds.
After a more careful inspection of the crystal structure,
the most interesting observation perhaps is related to the
intermolecular packing. While crown ether groups and H-
bonding segments both pack tightly among themselves, each
contributing to stabilizing energies of ꢀ17.34 kcalmolꢀ1 and
ꢀ19.93 kcalmolꢀ1 (Figure 1b), respectively, the straight decyl
(C10H21) side chains remain essentially non-packed (Fig-
ure 1a), with a small overall interaction energy of ꢀ4.23 kcal
molꢀ1 per chain. This packing energy is 8.75 kcalmolꢀ1 less
than the binding energy of ꢀ12.98 kcalmolꢀ1 per tightly
packed decane molecule.
alkyl chains possess a diameter of 4.1 ꢀ (Figure 2a), which is
0.7 ꢀ smaller than the H-bonding-enforced inter-chain sep-
aration distance of 4.8 ꢀ. Thus, existence of some void spaces
among vertically aligned alkyl chains is inevitable, a reason
why the end methyl group in 6V10 points toward the adjacent
alky chain to fill the void as much as possible (Figure 1).
Given a molecular size of 6.0 ꢀ for the tert-butyl group
(Figure 2a), we envisioned a possibility to interpenetrate one
dimensionally aligned alky chains alternatively using the tert-
butyl groups, forming hetero-ensembles (6F10·IFT)n from
homo-ensembles (IFT)n, which likely can transport anions,[52]
and (6F10)n, which likely can transport cations,[26] via a social
self-sorting process. We further envisioned that ensembles
(6F10·IFT)n, consisting of alternating cation- and anion-
binding motifs, might behave like a three-in-one transporters
capable of mediating transmembrane transport of cations,
anions and zwitterionic species such as amino acids. Equally
significantly, hetero-channel (6F10·IFT)n, for the first time,
provides an interesting structural tool for quantitatively
addressing two fundamentally significant but difficult-to-
address issues related to (1) the comparative ion transport
efficacies between18-crown-6 and electron-deficient iodo
groups and (2) the impact on ion transport activity the
inter-chain separation distance and number of binding sites
may have.
This lack of packing among the straight alkyl chains can be
readily understood on the basis of molecular dimensionality.
Results and Discussion
Previously, we have shown that the cation-transporting
activities of mono-peptide channels derived from phenyl-
alanine are among the highest and the most selective.
The 1,2,4,5-tetrafluoro-3-iodobenzene motif, which is
sterically compatible with the molecular dimensionality of
the assembled mono-peptide channel, has been employed to
mediate transmembrane anion transport.[75] As a starting
point, we therefore decided to examine the pairing compat-
ibility involving decyl-containing 6F10 and tert-butyl group-
containing IFT (Figure 2b).
It is well-known that H-bond acceptors like N- and O-
atoms decrease the electron density of the H-bond donors (H-
atoms), producing a deshielding effect and resulting in a larger
chemical shift of the H-atoms. The stronger the H-bonds
generate stronger deshielding effects and larger chemical
shifts. Experimentally, upon mixing homo-ensembles (IFT)n
and (6F10)n at 100 mM, we observed chemical shifts of all four
H-atoms Ha–Hd downfield shifted, with significant differences
of 0.20 ppm for Ha, 0.28 ppm for Hc, and 0.07 ppm for Hd.
These changes in chemical shift clearly signify the formation
of more stable hetero-ensembles (6F10·IFT)n from homo-
ensembles (IFT)n and (6F10)n.
Figure 2. a) Computed molecular dimensionalities for methylene (CH2)
and tert-butyl groups at the level of wB97XD/6–311G**. b) Illustration
of applying tert-butyl groups to alternatively interpenetrate the straight
alkyl chains, generating more stable hetero-oligomeric peptidic ensem-
bles as three-in-one transporters for transporting cations, anions and
amino acids. c) Partial NMR spectra of homo-ensembles (IFT)n and
(6F10)n and hetero-ensembles (6F10·IFT)n, demonstrating the forma-
tion of (6F10·IFT)n upon mixing (IFT)n and (6F10)n.
Lastly, the ability of the crown ether and electron-
deficient iodide groups to interact with charged ammonium
and carboxylate ends of the amino acids was confirmed by 1H
and 19F NMR experiments, respectively. Due to the solubility
issue of zwitterionic amino acids, we used simple alkyl
ammonium (compound 1; Supporting Information, Fig-
ure S1a) and carboxylate (compound 2; Supporting Informa-
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tion, Figure S1b) compounds to represent the ammonium and
carboxylate ends of the amino acid. Upon mixing 10 equiv-
alents of compound 1 with 6F10·IFT, the chemical shift of
ammonium H-atoms of 1 displays an upfield shift of 0.2 ppm
(Supporting Information, Figure S1a), suggesting the binding
of ammonium group of 1 by the crown ether of 6F10·IFT.
Similarly, in the presence of 10 equivalents of compound 2,
the chemical shift of fluorine atoms of 6F10·IFT displays
a downfield shift of 0.4 ppm (Supporting Information, Fig-
ure S1b), indicating the binding of carboxylate group by the
electron-deficient iodide group of 6F10·IFT. Given the
limited cavity size of the crown ether, we believe the most
likely scenario concerning amino acid transport involves the
binding of the amino acid by 6F10·IFT (Figure 2b), followed
by the alternative hopping of the ammonium and carboxylate
groups from one binding site to the other.
The main purpose of the following is to fully establish the
formation of hetero-ensembles (6F10·IFT)n in solution,
exhibiting ion transport behaviors distinctively different from
homo-ensembles (IFT)n and (6F10)n. Based on the hydro-
phobic membrane thickness of 28 ꢀ for EYPC (egg yolk L-a-
phosphatidylcholine) lipid membrane[58] and an inter-chain
distance of 4.8 ꢀ in these ensembles, roughly six molecules
are required to span the hydrophobic membrane region.
Accordingly, we will use (6F10·IFT)3, (IFT)6 and (6F10)6 to
represent the respective most likely ensembles in the
membrane.
pendent fluorescence at excitation wavelengths of 403 and
460 nm. EYPC lipids were used to prepare large unilamellar
vesicles (LUVs) of about 120 nm in diameter in buffer
(10 mM HEPES, 50 mM Na2SO4, pH 7.0), with intravesicular
region encapsulating the pH-sensitive HPTS (1 mM). The
HPTS-containing LUV suspension was diluted to the same
type of buffer having pH 8.0 to create a pH gradient for ion
transport study via Na+/H+ antiport mechanism. At the same
channel concentration of 7 mM, channel (6F10·IFT)3, having
a fractional Na+ transport activity (RNaþ ) of 51%, is substan-
tially more active than (6F10)6 (RNaþ = 37%). As expected,
the anion-transporting channel (IFT)6 does not transport Na+
ions, having a RNaþ value of 3.3% that is merely 1% more
than the background signal. These data confirm the cation-
transporting ability of (6F10·IFT)3 to be maintained after
enlarging the vertical separation distance between two
adjacent crown ether units from 4.8 ꢀ in (6F10)6 to 9.6 ꢀ in
(6F10·IFT)3. This is consistent with the earlier finding by
Fyles, showing that, within a lipid bilayer environment, the
maximum distance between two binding sites a sodium ion
can travel is 11 ꢀ.[76]
The anion transport properties of (6F10·IFT)3 and (IFT)6
were studied and compared using a chloride-sensitive SPQ
assay (Figure 3b), taking advantage of the fact that the
fluorescence intensity of SPQ dye decreases with increasing
concentration of chloride anions. At the same channel
concentration of 2 mM, hetero-channel (6F10·IFT)3 apparent-
ly is more active than anion channel (IFT)6 by 14% in
fractional chloride transport. Similarly, these data confirm the
anion-transporting ability of (6F10·IFT)3 to be maintained
after enlarging the vertical separation distance between two
The cation transport properties of these three types of
ensembles were studied and compared using a pH-sensitive
HPTS assay (Figure 3a). This assay relies on the HPTS dye
(8-Hydroxypyrene-1,3,6-trisulfonic acid), exhibiting pH-de-
Figure 3. a) pH-sensitive HPTS assay for comparing Na+ transport activities. b) Chloride-sensitive SPQ assay for comparing chloride transport
activities. c) and d) presents single channel currents and K+/Clꢀ selectivity values for home-ensembles (6F10)6 and hetero-ensembles (6F10·IFT)3.
In (a), the final ion transport trace was obtained, after subtracting background intensity at t=0, as a ratiometric value of I460/I403, and further
normalized based on the value of I460/I403 after addition of triton. The fractional changes RH+ was then calculated for each curve using the
normalized value of I460/I403 at 300 s before the addition of triton, with that of I460/I403 at t=0 s as 0% and that of I460/I403 at t=300 s (obtained
after addition of triton) as 100%. [total lipid]=82 mM. In (b), the emission of SPQ was monitored at 430 nm with excitation at 360 nm for 300 s.
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adjacent anion-transporting units from 4.8 ꢀ in (6F10)6 to
time the relative ion-transporting ability between 18-crown-6
9.6 ꢀ in (6F10·IFT)3.
and 1,2,4,5-tetrafluoro-3-iodobenzene motifs has been quan-
titatively assessed within the identical structural and exper-
imental settings.
We have conducted self-quenching carboxyfluorescein
(CF, 50 mM) assay to evaluate the membrane integrity in the
presence of 6F10·IFT (Supporting Information, Figure S2).
While highly fluorescent at low concentration, CF molecules
mostly self-quenches at high concentration of 50 mM. Com-
pared to high fluorescence intensities that reach 43% and
95% for melittin at 0.45 and 1.2 mM, 6F10·IFTat 20 mM elicits
only < 3% difference relative to the background signal,
confirming membrane integrity in the presence of 6F10·IFT
and that the observed ion transports are due to the ability of
6F10·IFT to transport these ions.
To quantitatively assess the impacts on ion transport
properties (transport rate, selectivity and channel stability) an
alternated array of cation- and anion-binding units may have,
we conducted single channel current measurements for
(6F10·IFT)3 and (6F10)6 (Figure 3c,d). There are three note-
worthy points to discuss.
First, incorporating anion-binding unit expectedly de-
creases the K+/Clꢀ selectivity from 27.6 for (6F10)6 to 4.7 for
(6F10·IFT)3. Still, (6F10·IFT)3 behaves mostly like a cation
channel, interestingly pointing out a higher ability of the 18-
crown-6 in transporting K+ cations than that of the electron-
deficient iodo group in chloride transport. More precisely, the
18-crown-6 group transports K+ ions 4.7 times as efficient as
the chloride transport mediated by the 1,2,4,5-tetrafluoro-3-
iodobenzene group. To our best knowledge, this is the first
Second, if we assume that the currents observed at
150 mV largely correspond to those arising from cations,
single channel currents of 2.7 pA for (6F10)6 and 0.65 pA for
(6F10·IFT)3 indicate that a transmembrane pathway, made up
of six vertically aligned crown ether units with an inter-chain
distance of 4.8 ꢀ, transports K+ ions at a rate about 4 times as
fast as that having only three such units of a wider separation
distance of 9.6 ꢀ. Alternatively, doubling the inter-chain
distance between the ion binding sites while eliminating 50%
of ion binding sites attenuates the ion transport ability by
76%.
Third, from the single channel currents recorded at
different voltages (Figure 3c,d), the channel total opening
times are 1.6 s out of a total recording time of 79.9 s for
(6F10)6 and 42.9 s out of 66.8 s for (6F10·IFT)3, roughly
corresponding to channel opening probabilities of 2.0% for
(6F10)6 and 64.2% for (6F10·IFT)3. These drastically differ-
ent channel opening probabilities fully validate our strategy
of using sterically compatible groups (straight alkyl chains
and tert-butyl groups) to create hetero-ensembles with high
structural stability.
A novel calcein-based fluorescence assay recently devel-
oped by Gale was adopted in this work for evaluating Gly
transport ability of various channels (Figure 4a).[74] The
Figure 4. a) Illustration of amino acid-sensitive calcein assay for assessing the ability of amino acid transporters to transport glycine (lex =495 nm,
lem =515 nm). b) Glycine transport curves for various channels at 20 mM. c) 13C NMR spectra that verify the glycine transport by hetero-channel
(6F10·IFT)3. d) Glycine transport curves for (6F10·IFT)3 and Gale’s dynamic covalent transporter system that employs 1 (1 mol%) and 3
(50 mol%).
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principle of this assay is based on the competitive binding of
intravesicular Cu2+ ions bound to calcein dye molecules by
amino acids such as Gly transported into LUVs via synthetic
amino acid transporters. Since it is Cu2+ ions, not amino acids,
that quench the fluorescence of calcein dye, increasing
removal of Cu2+ ions from calcein thereby increasingly
restores the quenched calcein fluorescence, leading to en-
hancement in fluorescence (DI).
From data presented in Figure 4b, hetero-channel
(6F10·IFT)3 evidently produces a significant fluorescence
enhancement with respect to both (6F10)6 and (IFT)6, and this
enhancement continues after 600 s. Interestingly, paring 6F10
with IF10 (obtained by replacing the tert-butyl group in IFT
with a n-decyl group) forms hetero-channel (6F10·IF10)3 that
is also able to transport Gly, but with transport activity
starting to level off around 300 s. Paring IFTwith either 6F12,
having a longer dodecyl chain, or 6F8, having a shorter octyl
chain, results in worse performance in Gly transport. Replac-
ing 18-crown-6 group in 6F10 with the 15-crown-5 group
generates 5F10, which similarly does not lead to improved
Gly transport when paired with IFT. Taken together, hetero-
channel (6F10·IFT)3 outperforms all the other combinations
involving 6F8, 6F12, 5F10, and IF10.
13C NMR spectra using 13C labeled Gly (Gly-13C) also
used by Gale[74] provide more convincing evidence for Gly
transport mediated by (6F10·IFT)3 (Figure 4c). The para-
magnetic Mn2+ ions present in the extravesicular region exerts
influences on the 13C NMR signal from extravesicular Gly-
13C, but not intravesicular Gly-13C signal. If transporter is
able to facilitate influx of Gly-13C, new 13C signal is expected
to be seen. Consistent with this expectation, we did observe
a new discernable 13C signal in the presence of 0.3 mol%
(6F10·IFT)3, while prior to its addition, only a broad signal
was obtained in the presence of 0.5 mM Cu2+ ions and 50 mM
Gly-13C.
Gale and his co-workers recently reported an elegant
transporter system exploiting both H-bonds and dynamic
covalent bond for Gly transport (Figure 4d).[74] After testing
a number of molecules, the best combination involves
squaramide molecule 1, which forms H-bonds with the
carboxylate group of Gly, and aldehyde molecule 3, which
forms a dynamic covalent imine bond with the ammonium
group of Gly. The resultant tripartite complex 1·Gly·3 is
mostly hydrophobic and crosses the membrane by a simple
diffusion process, with the fastest Gly influx occurring in the
presence of 1 mol% 1 and 50 mol% 3. Compared to Galeꢁs
dynamic transporter, hetero-channel (6F10·IFT)3 induces
much faster Gly transport during the first 60 s, and achieves
a similar overall performance over a duration of 600 s.
Following the same assay having 0.2 mM CuSO4 in the
extravesicular region (Figure 4a), we have examined another
eleven amino acids. Results summarized in Figure 5a reveal
six amino acids including Gly to be transportable. However,
after or even before subtracting the background signals, the
changes in fluorescence intensity of calcein for six amino acids
all are negative (See Val, Ile, Met, Pro, Leu, and Phe in
Figure 5a and the Supporting Information, Figure S3d, re-
spectively). This is surprising given that such changes
certainly are expected to be ꢁ 0 even if channel (6F10·IFT)3
Figure 5. a) Net changes in fluorescence intensity of calcein using the
assay conditions in Figure 4a. b) Illustration of new assay conditions,
removing 0.2 mM CuSO4 from the extravesicular region, with represen-
tative findings summarized in (c)–(e). Blank refers to the signals
obtained in the absence of channel molecules. All data are the average
of three independent measurements.
completely lacks an ability to transport these amino acids. The
most plausible explanation is that, during the channel-
mediated transport of amino acid, the electron-deficient
iodide groups of the channel interact rather weakly with the
carboxylate group of the amino acid, enabling it to interact
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with and cause influx of Cu2+ ions to quench the fluorescence
of calcein.
With the above reasoning in mind, we decided to remove
Keywords: amino acid transporters · crown ethers ·
halogen bonds · supramolecular chemistry ·
synthetic ion channels
0.2 mM CuSO4 from the extravesicular region (Figure 5b).
Under these conditions, we further removed 30 mM amino
acid to ascertain that the passive efflux of Cu2+ ions driven by
the concentration gradient or mediated by channel
(6F10·IFT)3 are maintained at the low levels (Figure 5c). It
might be worth pointing out that, during the freeze/thaw
cycles for LUV preparation, heating the lipid solution at 558C
water bath for 2 minutes is very necessary to achieve low
background signal shown in Figure 5c under the concentra-
tion gradient of Cu2+ ions that might lead to efflux of Cu2+.
We then conducted systematic evaluations of 12 amino
acids using the new assay shown in Figure 5b. In line with our
expectations, channel (6F10·IFT)3 indeed displays measura-
ble ability to transport all 12 amino acids (Figure 5d;
Supporting Information, Figures S4, S5). To allow for
straightforward and better comparison of the transport
efficiencies for these amino acids, we defined the fractional
activity as (DIchannelꢀDIblank)/DIblank) wherein DIchannel and
DIblank are changes in fluorescence signals obtained in the
presence and absence of channel (6F10·IFT)3 (Figure 5e).
Based on the calculated fractional activities, amino acid Ser
stands out as the most preferred substrate, followed by Gly,
Met and Ala, for transmembrane transport by (6F10·IFT)3.
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Social self-sorting of functionalized peptidic scaffolds has
been relatively rare and thus far difficult to achieve.[77]
Herein, we have devised and demonstrated a novel bottom-
up crystal structure-guided molecular strategy, allowing for
social self-sorting to readily take place by pairing straight
alkyl chains with sterically compatible tert-butyl groups. This
process generates more stable hetero-oligomeric ensembles
(6F10·IFT)n from the corresponding homo-ensembles (6F10)n
and (IFT)n. The resultant alternatively arrayed cation-binding
crown ethers and electron-deficient anion-binding iodide
groups, which are aligned to the same side, exhibit better ion
transport activities than the homo ensembles, and more
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Manuscript received: January 31, 2021
Revised manuscript received: March 22, 2021
Accepted manuscript online: March 23, 2021
Version of record online: && &&
,
&&&&
&&&&
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These are not the final page numbers!

Angewandte
Research Articles
Chemie
Research Articles
3-in-1 Transporters
We present a broadly applicable social
self-sorting strategy, enabling cation-
binding crown ether groups and anion-
binding fluorobenzene groups to be
alternatively packed. This generates a 3-
in-1 transporter system, promoting
transmembrane transport of cations,
anions, and zwitterionic species such as
amino acids.
J. Shen, R. Ye, H. Zeng*
&&&& — &&&&
Crystal Packing-Guided Construction of
Hetero-Oligomeric Peptidic Ensembles
as Synthetic 3-in-1 Transporters
Angew. Chem. Int. Ed. 2021, 60, 2 – 9
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org
&&&&
These are not the final page numbers!