Buckyball-Based Spherical Display of Crown Ethers for de Novo Custom Design of Ion Transport Selectivity

Custom Design of Ion Transport Selectivity

Article

Buckyball-Based Spherical Display of Crown Ethers for De Novo

Ning Li, Feng Chen, Jie Shen, Hao Zhang, Tianxiang Wang, Ruijuan Ye, Tianhu Li, Teck Peng Loh,

Yi Yan Yang, and Huaqiang Zeng*

Cite This: https://dx.doi.org/10.1021/jacs.0c09655

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sı Supporting Information

ABSTRACT: Searching for membrane-active synthetic analogues

that are structurally simple yet functionally comparable to natural

channel proteins has been of central research interest in the past

four decades, yet custom design of the ion transport selectivity still

remains a grand challenge. Here we report on a suite of buckyball-

based molecular balls (MBs), enabling transmembrane ion

transport selectivity to be custom designable. The modularly

tunable MBm-Cn (m = 4−7; n = 6−12) structures consist of a C -

fullerene core, ﬂexible alkyl linkers Cn (i.e., C6 for n-C H group),

and peripherally aligned benzo-3m-crown-m ethers (i.e., m = 4 for

benzo-12-crown-4) as ion-transporting units. Screening a matrix of

6 such MBs, combinatorially derived from four diﬀerent crown

units and four diﬀerent Cn linkers, intriguingly revealed that their

transport selectivity well resembles the intrinsic ion binding aﬃnity of the respective benzo-crown units present, making custom

design of the transport selectivity possible. Speciﬁcally, MB4s, containing benzo-12-crown-4 units, all are Li -selective in

transmembrane ion transport, with the most active MB4-C10 exhibiting an EC (Li ) value of 0.13 μM (corresponding to 0.13 mol

of the lipid present) while excluding all other monovalent alkali-metal ions. Likewise, the most Na selective MB5-C8 and K

selective MB6-C8 demonstrate high Na /K and K /Na selectivity values of 13.7 and 7.8, respectively. For selectivity to Rb and

Cs ions, the most active MB7-C8 displays exceptionally high transport eﬃciencies, with an EC (Rb ) value of 105 nM (0.11 mol

) and an EC (Cs ) value of 77 nM (0.079 mol %).

INTRODUCTION

evolutionally selected protein channels. For instance, the KscA

channel, as one of the most well-studied membrane-embedded

■

Transmembrane transport of metal cations exquisitely

regulated by natural channel proteins is ubiquitous in nature,

which underlies diverse physiological processes to sustain

natural channel proteins, can transport ∼10 K ions per

second, with an extraordinarily high K /Na selectivity of

−3

∼10000. In the ﬁeld of synthetic cation transporters, exciting

life.

Signiﬁcant progress has been made in recent years in

advancement has been achieved regarding the transport

revealing the structural conformation of these naturally evolved

biomacromolecules and also in understanding the mechanism

by which they can traﬃc ion ﬂux eﬃciently and selectively.

eﬃciency on par with the natural systems, but devising

artiﬁcial transporters with high and designable cation

selectivity still represents a daunting task for the research

community.

−7

Such knowledge has further enabled and inspired scientists,

especially synthetic chemists, to design artiﬁcial versions of

functional ion transporters that not only deepen our

understanding of transmembrane transport but also pave the

way for their potential applications in the pharmaceutical and

Crown ethers (CEs), discovered in the late 1960s, have

been experiencing extensive investigative eﬀorts for the design

of a plethora of artiﬁcial membrane transporters over the last

7,19−28

−16

few decades.

This is primarily built upon their

chemical industries.

_I_n_m_o_s_t_b_i_o_l_o_g_i_c_a_l_s_y_s_t_e_m_s_,_N_a+ _a_n_d_K+ perform in

structural simplicity and unique coordination capability toward

combination as the electrolyte in both intra- and extracellular

environments, exerting pivotal regulatory roles in vital

physiological functions such as muscle contraction, neural

signal transmission, and heartbeat. To perform such functions

properly, these cations have to move across the intrinsically

impermeable cell membrane in both an eﬃcient and selective

manner. This is exactly what nature has achieved using

Received: September 8, 2020

XXXX American Chemical Society

Journal of the American Chemical Society

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Figure 1. Molecular design of buckyball-based molecular balls as ion-selective transporters. (a) Molecular structures of designed MBs with

diversiﬁcations in ﬂexible alkyl linker lengths and peripheral crown ether units for a combinatorial identiﬁcation of selective and eﬃcient MBs. (b)

Illustrations of MB’s three-dimensional conﬁguration and distribution of 12 side arms. (c) Schematic illustration of MBs participating in

transmembrane transport of alkali-metal ions with selectivity imparted by the crown ether’s intrinsic ion-binding selectivity, with the proposed ion

transport mechanism shown in the square box on the left.

the biologically relevant alkali-metal ions. Although numerous

studies have shown that binding of CEs with alkali-metal ions

in solution largely follows the so-called size-ﬁt rule (i.e., 12-

crown-4, 15-crown-5, 18-crown-6, and 21-crown-7 favoring

> Li⁺for H-bond-directed stacks of 18-crown-6, largely

deviating from their solution binding aﬃnities. Previous

−

studies on Cl anionophores have shown that the tight-

binding-associated retarding eﬀect is not predictable either, in

29−33

−

Li , Na , K , and Cs ions, respectively)

such a trend was

which anionophores of higher Cl binding aﬃnity can lead to

found to be absent in many other examples. Speciﬁcally in

the realm of synthetic ion transporters, it is also impractical to

reliably predict the ion transport selectivity of the CE-based

backbone-dependent higher or lower transport eﬃciencies

and hence selectivities. Therefore, it would be of high interest

and value to identify ion-transporting scaﬀolds that could

facilitate a direct translation of solution binding aﬃnity to

transmembrane ion transport selectivity without compromising

the transport eﬃciency.

17,19,20,24,25

transporters from their solution binding proﬁle.

Intuitively, favorable ion binding should beneﬁt the ion-

capturing process but could be detrimental to ion release to the

next binding site or the other side of the membrane. For

example, a number of 15-crown-5-based synthetic transporters

Herein we present such a distinctive class of C -fullerene-

based molecular balls (MBs, Figure 1a) carrying CEs on the

periphery and exhibiting excellent transport selectivity toward

alkali-metal ions that closely follows the CE ion binding aﬃnity

in solution. In particular, upon insertion into a lipid membrane,

were found to be capable of selectively transporting K ions,

17,19,20,24

instead of Na .

Similarly, Barboiu and co-workers

revealed a transport selectivity order of Cs > Rb > K > Na

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

MB4-C10 containing 12 benzo-12-crown-4 units and n-

C10H₂₀linkers allows Li to rapidly pass through while

excluding nearly all other monovalent alkali-metal ions.

Similarly, MB5-C8, MB6-C12, and MB7-C8 exhibit good to

excellent transport selectivities toward Na , K , and Rb /Cs

ions, respectively. In addition, these MBs also display high

transport eﬃciency, with EC₅₀values in the range of 0.079−

.48 mol % relative to lipid molecules present in the

membrane. Analogous supramolecular spherical assemblies

have attracted extensive interest in diverse ﬁelds such as gene

7−39

delivery and regulation,

protein binding, and antiviral

activity. To this end, our unique design of MBs as synthetic

ion transporters oﬀers an unprecedented approach toward

attaining both high transport eﬃciency and especially custom

designable ion selectivity, an unmet challenge that has been

long sought after.

RESULTS AND DISCUSSION

■

The structure of these MBs is modularly tunable, consisting of

a C -fullerene core, ﬂexible alkyl linkers, and CEs as the ion

capture and transport units at the molecular periphery (Figure

a). Four types of CEs were selected in this study, i.e., 12-

crown-4, 15-crown-5, 18-crown-6, and 21-crown-7, as they are

known to favorably bind with alkali-metal ions from Li to Cs

(

ionic radius of 0.76−1.67 Å) via host−guest complexation.

In experiments using the chloroform−water biphasic picrate

extraction method developed by Cram, we obtained the

binding constants of the corresponding benzo versions, i.e.,

benzo-12-crown-4, -15-crown-5, -18-crown-6, and -21-crown-

Figure 2. ¹³C NMR spectra of MB4-C10, MB5-C8, MB6-C12, and

, revealing their preferential binding with Li , Na , K , and

MB7-C8 in DMSO-d or CDCl . Representative signals assigned and

Cs /Rb ions (Scheme S4 and Table S1), respectively. Alkyl

chains were then used to link the fullerene core and the benzo-

CE units, in order to establish the structural ﬂexibility and also

to enhance the lipophilicity of the MB. Since linkers may aﬀect

labeled are in full agreement with the octahedral symmetry of the

molecules.

17,21

the transport performance,

we further screened four alkyl

whereas MB6-C12 and all MB7s are tarlike heavy oils. These

MBs are found to be soluble in chloroform, dichloromethane,

dimethylformamide, and dimethyl sulfoxide but only poorly

soluble in acetonitrile or methanol.

chains of diﬀerent lengths (i.e., n-C H , n-C H , n-C H ,

10 20

and n-C H ), giving MBs with estimated physical diameters

of 38−53 Å at their most extended state (excluding the

amphiphilic CEs, Figure 1a). They are dimensionally

comparable to the thickness of typical lipid bilayer membranes,

usually about 30−35 Å thick for the hydrophobic region and

In the literature, ¹³C NMR spectroscopy has been

established as a particularly useful tool for the structure

characterization and purity conﬁrmation of C -fullerene

0−55 Å overall, including the polar head groups and midpolar

hexakis adducts, as two signatory signals of the sp carbons

of C₆₀can usually be observed, in accordance with the

regimes.

7,43

The actual synthesis of these MBs was accomplished via

three steps, including (i) reacting bromo alcohols with malonic

acid to obtain the V-shaped alkyl linkers, (ii) attaching such

linkers onto C -fullerene via the Bingel reaction in a 6:1 ratio,

octahedral symmetry of the C₆₀core.

The spectrum of

MB4-C10, for example, shows such sp signals at δ 140.8 and

145.0 ppm (Figure 2 and Figure S2 ). The C₆₀sp carbon and

the malonate bridgehead carbon signals emerge at δ 66.8 and

45.7 ppm, respectively. In addition, two carbonyl signals (δ

165.1 and 162.7 ppm) were also observed, which are in full

consistency with the proposed octahedral molecular symmetry.

and (iii) introducing 12 CE units at the molecular periphery to

complete the sphere construction. The Bingel addition of

malonates onto the fullerene core is octahedrally symmetric,

giving MBs with 12 CE units evenly distributed on the surface

Similar C NMR spectra were obtained for all other MBs as

(

Figure 1b). In total, a matrix of 16 MBs (combination of four

linkers and four CEs, expected molecular weight in the 5534−

128 Da range) were prepared. Although their identity and

high purity of >90% could be conﬁdently conﬁrmed by HPLC

well, and their H NMR spectra also display a full set of

expected signals with satisfactory integration ratios, although

the peak multiplicity is lost in some cases (see the Supporting

Information for more details). In view of such highly

symmetrical structures with ﬂexible alkyl linkers and ion-

capturing CEs, we envisioned that the appropriately sized MBs

might be able to perform as eﬃcient transporters to move

alkali-metal ions across a lipid bilayer membrane in the

presence of an ionic concentration gradient (Figure 1c). ₄

Figure S1 ) and H and C NMR spectroscopy (Figure 2 and

Figure S2 as well as spectra in the Supporting Information ),

the possibility of incomplete functionalization of C -fullerene

cannot be exclusively ruled out. No molecular peaks were

observed in the MALDI-TOF mass spectra, suggesting their

easy fragmentation under laser beam irradiation. It is worth

noting that MB4s, MB5s, and some of the MB6s with short

linkers (MB6-C6, MB6-C8, and MB6-C10) are dark solids,

4,45

The well-established ﬂuorescence-based HPTS assay

was adapted in this study to assess the ion transport selectivity

and eﬃciency of these MBs (Figure 3a). In this assay, large

Journal of the American Chemical Society

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Figure 3. LUV-based lipid bilayer experiments for elucidating ion transport activity and selectivity. (a) Schematic illustration of the pH-sensitive

HPTS assay for ion transport study. Diﬀerent extravesicular salts are used to facilitate the comparison of ion transport activities. (b−e) M ion

transport selectivity of the most active MBs in each subgroup selective to diﬀerent alkali-metal ions, determined over 300 s with an extravesicular

environment of 100 mM MCl (M = Li , Na , K , Rb , Cs ): (b) for MB4-C10; (c) for MB5-C8; (d) for MB6-C12; (e) for MB7-C8. The

normalized ﬂuorescence intensity R_M⁺is calculated by the formula R_M= (I_M− I )/(I

intensities at 300 s before and after the Triton addition, respectively, and I is the background intensity. While Blank (LiCl, 13.7%) in (b) means

that the background transport percentage of Li ion in absence of any MB is 13.7%, NaCl (2.1%) in (b) refers to a normalized ion transport activity

− I ), in which I and I_T_r_i_t_o_nare the ﬂuorescence

Triton

value in the presence of the transporter MB4-C10, after subtracting the MB4-C10-mediated transport percentage of 14.6% from the background

signal Blank (NaCl, 12.8%) and further normalization (e.g., 2.1% = (14.6% − 12.8%)/(100% − 12.8%)). [Total LUV lipid] = 97.5 μM.

unilamellar vesicles (LUVs; about 120 nm in diameter) were

fabricated using EYPC (egg yolk L-α-phosphatidylcholine),

trapping 10 mM HEPES buﬀer, 100 mM NaCl, and 1.0 mM

pH-sensitive HPTS dye (8-hydroxyprene-1,3,6-trisulfonic

acid) in the intravesicular region at pH 7.0. The extravesicular

good transport eﬃciency. The most eﬃcient candidate in each

subgroup selective to a particular alkali-metal ion was identiﬁed

to be MB4-C10, MB5-C8, MB6-C12, and MB7-C8, and their

EC (M ) value were determined as 0.13 μM, 0.47 μM, 0.40

μM, and 105 nM/77 nM for Li , Na , K and Rb /Cs ions,

respectively.

environment was set to be 100 mM MCl (M = Li , Na , K ,

Rb , Cs ) and 10 mM HEPES at pH 8.0. In such an

asymmetric system, the pH gradient provides the driving force

to move the alkali-metal ions toward the intravesicular region.

This cation translocation is associated with either proton eﬄux

or hydroxide inﬂux for charge neutralization, resulting in an

intravesicular pH increase that can be seen macroscopically by

monitoring the ﬂuorescence intensity of the encapsulated

HPTS dye.

Ion transport selectivity of these MBs can be quantiﬁed

using the ratio of fractional transport activity R_Min the HPTS

assay, given that R_Mis obtained at the MB concentration

where the activity for the most active ion reaches 95−100%

over the 300 s data recording period. We and others have

veriﬁed the reliability of such an R -based approach to ion

selectivity quantiﬁcation, with reference to the classic planar

19,46

lipid bilayer method.

The selectivity proﬁles for the most

Initial screening using the HPTS assay revealed that all MBs

are able to transport alkali-metal ions across the LUV

membrane. More importantly, their transport selectivity is

highly dependent on the choice of CE units in the molecular

structure. The data compiled in Table 1 and Figure 3 show

eﬃcient MB4-C10, MB5-C8, MB6-C12, and MB7-C8 are

shown in Table 1 and in Figure 3b−e. Similar patterns were

also observed on other MBs containing respective CEs,

although their ion-discriminating capabilities diﬀer (Figures

S3 −S18).

that those containing 12-crown-4 preferably transport Li , and

At 0.45 μM, MB4-C10, containing Li -selective benzo-12-

the 15-crown-5-, 18-crown-6-, and 21-crown-7-containing MBs

crown-4 units (Table S1) and n-C H linkers, induces the

are selective to Na , K , and Rb /Cs ions, respectively.

The classic Hill analyses were performed to determine the

EC₅₀values for these MBs, at which 50% of the maximal

transport activity is reached. As shown in Table 1, the majority

show EC₅₀values of around 5.0 μM or below, indicating their

maximal transport activity for Li (R = 99.8%), yet no

obvious activity was seen for other alkali metal ions (R = 0−

2.1%, M = Na , K , Rb , Cs ; Figure 3b). The transport

selectivity, therefore, calculated by the R /R ratio, was

deemed to be greater than 47.5. In 2011, Otis et al. reported

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

Article

Table 1. Ion Transport EC Values (Half Maximal Eﬀective

Concentration, in μM) and Selectivities of All MBs

despite both being critically important electrolytes in biological

systems. This is potentially due to the higher dehydration

31,58

Calculated from the EYPC-LUV Experimental Data Using

energy of Na ions,

energetically less favorable in comparison to K ions.

Accordingly, many 15-crown-5-based transporters have dem-

making its membrane penetration

Hill Analysis

onstrated ion transport selectivity toward K , instead of

17,19,20,24

Na .

ion ﬁsher, consisting of a lipid anchoring cholesterol moiety

ﬁshing rod), ﬂexible alkyl chain (ﬁshing line), and benzo-15-

Previously, a 15-crown-5-containing molecular

(

crown-5 unit (ﬁsh hook), was reported to be a selective CE-

based Na⁺ion transporter. It featured a low Na /K

selectivity of 3.6 and a low transport activity (EC (Na ) >

5% of the membrane lipid present). With a Na /K selectivity

of 13.7 and EC (Na ) value of 0.47 μM (0.48 mol % relative

to lipid), this MB5-C8 is undoubtedly among the best Na -

56,57,59

selective synthetic transporters to date.

Such Na /K

selectivity is even comparable to that of some of the natural

60,61

Na channels.

Following the same trend, the benzo-18-crown-6-containing

MB6-C12 shows excellent transport selectivity to K ions

(

Figure 3d), attaining an R value of 99.6% at 1.75 μM. For

comparison, R_N_aobtained under identical experimental

conditions is merely 14.3%, implying a K /Na selectivity of

.0. Although such selectivity is inferior to the highest example

in the literature (18.0 for the molecular ion ﬁsher design

described above ), its transport eﬃciency (EC = 0.41 mol

The calculation was accomplished by ﬁtting the transport activity

versus MB concentration using the Hill equation Y = 1/(1 + (EC /

%) is about 2 times higher (EC₅₀= 1.1 mol % for the

molecular ion ﬁsher), making it an overall excellent K -

[

MB]) , giving the EC values and Hill coeﬃcient n. The R values

for all ﬁttings were greater than 0.90. The total lipid concentration

selective synthetic transporter. As discussed earlier, favorable

ion binding does not necessarily lead to superior ion transport

utilized was 97.5 μM in these tests.^E^C⁵0 values cannot be

experimentally determined, as precipitations occur before 50% of

selectivity for K ions. For instance, Sun et al. reported on H-

the maximal transport activity is reached. Li /M , Rb /M , and Cs /

bond-directed stacks of cholesteryl-18-crown-6 complexes in

M refer to the transport selectivity of Li , Rb and Cs over other

alkali-metal ions, respectively. The boldface entry in each row of

EC (M ) represents the highest ion transport activity of MBs

2015, which feature clear Cs selectivity followed by Rb , K ,

Na , and Li in descending order. It is also worth mentioning

that the K -selective MB6-C12 displays K /Li , K /Cs , and

K /Rb selectivities of 26.9, 5.0 and 1.8, reminiscent of the

containing the same crown units but diﬀerent alkyl linkers.

MD simulation results of ion binding aﬃnity of 18-crown-6 in

on a 12-crown-4-based helical peptide, but its Li selectivity

aqueous solution. A similar transport activity (EC50(K ) =

+ +

was not well characterized. Lithium salts have been found to

be eﬀective in treating manic depression and also in reducing a

0.52 and 0.86 μM) and K /Na selectivity (R /RNa = 7.8 and

7.7) can also be achieved with MB6-C8 and MB6-C10,

47,48

patient’s risk of attempting suicide,

whereas a major

respectively (Table 1 and Figures S12 and S13). Together with

drawback of such lithium therapy is the low penetration rate of

lithium through biological membranes. This leads to delayed

onset and decreased eﬀect of the pharmaceutical action, usually

making relatively large doses necessary that could further cause

undesirable side eﬀects. Given an EC (Li ) value of 0.13

μM and Li /M selectivity of greater than 47.5, we anticipate

that MB4-C10 may have potential value as potent codrugs in

such lithium therapy. Similar excellent Li selectivity is also

seen with MB4-C8 and MB4-C12, although their transport

eﬃciency varies with EC (Li ) values of 1.01 and 0.16 μM,

respectively (Table 1). Other potential applications of such

MB6-C12, they form a group of highly active synthetic K

transporters with high K /Na selectivity.

Likewise, MB7-C8 containing 21-crown-7 as the ion capture

unit favorably transports the large Rb and Cs ions with

almost identical selectivity and eﬃciency (Figure 3e). In the

literature, examples of 21-crown-7-based ion transporters are

relatively rare, in comparison to other CEs. Voyer et al. once

reported a 21-crown-7-appended helical peptide as an ion

channel, which unfortunately is unable to diﬀerentiate alkali-

metal ions. Otis et al. also showed similar 21-crown-7-based

α-helical peptides acting as transmembrane ion channels with a

highly eﬃcient, Li -selective synthetic transporters could

include electrolytes for lithium batteries, production of ion-

selectivity order of Cs > K > Na , despite their moderate

transport eﬃciency. In comparison, MB7-C8 displays

51,52

selective electrodes,

lithium from the ocean.

lubricants, and even extraction of

preferable transport of Cs and Rb ions (R = 95.1% and

4,55

R_R_b= 91.1% at 1.0 μM), with Cs /K , Cs /Na , and Cs /Li

selectivities of 1.6, 4.5, and 3.3, respectively. The EC values

MB5-C8, containing Na -selective benzo-15-crown-5 (Table

S1) and n-C H linkers, selectively transports Na ions, giving

of MB7-C8 toward Rb and Cs ions, determined by Hill

analysis, are 105 and 77 nM, respectively, corresponding to

transporter/lipid ratios of about 1/928 and 1/1266, or 0.11

and 0.079 mol % relative to lipid. This makes MB7-C8, to the

best of our knowledge, the most eﬃcient Rb⁺and Cs

synthetic transporter to date.

a fractional activity R of 103.1% at 3.0 μM (Figure 3c). Its

corresponding R_Mvalues for Li , K , Rb and Cs are 8.8%,

.5%, 4.2%, and 1.1% respectively, indicative of an excellent

Na selectivity. In the literature, Na transporters have been far

less well-developed than K -selective transporters,

21,56,57

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

One may have noticed the diﬀerences in ion transport

selectivity of MBs bearing the respective crown units described

above (Figure 3b−e), which are well aligned with the solution

binding selectivities of benzo-CEs (Table S1). In general, MBs

containing small CEs (i.e., 12-crown-4 and 15-crown-5)

demonstrate much better selectivity than those containing

large CEs (i.e., 18-crown-6 and 21-crown-7). This trend agrees

with previous observations that the binding aﬃnity of diﬀerent

CEs toward their preferred alkali-metal ions was more

pronounced for small ions in comparison to large ions.

The structural ﬂexibility of the CE macrocycle may account for

such an incongruity. Apart from the preferred ones, large CEs

can readily deform and adapt their macrocycles for binding

with small alkali-metal ions, whereas the opposite situation

i.e., small CEs binding with large ions) is unlikely and might

require alternative binding conﬁgurations, such as a sandwich-

(

like complex involving two CEs and one cation. This may

explain the exceptional selectivity of MB4-C10 for Li and

MB5-C8 for Na ions, followed by the high selectivity of MB6-

C8 for K ions and the low selectivity of MB7-C8 for Cs /Rb

ions (Table 1). To further shed light on the ion transport

mechanism, we prepared the 18-crown-6-based control

compound MB6-R, having a rigid 1,2,3-triazole moiety in the

alkyl linker of ∼14.4 Å (Figure S19). This length is comparable

to that of n-C H (∼13.2 Å) in MB6-C6 and n-C H linkers

(

∼15.6 Å) in MB6-C8. However, no detectable K transport

activity emerged with MB6-R at a concentration as high as 5.0

μM, while the similar-sized MB6-C6 and MB6-C8 exhibit

transport activities of 46.3% and 100% at 5.0 and 2.5 μM,

respectively (Figure S19 ). These comparative data clearly

reveal the indispensable role of ﬂexibility of the alkyl linkers for

these MBs to perform as ion transporters. One possible ion

transport pathway can therefore be proposed (Figure 1c),

involving (i) ion capture at the membrane-water interface, (ii)

ion relay between adjacent CEs facilitated by swinging of the

ﬂexible alkyl linker, and (iii) ion release upon reaching the

Figure 4. Schematic illustration and experimental results of SPQ and

CF dye leakage assays. (a) SPQ assay to conﬁrm the inability of MBs

to transport Cl anions. [MB6-C12] = [MB7-C8] = [L8] = 2.0 μM;

−

[

MB4-C10] = [MB5-C8] = 0.1 μM. (b) CF dye leakage assay to

conﬁrm the LUV integrity in the presence of MBs at 2.0 μM.

of 0.1 μM was therefore utilized, at which MB4-C10 and MB5-

C8 induced a fractional transport activity R

value of 34.9%

opposite side.

and R_N_avalue of 19.4% in the HPTS assay, but no obvious

These MBs share certain similarities with both conventional

channel- and carrier-class transmembrane ion transporters. On

one hand, they span across the entire membrane with openings

to both the intra- and extravesicular regions simultaneously, an

inherent characteristic to channels. On the other hand, there

exist no well-deﬁned passages for ions to pass through and the

transporting components (alkyl linkers and CEs) are free to

move within certain regions of the lipid membrane, resembling

how carriers ferry ions across. Single-channel current measure-

ments were therefore carried out to determine which

mechanism (channel or carrier) MB is following to transport

alkali-metal ions. Despite the extensive eﬀorts devoted, our

failure to capture any single-current signals suggests that these

MBs might perform more like ion carriers.

SPQ ﬂuorescence quenching was observed. When this is

combined with the fact that these MBs are only weakly active

for their unfavorable alkali-metal ions, the contradistinctive

data clearly conﬁrm the incapability of MBs to transport

anions, and the M /H antiport should be the main

transporting species.

We further conducted a carboxyﬂuorescein (CF) dye

leakage assay to conﬁrm the LUV membrane integrity in the

presence of these MBs (Figure 4b). The CF-containing

LUVs were prepared with the intravesicular region containing

50 mM CF dye, 10 mM HEPES, and 100 mM NaCl at pH 7.5.

The extravesicular region only containsed HEPES and NaCl at

identical pH. At such a high concentration, trapped CF dyes

largely remain as nonﬂuorescent dimers, whereas a dramatic

ﬂuorescence increase will emerge if there is any CF dye leakage

or membrane destruction taking place. As shown in Figure 4b,

MB4-C10, MB5-C8, MB6-C12, and MB7-C8 at 2.0 μM do

not induce any ﬂuorescence increase, and a similar conclusion

can also be drawn for other MBs (Figure S20). Conversely,

melittin, a pore-forming peptide as the positive control in this

assay, causes signiﬁcant ﬂuorescence increases of 77.2% and

94.0% at lower concentrations of 100 and 200 nM,

respectively. These results unambiguously conﬁrm that the

LUV membrane integrity is maintained in the presence of MBs

and that the ion transport activities observed are not due to

any MB-induced membrane-lysing eﬀect.

To conﬁrm alkali-metal ions as the main transporting

species, we introduced the chloride-sensitive SPQ (6-methoxy-

N-(3-sulfopropyl) quinolinium) dye into the LUV-based assay

(

Figure 4a). The intravesicular region contains 0.5 mM SPQ

and 200 mM NaNO salt, and the extravesicular environment

is set to be 200 mM NaCl. To establish the eﬃcacy of this SPQ

assay, the previously reported anion channel L8 was used as

the positive control, which caused 62.0% SPQ ﬂuorescence

quenching at 2.0 μM. At the same concentration, no detectable

quenching was observed with MB6-C12 or MB7-C8.

Unfortunately, MB4-C10 and MB5-C8 precipitated out at

.0 μM in the buﬀer-free salt solution. A lower concentration

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

https://pubs.acs.org/doi/10.1021/jacs.0c09655.

Article

temperature for 45 min to give a milky suspension. The mixture was

CONCLUSIONS

■

then subject to 10 freeze−thaw cycles: freezing in liquid N for 1 min

In summary, we have successfully prepared a suite of modularly

structured buckyball-based molecular balls and for the ﬁrst

time demonstrated that solution-based cation binding

selectivity can be reliably translated into membrane-based

transport selectivity. More precisely, identiﬁed to be the most

active in their respective group containing the same crown

units (EC₅₀= 0.079−0.48 mol % relative to lipid molecules),

MB4-C10, MB5-C8, MB6-C12, and MB7-C8 selectively

and heating at 55 °C in a water bath for 2 min. The vesicle suspension

was extruded through a polycarbonate membrane (0.1 μm) to

produce a homogeneous suspension of large unilamellar vesicles

(LUVs) of about 120 nm in diameter with HPTS trapped inside. The

extravesicular HPTS dye was separated from the LUVs using size

exclusion chromatography (stationary phase, Sephadex G-50, GE

Healthcare, USA; mobile phase, 10 mM HEPES buﬀer with 100 mM

NaCl, pH 7.0) and diluted with the mobile phase to yield 5.0 mL of

6.5 mM lipid stock solution. The LUV solution was stored in 4 °C

transport Li , Na , K , and Rb /Cs ions, respectively, with

transport selectivity well aligned with the intrinsic ion binding

aﬃnity of the respective crown ethers. Our ﬁndings thus

uncover the biocompatible C -fullerene as an excellent

refrigerator until use. The HPTS-containing LUV suspension (30 μL)

was added to a HEPES buﬀer solution (1.96 mL, 10 mM HEPES, 100

mM MCl at pH 8.0, where M = Li , Na , K , Rb , Cs ) to create a

pH gradient for ion transport studies. A solution of MB in dimethyl

sulfoxide or dimethylformamide (those with n-C12 linkers) at

diﬀerent concentrations was then injected into the suspension with

gentle stirring. Upon addition, the emission of HPTS was immediately

monitored at 510 nm with excitations at 403 nm recorded for 300 s

using a ﬂuorescence spectrophotometer (Hitachi, Model F-7100,

Japan). At 300 s, an aqueous solution of Triton X-100 (20 μL, 20% v/

v) was injected to induce the maximum change in dye ﬂuorescence

emission. The ﬁnal transport trace was obtained by normalizing the

ﬂuorescence intensity using the equation I = (I − I )/(I − I ),

platform to construct synthetic ion transporters with

unprecedented custom-designable ion transport selectivity,

which could provide a de novo basis for rationally designing

diverse types of artiﬁcial ion transporters with high transport

selectivity to support interesting applications within the

context of lipid or biomimetic membranes.

METHODS

■

where I = fractional emission intensity, I = ﬂuorescence intensity at

Typical Reaction Conditions. Malonic acid (520 mg, 5.0 mmol),

bromo alcohol (1.99−2.92 g, 11 mmol), and 4-dimethylaminopyr-

idine (244 mg, 2.0 mmol) were dissolved in a mixed solvent of

dichloromethane (10 mL) and dimethylformamide (10 mL). After

the mixture was cooled to 0 °C using an ice bath, N-(3-

time t, I = ﬂuorescence intensity after addition of Triton X-100, and

I₀= initial ﬂuorescence intensity. The fractional change R_Mwas

calculated for each curve using the normalized value of I at 300 s

before the addition of Triton, with the blank set as 0% and that of

Triton as 100%. Fitting the fractional transmembrane activity R_Mvs

(

dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (2.90

transporter concentration used the Hill equation: Y = 1/(1 + (EC /

g, 15 mmol) was added, and the reaction mixture was warmed up

overnight with stirring. Removal of solvent in vacuo gave the crude

product, which was washed using deionized water three times, dried

over Na SO , and then subjected to column chromatography to aﬀord

[

MB]) ) gave the EC values.

ASSOCIATED CONTENT

sı Supporting Information

■

the malonate linker with yields of 30−52%. The obtained linker

(

628−874 mg, 1.46 mmol), C -fullerene (105 mg, 0.15 mmol), and

The Supporting Information is available free of charge at

CBr (4.84 g, 15 mmol) were dissolved in 1,2-dichlorobenzene (15

mL), to which 1,8-diazabicyclo[5.4.0]undec-7-ene (445 mg, 2.9

mmol) was added dropwise. The reaction mixture was stirred at room

temperature for 3 days. Filtration of the reaction mixture gave a dark

red solution that was subjected to column chromatography to aﬀord

the C -linker complex with yields of 69−75%. A suspension of the

Synthetic procedures, a full set of characterization data

including H NMR, C NMR, and MS, and a complete

set of ion transport studies (PDF )

C -linker complex (224−292 mg, 0.068 mmol), 4′-carboxybenzo-

crown (239−356 mg, 0.89 mmol), and K CO (246 mg, 1.8 mmol) in

AUTHOR INFORMATION

■

dimethylformamide or acetonitrile (15 mL) was heated at 85 °C.

Removal of solvent in vacuo gave the crude product, which was

successively washed with deionized water and methanol three times

each to aﬀord MBs as dark solids or tarlike heavy oils with yields of

Corresponding Author

Huaqiang Zeng − Institute of Advanced Synthesis and Yangtze

River Delta Research Institute, Northwestern Polytechnical

University, Xi ’an, Shaanxi 710072, People ’s Republic of

China; orcid.org/0000-0002-8246-2000;

Email: hqzeng@nwpu.edu.cn

4−96%.

NMR and MALDI-TOF Measurements. NMR spectra were

acquired with a Bruker ACF-400 (400 MHz) spectrometer. The

solubility of compounds with C12 linkers is only moderate in

dimethyl sulfoxide, and chloroform was found to dissolve them well.

Authors

Deuterated dimethyl sulfoxide (DMSO-d , 99.5%) and chloroform

Ning Li − The NanoBio Lab, Singapore 138669;

(

CDCl , 99.8%) were purchased from Cambridge Isotope Labo-

orcid.org/0000-0003-0179-1425

ratories, Inc., and used as received without further puriﬁcation.

MALDI-TOF mass spectra were acquired with a JMS-S3000 Spiral

TOF instrument (JEOL Ltd., Japan) in the positive linear

conﬁguration. System parameters were as follows: 20 kV accelerating

potential, 250 Hz laser frequency, 500 ns delay time. The matrix was a

saturated solution of 1,8,9-trihydroxyanthracene (dithranol) in

dichloromethane.

^I^oⁿ^T^r^aⁿ^s^p^o^r^t^S^t^u^d^y^aⁿ^d^E^C⁵0 Measurements Using the

HPTS Assay. Egg yolk L-α-phosphatidylcholine (EYPC, 1.0 mL, 25

mg/mL in chloroform, Avanti Polar Lipids, USA) was loaded into a

round-bottom ﬂask, and the solvent was removed under reduced

pressure at 30 °C. After the resulting ﬁlm was dried under high

vacuum overnight at room temperature, the ﬁlm was hydrated with

HEPES buﬀer solution (1.0 mL, 10 mM HEPES, 100 mM NaCl, pH

Feng Chen − The NanoBio Lab, Singapore 138669

Jie Shen − The NanoBio Lab, Singapore 138669

Hao Zhang − Institute of Advanced Synthesis and Yangtze

River Delta Research Institute, Northwestern Polytechnical

University, Xi ’an, Shaanxi 710072, People ’s Republic of

China; orcid.org/0000-0002-7961-3322

Tianxiang Wang − School of Physical & Mathematical

Sciences, Nanyang Technological University, Singapore

637371

Ruijuan Ye − Institute of Advanced Synthesis and Yangtze

River Delta Research Institute, Northwestern Polytechnical

University, Xi’an, Shaanxi 710072, People’s Republic of

China

.0) containing the pH-sensitive HPTS dye (1.0 mM) at room

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

orcid.org/0000-0002-2936-337X

Article

Tianhu Li − Institute of Advanced Synthesis and Yangtze River

Delta Research Institute, Northwestern Polytechnical

University, Xi’an, Shaanxi 710072, People’s Republic of

China

Teck Peng Loh − Institute of Advanced Synthesis and Yangtze

River Delta Research Institute, Northwestern Polytechnical

University, Xi’an, Shaanxi 710072, People’s Republic of

China; School of Physical & Mathematical Sciences,

Nanyang Technological University, Singapore 637371;

(13) Montenegro, J.; Ghadiri, M. R.; Granja, J. R. Ion Channel

Models Based on Self-Assembling Cyclic Peptide Nanotubes. Acc.

Chem. Res. 2013, 46 , 2955.

(14) Fyles, T. M. How Do Amphiphiles Form Ion-Conducting

Channels in Membranes? Lessons from Linear Oligoesters. Acc. Chem.

Res. 2013, 46, 2847.

15) Otis, F.; Auger, M.; Voyer, N. Exploiting Peptide Nanostruc-

tures To Construct Functional Artificial Ion Channels. Acc. Chem. Res.

013, 46, 2934.

16) Gong, B.; Shao, Z. Self-Assembling Organic Nanotubes with

Precisely Defined, Sub-nanometer Pores: Formation and Mass

Transport Characteristics. Acc. Chem. Res. 2013, 46, 2856.

(17) Ren, C.; Chen, F.; Ye, R. J.; Ong, Y. S.; Lu, H.; Lee, S. S.; Ying,

J. Y.; Zeng, H. Q. Molecular Swings as Highly Active Ion

Transporters. Angew. Chem., Int. Ed. 2019 , 58 , 8034.

Yi Yan Yang − Institute of Bioengineering and Nanotechnology,

The Nanos, Singapore 138669; orcid.org/0000-0002-

871-5448

Complete contact information is available at:

https://pubs.acs.org/10.1021/jacs.0c09655

18) Pedersen, C. J. Cyclic polyethers and their complexes with

metal salts. J. Am. Chem. Soc. 1967, 89 , 2495.

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Conducting Highly Selective K -Channels via Modularly Tunable

Notes

Directional Assembly of Crown Ethers. J. Am. Chem. Soc. 2017, 139,

The authors declare no competing ﬁnancial interest.

12338.

(

20) Feng, W.-X.; Sun, Z.; Zhang, Y.; Legrand, Y.-M.; Petit, E.; Su,

ACKNOWLEDGMENTS

■

C.-Y.; Barboiu, M. Bis-15-crown-5-ether-pillar[5]arene K -Responsive

Channels. Org. Lett. 2017, 19, 1438.

This work was supported by the Institute of Advanced

Synthesis, Northwestern Polytechnical University, and Nano-

Bio Lab (Biomedical Research Council, Agency for Science,

Technology and Research, Singapore).

(21) Ye, R. J.; Ren, C.; Shen, J.; Li, N.; Chen, F.; Roy, A.; Zeng, H.

Q. Molecular Ion Fishers as Highly Active and Exceptionally Selective

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