A unimolecular G-quadruplex that functions as a synthetic transmembrane Na+ transporter

Article abstract of DOI:10.1021/ja056888e

We describe the covalent post-modification of a hydrogen-bonded assembly with the subsequent formation of a potent transmembrane Na⁺ ion transporter. Olefin metathesis is used to cross-link all 16 guanosine subunits in a lipophilic G-quadruplex

Full text of DOI:10.1021/ja056888e

Published on Web 12/10/2005
A Unimolecular G-Quadruplex that Functions as a Synthetic Transmembrane
Na+ Transporter
Mark S. Kaucher, William A. Harrell, Jr., and Jeffery T. Davis*
Department of Chemistry and Biochemistry, UniVersity of Maryland, College Park, Maryland 20742
Received October 8, 2005; E-mail: jdavis@umd.edu
There is a growing appreciation that nucleobases can be used to
build supramolecular systems with potential applications.1 We,2 and
others,3 have proposed that the G-quartet, a motif found in nucleic
acid assemblies,4 may serve as a scaffold for building synthetic
transmembrane ion channels. As the first step toward achieving
that goal we show that a strategy combining noncovalent synthesis
and post-assembly modification of a G-quadruplex generates a
unimolecular ion transporter that moves Na+ across a phospholipid
bilayer.5 Synthetic pores and transmembrane transporters may
ultimately provide new sensors and antimicrobial agents.6
We have previously shown that 5′-tert-butyldimethylsilyl-2′,3′-
Figure 1. CD spectra of unimolecular G-quadruplex 3 (0.05 mM) in 10
isopropylidene guanosine forms a lipophilic G-quadruplex of
formula [G]16‚3K+‚Cs+‚4pic-.7 One remarkable feature of this
structure are four collinear cations that give the appearance of an
ion channel. This G-quadruplex has dimensions (26 Å × 30 Å ×
30 Å) that approach those needed to span a bilayer membrane. For
instance, the gramicidin dimer, a Na+ channel, has been estimated
to be 26 Å thick.8
Despite the thermodynamic stability of this noncovalent as-
sembly, individual guanosine subunits are in dynamic equilibrium
between “monomer” and hexadecamer when lipophilic G-quadru-
plexes are in solution.9 To circumvent problems posed by such
kinetic instability we decided to use olefin metathesis to cross-link
subunits that had been organized within a G-quadruplex (Scheme
1).10 We reasoned that such post-assembly modification might
mM sodium phosphate (pH 6.4) with (a) and without (b) EYPC liposomes
(100 nm).
protons (δ 11.83 and 11.70) and N2 amino protons (δ 9.72 and
9.56) were characteristic of a D4-symmetric hexadecamer 2 of
formula [G 1]16‚4K+‚4DNP-. Finally, diffusion NMR experiments
in CD2Cl2 confirmed that this species was indeed a hexadecameric
G-quadruplex 2.9,12
Olefin metathesis of the self-assembled G-quadruplex 2 (8 mM),
using Grubb’s second-generation catalyst, (H2IMes)(PCy3)(Cl2)-
RudCHPh (10 mol % per alkene unit),13 was complete within 48
h in CH2Cl2 at 35 °C. Metathesis was monitored by tracking the
1
disappearance of the H NMR signals for the terminal olefins in
G-quadruplex 2 and the appearance of new signals for the secondary
olefins (cis/trans mix) within the metathesis product 3. Importantly,
no olefin metathesis was observed when “monomeric” G 1 (130
mM) was treated with Grubb’s catalyst under similar conditions.
Thus, cation-templation of the noncovalent G-quadruplex 2 is
necessary for the subsequent covalent capture step.
Scheme 1
The structure of the metathesis product 3 was confirmed using
mass spectrometry, diffusion NMR, and CD spectroscopy. ESI-
MS showed a parent peak at m/z ) 2766.3 consistent for a triply
charged species of formula C384H400N80O128‚3K+ and molecular
weight of 8299 amu. Other ESI-MS experiments also showed that
3 runderwent Na+/K+ cation exchange, providing the first hint that
this unimolecular G-quadruplex could mediate ion exchange.
Pulsed-field gradient NMR experiments in DMSO-d6 confirmed
that metathesis product [G]16 3 was much larger than both precursor
G 1 and adenosine standard A 4. Thus, the ratio of the experimental
diffusion coefficients (Ds(A 4)/Ds[G]16 3) ) 0.40 was close to the
value expected for a hexadecamer (0.37).9 Finally, the metathesis
provide a unimolecular G-quadruplex that would be stable and
functional within the hydrophobic phospholipid membrane. As
detailed below, this synthetic strategy appears to have been
successful.
The precursor, 5′-(3,5-bis(allyloxy)benzoyl)-2′,3′-isopropylidene
G 1 was prepared in two steps from guanosine. G 1 was outfitted
with two meta-substituted allyl ethers, with the notion that such a
pattern would permit olefin metathesis both within an individual
G-quartet and between layers. Also, the 3,5-diallyl ethers would
be unlikely to undergo intramolecular cyclization to give a strained
cyclophane. Solid-liquid extraction of potassium 2,6-dinitrophen-
olate (K+DNP-) by G 1 in CH2Cl2 gave quantitative formation of
the G-quadruplex 2 as determined by CD and NMR spectroscopy.
G-quadruplex 2 had the characteristic positive CD band, centered
at 258 nm, for a structure with stacked G-quartets.7,11 The two sets
of 1H NMR signals in a 1:1 ratio, the 4:1 ratio of NMR signals for
G 1 and DNP- anion, and signals for hydrogen-bonded N1 amide
1
product [G]16 3 gave characteristic H NMR signals (NH amide
signals at δ 11.76 and 11.64) and CD spectra (λmax ) 260 nm) in
CD2Cl2 for a lipophilic quadruplex (see Supporting Information).
Significantly, metathesis product [G]16 3 gave the diagnostic CD
spectrum for a G-quadruplex when added to an aqueous solution
of EYPC liposomes (Figure 1), indicating that [G]16 3 can fold
into this secondary structure within the phospholipid bilayer.14 In
contrast, no active CD bands were observed for [G]16 3 when it
was added to a solution of 10 mM sodium phosphate that did not
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J. AM. CHEM. SOC. 2006, 128, 38-39
10.1021/ja056888e CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. A series of 23Na NMR spectra 10 min after addition of (a)
metathesis product 3, (b) G 1, (c) DMSO blank, and (d) gramicidin to a
solution of EYPC liposomes (200 nm) that initially contained 100 mM LiCl,
10 mM lithium phosphate suspended in an extravesicular buffer containing
100 mM NaCl, 10 mM sodium phosphate. Transport of Na+ across the
bilayer is indicated by a 23Na NMR peak at δ 0.24 ppm.
Figure 2. Transport of Na+ as determined in a pH gradient assay. EYPC
liposomes (100 nm) containing HPTS dye (0.1 mM) in 100 mM NaCl, 10
mM sodium phosphate (pH 6.1) were suspended in 100 mM NaCl, 10 mM
sodium phosphate (pH 6.1). The compounds, G 1, G-quadruplex 3, or
gramicidin, were added at t ) 0 s as DMSO solutions to give a 1:100 ligand-
to-lipid ratio. The addition of NaOH solution at t ) 40 s established a pH
gradient of about 1 pH unit. At t ) 430 s the liposomes were destroyed
with Triton-X detergent. Measurement of the fluorescence of the trianionic
and tetraanionic forms of HPTS dye allowed determination of the liposomal
pH.
ions across phospholipid bilayer membranes. We are now focused
on determining the cation selectivity (Na+ vs K+) and mechanism
of transport (carrier vs channel) for this transmembrane ion
transporter.
contain liposomes, indicating that G 3 is either unstructured or
insoluble in aqueous solution.
Acknowledgment. We thank the Department of Energy (BES,
Separations and Analysis Program) for financial support.
Having shown that metathesis product [G]16 3 forms a G-
quadruplex in phospholipid liposomes, we next evaluated its ability
to function as a transmembrane ion transporter. Initial studies used
a standard base-pulse assay to indirectly measure Na+ transport
across liposomal membranes.15 Liposomes (100 nm) containing the
pH-sensitive dye, HPTS, were suspended in a solution of 75 mM
Na2SO4, 10 mM sodium phosphate (pH ) 6.0). As shown in Figure
2, addition of exogeneous base led to a rapid increase in the internal
pH of these liposomes when they were in the presence of metathesis
product [G]16 3 (1 mol %). In sharp contrast, no pH change occurred
when either G 1 or the noncovalent assembly [G 1]16‚4K+‚4DNP-
was added to the HPTS-loaded liposomes. Gramicidin was a
positive control in these experiments. The pH increase mediated
by [G]16 3 is consistent with Na+ influx across the phospholipid
bilayer.
Direct evidence for transmembrane cation transport down a Na+
concentration gradient, as facilitated by unimolecular G-quadruplex
3, was obtained from 23Na NMR experiments.16 EYPC liposomes
(200 nm) containing 130 mM LiCl in 10 mM lithium phosphate
(pH ) 6.4) were suspended in a solution containing 100 mM NaCl,
10 mM sodium phosphate (pH ) 6.4). Addition of the NMR shift
reagent, Dy(PPPi)2-7, caused the “outer” 23Na peak to move upfield
to δ -7.00, distinguishing it from any “internal” 23Na at δ 0.24.
After incubation of the LiCl-filled liposomes with the metathesis
product [G]16 3 (0.1 mol %) for 10 min, 23Na NMR analysis showed
that equilibrium had been achieved between the internal and external
Na+ populations (Figure 3). Importantly, under the same conditions
no Na+ transport was mediated by the precursor G 1 or the
noncovalent assembly 2 [G 1]16‚4K+‚4DNP- (1 mol %). Again,
gramicidin served as a positive control in these Na+ transport
experiments.
Supporting Information Available: Experimental protocols and
selected spectroscopic data. This material is available free of charge
via the Internet at http://pubs.acs.org.
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In conclusion, we have used a strategy that combines noncovalent
synthesis and covalent capture to prepare a functional supramo-
lecular assembly, unimolecular G-quadruplex 3, in just two steps
from a guanosine derivative. The unimolecular G-quadruplex 3
apparently folds into a conformation that allows it to transport Na+
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