Evidence for multiple alkali metal cation complexation in membrane-spanning ion transporters

Article abstract of DOI:10.1039/b005442i

The polynitrogen-containing cation transporters 1-4 are shown by electrospray mass spectrometry to form multicationic species. In the absence of Na⁺, protonated species dominate but increasing the sodium concentration leads to all binding sites

Full text of DOI:10.1039/b005442i

Evidence for multiple alkali metal cation complexation in membrane-spanning
ion transporters
Hossein Shabany,a Clare L. Murray,a Charles A. Gloeckner,b Michael A. Grayson,b Michael L. Grossb and
George W. Gokel*a
a Bioorganic Chemistry Program and Dept. of Molecular Biology & Pharmacology, Washington University School of
Medicine, 660 South Euclid Ave., Campus Box 8103, St. Louis, MO 63110, USA.
E-mail: ggokel@molecool.wustl.edu
b Department of Chemistry, Washington University, St. Louis, MO 63130, USA
Received (in Columbia, MO, USA) 28th June 2000, Accepted 16th October 2000
First published as an Advance Article on the web
The polynitrogen-containing cation transporters 1–4 are
shown by electrospray mass spectrometry to form multi-
cationic species. In the absence of Na+, protonated species
dominate but increasing the sodium concentration leads to
all binding sites being occupied.
The study of cation-conducting channels currently presents an
enormous challenge to both chemists and biologists.1 An
important goal for the former is to prepare compounds that are
inherently simpler than protein, or even peptide, channels2 that
transport cations at substantial rates across phospholipid bilayer
membranes. With functionality achieved, experimental study of
the systems should offer insight into the details of such
phenomena as ion transport, selectivity, and gating kinetics. A
major question concerns the chemical details of cation transport
within the pore. A postulate that K+ transport and selectivity
were influenced by cation–p interactions within the pore3 was
disproved by site-directed mutagenesis.4 The chemical roles of
polyarenes in synthetic channels5 and of the central ion capsule6
in the KcsA K1 channel of Streptomyces lividans7 beg further
exploration of alkali metal cation interactions in low polarity
media. In a previous study, electrospray mass spectrometric
results demonstrated that all three macrocyclic rings of a
hydraphile channel-former could simultaneously bind cations.8
We now report that the novel tetramacrocyclic9 and nitrogen-
heterocycle-containing10 channel-formers reported in the pre-
ceding two communications can bind a full complement of
alkali metal ions by using crown ethers but not piperazine or
bipiperidyl donors.
Cation transport through a phospholipid bilayer for the
hydraphile family has been demonstrated by using fluorescence
(H+),11 23Na NMR (Na+),12 and planar bilayer conductance
methods (Na+).13 The ability of hydraphile channels to span the
bilayer14 has been confirmed and the optimum length for this
family of channels has been established.15 The compounds
reported here are elaborations of the previously known systems
and all exhibit effective Na+ transport in a phospholipid bilayer
(NMR method). Thus, under comparable conditions, the
transport rates in mixed phosphatidyl choline–phosphatidyl
glycerol bilayer liposomes, the following Na+ transport rates
were observed at room temperature: 1, 80%; 2, 210%; 3, 50%;
and 4, 340%. In all cases, the values are expressed as a percent
of the rate observed for the dansyl-sidearmed channel, 5
(100%).
to give PhCH2 < N18N > (CH2)11CO2Me. Hydrolysis (2 M
NaOH, 93%) followed by treatment with (COCl)2 (2 M solution
in CH2Cl2, 2 h) gave PhCH2 < N18N > (CH2)11COCl, which
was allowed to react directly in CH2Cl2 with H < N18N > H
(Et3N, 6 equiv., cat. DMAP, to give the tris(crown) diamide as
a yellow oil (65%). The tris(crown) diamide was debenzylated
Compounds 211 and 514 were prepared as previously reported
and the synthesis of 4 is described in the preceding communica-
(H2–Pd/C,
[H < N18N > (CH2)11CO]2 < N18N > (yellow oil, 95%). A
similar strategy was used to prepare
60
psi,
abs.
EtOH)
to
give
tion.9 Compound
1
was prepared by treatment of
PhCH2 < N18N > H with Br(CH2)12Br (Na2CO3, KI, 2 h) in
boiling PrCN to give PhCH2 < N18N > (CH2)12Br as a yellow
oil (39%). Further reaction of the above with 4,4A-bipiperidine
(procedure as above, 16 h) gave 1 as a colorless solid (8%, mp
55–56 °C). The preparation of 3 required a more elaborate
approach. Benzyldiaza-18-crown-6 was alkylated with
Br(CH2)11CO2Me (Na2CO3, cat. KI, BuCN, 20 mL, 24 h, 50%)
ClCO(CH2)11NC5H9C5H9N(CH2)11COCl, beginning with the
alkylation of 4,4A-bipiperidine by Br(CH2)11CO2Me/Na2CO3
(cat.
KI,
BuCN,
20
mL,
24
h)
to
give
MeO2C(CH2)11NC5H9C5H9N(CH2)11CO2Me (71%). This, in
turn, was hydrolyzed (2 M NaOH, 94%, colorless solid, mp
260 °C, decomposed) and treated with ClCOCOCl (excess, 2 M
DOI: 10.1039/b005442i
Chem. Commun., 2000, 2375–2376
This journal is © The Royal Society of Chemistry 2000
2375

Table 1 Electrospray mass spectrometric analysis of compounds 1–4
structures. In principle, if 4,4A-bipiperidyl could support cation
complexation directly, the complexes [1·3Na]3+ and [3·4Na]4+
would have been observed at m/z values of 424.7 and 442.1,
respectively. Peaks corresponding to such complexes were
sought but not observed.
An interesting observation is that the higher organization of 3
relative to 2 appears to result in a more stable tris(Na+) complex.
Thus, the [host·2Na]2++[host·3Na]3+ ion abundance ratios for 2
and 3 are 100+20 and 100+45. The ‘central’ macrocycle is
bis(amidated) in 3 and is expected to be a weaker donor for Na+.
It appears that the diamine opposite can help to stabilize this
complex relative to the situation in which the additional
structural element is absent.
The critical inference we draw from these data is that direct
interaction between the bipiperidyl unit of 1 and a cation is not
detectable, even in the low dielectric medium of electrospray
mass spectrometry. Extending this to the low polarity, insulat-
ing regime of a bilayer, we infer that bipiperidyl cannot directly
support cation complexation in that situation either. This does
not prove, but strongly supports, the notion that the function of
1 as a channel is due, in part, to the interaction of bipiperidyl
with intrapore water or waters of hydration rather than directly
with Na+.10
Ion
m/z
Rel. int. (%)
1
1204.98
1227.8
625.5
424.7
324.2
1299.01
1322.5
672.8
456.2
347.9
1675.3
1698.7
861.1
581.8
442.1
1713.5
1737.7
880.2
594.4
451.3
n/a
30
100
0
[1·1Na]+
[1·2Na]2+
[1·3Na]3+
[1·4Na]4+
2
0
n/a
72
100
20
0
n/a
30
100
45
0
n/a
38
100
87
47
[2·1Na]+
[2·2Na]2+
[2·3Na]3+
[2·4Na]4+
3
[3·1Na]+
[3·2Na]2+
[3·3Na]3+
[3·4Na]4+
4
[4·1Na]+
[4·2Na]2+
[4·3Na]3+
[4·4Na]4+
We thank the NIH (GM 36262) and NSF (CHE-9805840) for
grants that supported this work. The mass spectrometry research
resource is supported by the National Centers for Research
Resources of the NIH (Grant P41RR00954).
solution in CH2Cl2). The latter was treated with
[H < N18N > (CH2)11CO]2 < N18N > (Et3N, 6 equiv., cat.
DMAP, CH2Cl2, 48 h) to afford tetraamide 3 as a yellow oil
(30%).
Compounds 1–4 present an opportunity to directly assess the
interactions of the hydraphile’s modular elements and Na+.
Assuming that a crown macroring will bind a single Na+ ion, we
anticipate that we will observe ions corresponding to [1·2Na]2+,
[2·3Na]3+, [3·3Na]3+, and [4·4Na]4+. The projected maximum
complexation by 1 or 3 is based on the expectation that 4,4A-
bipiperidyl will not complex Na+. This is particularly important
considering the conclusions of the preceding communication
that the bipiperidyl unit can organize water but its nitrogen
donor groups are too far apart to effectively coordinate with
Na+.10
The mass spectrometric analyses were conducted using an
electrospray ion source (ESI-MS).16 The inlet temperature was
ca. 55 °C. Typically, 1.5 mg of channel was dissolved in 1 mL
of CHCl3 and the spray solution was prepared by adding 1 mL
of MeOH–CHCl3 1+1 (v/v) and 40 mL of 100 mM NaOH to 60
mL of the sample solution. After mixing, 20 mL of the sample
solution was loop injected by continuous infusion [10 mL min21
MeOH–CHCl3 (1+1, v/v)]. The instrument continuously
scanned (magnetic) at 20 s decade21 of mass over the range
from 2000 to 400 Da. The data thus obtained are shown in
Table 1.
For each structure, we have calculated the anticipated
molecular weights for adducts of 1–4 with 1, 2, 3 and 4 Na+
ions. In each case, the most abundant ion (base peak) observed
was the disodium adduct. In all cases, the maximum number of
Na+ ions complexed was equal to the number of macro-rings
and no higher order ions were detected. It is also interesting to
observe that the ion currents for the complexes containing either
three or four Na+ ions (2, 3 or 4) were significantly larger than
observed for either cation complex of 1.
The most significant finding of the present study is that in all
cases, the number of Na+ ions bound by each host molecule
corresponded to the number of macrocycles. In no case did the
number of Na+ ions exceed the number of macro-rings
indicating a generalized affinity of the cation for any of these
Notes and references
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Cambridge University Press, Cambridge, 1996; B. Hille, Ionic Channels
of Excitable Membranes, Sinauer Press, Sunderland, MA, 2nd edn.,
1992.
2 B. Bechinger, J. Membr. Biol., 1997, 156, 197.
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2000, 2, 37.
6 B. Roux and R. MacKinnon, Science, 1999, 285, 100.
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10 C. L. Murray, H. Shabany and G. W. Gokel, Chem. Commun., 2000,
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11 O. Murillo, S. Watanabe, A. Nakano and G. W. Gokel, J. Am. Chem.
Soc., 1995, 117, 7665.
12 O. Murillo, I. Suzuki, E. Abel, C. L. Murray, E. S. Meadows, T. Jin and
G. W. Gokel, J. Am. Chem. Soc., 1997, 119, 5540.
13 E. Abel, E. S. Meadows, I. Suzuki, T. Jin and G. W. Gokel, Chem.
Commun., 1997, 1145.
14 E. Abel, G. E. M. Maguire, E. S. Meadows, O. Murillo, T. Jin and G. W.
Gokel, J. Am. Chem. Soc., 1997, 119, 9061; E. Abel, G. E. M. Maguire,
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9043.
15 C. L. Murray and G. W. Gokel, Chem. Commun., 1998, 2477.
16 Only sectors one and two, a reversed geometry BE, extended mass range
configuration (Vacuum Generators ZAB-T), were used for the mass
analysis. The electrospray ion source operated as follows: spray needle
voltage, 8940 V; counter electrode, 5255 V; sampling cone, 4200 V;
accelerating voltage, 4120 V; source temperature, 75% power (ca. 55
°C). Sample preparation was as follows: 1.5 mg of sample was dissolved
in 1 mL chloroform.
2376
Chem. Commun., 2000, 2375–2376