DOI:
10.1039/A903825F
(Feature Article)
Chem. Commun., 2000, 1-9
Hydraphiles: design, synthesis and analysis of a family
of synthetic, cation-conducting channels
Received 12th May 1999, Accepted 17th August 1999
First published on UnassignedUnassigned24th December 1999
Abstract
The concept of channels has been with us more than a century.
For half a century, biologists have studied the remarkable workings of
protein and peptide channels that permit various cations and small
molecules to pass through the phospholipid bilayer membrane. During the
past decade, attempts have been made by chemists and biochemists to examine
the action of channel compounds from the chemical point of view and to
model their function using synthetic structures. What follows is a
description of our own efforts to design, synthesize, and characterize a
cation transporter that functions in a phospholipid bilayer.
| George Gokel was born in New York City but moved as a child to
Florida where he grew up. He studied chemistry at Tulane University in New
Orleans and earned a doctorate in chemistry at the University of Southern
California in Los Angeles. After post-doctoral work with Donald Cram at
UCLA and a short stint at the DuPont Chemical Co., Dr Gokel began his
academic career. He has held positions in chemistry departments at the
Pennsylvania State University, the University of Maryland, and the
University of Miami. He is currently Professor in the Department of
Molecular Biology and Pharmacology and Director of the Bioorganic Chemistry
Program at the Washington University School of Medicine in St.
Louis. |
Introduction
During the second half of the 20th century, there have been three
important trends in organic chemistry. By the 1950s, the study of physical
organic chemistry had moved to the forefront of the science. The study of
steric and electronic effects and their influence on mechanism was at
center stage. The refinement of physical organic principles provided the
critical underlayment for the systematic development of synthetic
methodology and strategy. Synthetic chemistry was built on the dual
foundations of imagination and physical organic chemistry and reached
ascendancy during the 1970s and 1980s. The importance of these two areas
continues to be profound. Our understanding of mechanism and our ability to
synthesize essentially whatever we can envision has spurred the organic
chemist’s imagination into supramolecular chemistry, particularly
into the realms of bioorganic chemistry and materials development. Both of
these areas face a similar challenge. In short, it is to design a compound
that has a desired property or function without knowing precisely how such
function is controlled. We have referred to the effort to design compounds
having specific functions rather than specific structures as
‘property-directed synthesis’.1
This article is about our efforts to design compounds that perform as
transmembrane channels2 in phospholipid
bilayers.At the beginning of our effort to design functional synthetic channel
compounds, we confronted a difficulty faced by everyone who attempts to
mimic nature. The problem is to design a chemical compound that will
function as the natural material does even though Nature’s mechanism
or mode of action may be imperfectly understood. This problem was
compounded in the case of channel models because many in the biological
community viewed proteins as the only authentic channels. Even peptides
that exhibited channel-like function were regarded by some as intriguing
but marginally useful.
Design strategy
The basic issue that must be considered in the design of a
cation-conducting channel is how to get the cation from one side of a
bilayer membrane to the other. Organic chemists have dealt with the issue
of transporting cations across various membranes by designing, preparing,
and using a variety of carrier molecules. These carriers function by
complexing a cation at one surface of a membrane, carrying it
‘ferry-boat style’ across the non-polar or insulator regime of
the membrane, and then releasing it at the opposite membrane surface. Crown
ethers have proved to be particularly successful in transporting cations
across bulk membranes. In this context, many combinations of macrocycles,
salts, and solvents have been studied.3Our early work with macrocycles led us to confront an interesting
problem. The cation complexation constant is given by
KS (usually as the decadic logarithm, i.e. log
KS). The equilibrium constant is determined by the
rates at which complexation and decomplexation occur, i.e.KS =
k1/k−1 =
kc/kd =
kcomplex/kdecomplex. Simple crown
ethers4 such as 18-crown-6 show fast binding
and release kinetics as required for successful carrier transport but
cation binding selectivity is relatively poor. The cryptands are strong
binders that show excellent cation selectivity but their binding and
release kinetics are poor. We thus developed the family of compounds we
named ‘lariat ethers’5 that
could achieve the three-dimensional binding arrangement characteristic of
cryptands and that also would exhibit good binding dynamics.6
The
use of compounds that combine structural features thought to be important
with flexibility (and therefore adaptability) was a cornerstone of our
channel design philosophy. What were the critical structural features? The
first consideration was whether or not the channel would span the bilayer.
This is a particularly intriguing question because in 1989, when the
original design work was underway, the thickness of a bilayer was at least
subject to interpretation if not unclear. Actually, there are three
identifiable regions within the bilayer as shown in Fig. 1. These are the insulating regime or
‘hydrocarbon slab’, the polar headgroups, and the midpolar
regime. The overall thickness of the membrane will depend upon the
identities of the headgroups and the fatty acids.7
|
| Fig. 1 | |
Chemists generally consider the ‘membrane thickness’ to be
the entire width of the bilayer. This is known from X-ray structures of
liposomes to be about 40 Å or more.8
Biologists often regard the thickness of a membrane to be 30–35
Å since this is the value obtained from electrophysiological
measurements that corresponds to the insulator regime.9 The two values are different but both are correct
in their context. The question of what, exactly, requires to be spanned by
a channel model compound clearly remains. Should the channel’s length
be 30 or 50 Å?10 Is the
‘correct’ length somewhere in between?
This issue illustrates a fundamental problem in modeling biological
function. We may choose a span of either 30 or 40 Å to incorporate
into our design. Assume we choose 40 Å, complete the design and
synthesis, and then assess transport activity. If no cation transport is
observed, does that mean that the length is wrong or that some other design
feature is inappropriate? The length may be changed to 35 Å. If no
cation transport occurs, should lengths of 30, 45 Å, etc. be
tried? No variation in length will make the molecule function if some
critical feature different from span is ill-designed. Combinatorial
approaches could lead to optimization of this length but only after a
functional design is in hand.
Several variables can immediately be recognized as bearing on channel
function. These include the presence of donor groups, the
‘relays’, headgroups, and the conceptual models for the
channel. Each of these variables has aspects that must be considered in the
design of a synthetic channel. The consideration must, in the channel case,
be done without having an adequate picture of how the wonderful and complex
proteins actually work.
Donor groups
It seems reasonable that donor groups such as O, N or S must be present
in a channel compound or how would the channel interact with a cation? In
the design of the channel, one must consider which donor groups to
incorporate. Do we wish only a few donors to be present or should they be
numerous? Perhaps the decision about numbers will be influenced by whether
the donors are strong or weak. The strength or weakness of a donor group
depends on the cation with which it interacts. For example, sulfur
(thioether) is a good donor for Zn2+ but less effective for
K+.Ether oxygens, like water, are good donor groups for alkali metals. In
that case, what sort of scaffolding should be used to organize the donor
groups? Should the donor groups be incorporated into a macrocycle?
18-Crown-6 is selective for K+ but will a channel incorporating
18-crown-6 also be selective for this cation? Indeed, can we think about
cation selectivity in channels in the same way we conceive of
complexation?11 Let us consider 18-crown-6
and its ability to complex Na+ and K+. In aqueous
solution, where binding constants are low, 18-crown-6 is selective for
K+ over Na+. The respective binding constants are:
KS (Na+) = 6.5; KS
(K+) = 118.12 This translates to
an 18-fold selectivity for K+ over Na+. As noted
above, KS =
kcomplex/krelease =
k1/k−1. The binding rates are
known for these two cases and they differ by 2-fold: k1
(Na+) = 2.2 × 108 M−1;
k1 (K+) = 4.4 × 108
M−1. The selectivity therefore lies principally in the
≡10-fold difference in cation release rates:
k−1 (Na+) = 3.4 × 107
s−1; k-1 (K+) = 3.7 ×
106 s−1.
The reaction rates are important because a channel is a dynamic
structure. The selectivity of a crown ether or cryptand is determined by
what cation is bound relative to another. Which cations are transported
rather than retained must define the selectivity of a channel. Thus, the
binding selectivity of 18-crown-6 for K+ over
Na+ may mean that a channel incorporating this macrocycle would
pass Na+ and thus show transport selectivity for it
rather than for K+. When we observed above that ‘sulfur
(thioether) is a good donor for Zn2+ but less effective for
K+’, the context was binding rather than
‘permitting’ the cation to pass by. Thioether might be a
‘good’ channel donor group for an alkali metal or alkaline
earth metal ion in the sense that it permits K+ or
Ca2+ but restricts Zn2+.
Headgroups
An amphiphile is a compound having two different affinities or
‘philicities’. The amphiphile sodium dodecyl sulfate has a
polar sulfonic acid that constitutes the ‘head’ and a 12-carbon
span that comprises the ‘tail’. It seems reasonable that a
channel would be an amphiphile since it must insert into a bilayer membrane
that is constituted of phospholipid amphiphiles. If a single molecule spans
the channel, it must be a twin-headed amphiphile.In a channel molecule, the headgroup is required to play a second role:
it must serve as, or lead to, a cation entry portal. One point of a
membrane is, after all, to prevent salts from getting into or out of a
cell. If the channel is to function, it must facilitate the entry and exit
of cations (anions, small molecules etc.) without disrupting the
membrane structure. It must create a controlled orifice in both the intra-
and extra-vesicular surfaces of the bilayer.
There are two obvious challenges in the design of headgroups for a
synthetic channel. First, where should the headgroup be placed relative to
membrane elements? One possibility is at the membrane surface but an
alternative is in the midpolar regime, which is the gateway to the
hydrocarbon slab. Second, how polar should the headgroup be? If it is fully
charged, should the field be positive or negative? It seems reasonable that
a negatively charged headgroup would attract a cation and a cationic
headgroup would favor an anion but this is more intuition than knowledge.
Information about headgroup preferences might be gleaned from the specifics
of protein channel structures. Although the amino acid sequences of many
protein channels are known, the three dimensional structures of few have
been established.
For most proteins, transmembrane segments are identified by subjecting
the amino acid sequence to hydropathy analysis.13 Typically, a computer program examines the
entire length of the protein searching for sequences of amino acids that
are hydrophobic. A transmembrane segment is about 20 amino acids if it is
α-helical and it is about 10 amino acids if it is a β-sheet.
Assuming an α-helical transmembrane segment (the common situation),
the program looks for a sequence that is expected to partition into a low
polarity medium. It is interesting to note that in an α-helix, each
amino acid spans about 1.5 Å. Thus, a 20 amino acid sequence
translates to a 30 Å span—the estimated thickness of the
insulating regime. From this discussion, the problem is apparent. If one
doesn’t know the exact structure of the protein, it is hard to guess
whether the polar residues, if any, are on the membrane surface or in the
midpolar regime. In a synthetic channel, should there possibly be
‘headgroups’ in both positions? If one is unsure of the
headgroup position(s), then guessing whether the environment is positively
or negatively charged is obviously a challenge.
Water14 is a ubiquitous element in
biology and certainly present in bilayer membranes as well as in many
proteins. What role will water play in transport? It seems reasonable to
think that cations will be only partly desolvated as they pass through the
membrane. Complete desolvation is a high-energy process and it is hard to
see why all of the water would be stripped from a cation on the periplasmic
side of the membrane when it must be rehydrated on the cytoplasmic surface.
If water is attached to a cation, how does this affect transport? Must we
consider the larger size of the fully hydrated cation rather than its
crystallographic diameter? Will discrete molecules of water remain attached
to individual ions or will there be exchange with the environment? How does
the presence of water affect the choice of donor groups? Amidst all of
these variables, one thing that seems clear is that it will be difficult
for any positively charged species to traverse a 30 Å, nonpolar span
without some interim stabilization.
Our original design for a cation-conducting channel is shown in Fig. 2. The questions posed above were dealt with as
follows. Diaza-18-crown-6 polyethers were selected to serve as both
headgroups and entry portals. It was known15 that the crowns could bind both Na+
(log KS = 2.99)16 and
K+ (log KS = 3.80). It was also known from
the early work of Kuwamura17 and of
Okahara18 that alkyl-substituted crown
ethers formed micellar aggregates when sonicated in aqueous suspension. We
demonstrated that twin-tailed diazacrowns could form stable liposomes,
suggesting that the crown would be effective as a head group.19
|
| Fig. 2 | |
The two distal macrocycles were expected to serve both as headgroups and
as entry portals. A K+ cation (ionic diameter ≡2.7
Å) can pass through the center of the macrocycle as can
Na+ (≡2 Å). The central macrocycle was also expected
to serve as a portal. Its role was predicted to be as a ‘relay
station’ for the transient cation. The polar interaction of the crown
with the transient cation at the least polar locale within the bilayer (the
midplane) was expected to provide transient stabilization so the
transmembrane journey could be completed. It was unknown at the time of the
design how this might be accomplished within a protein channel.20
Dodecyl groups were chosen to be the hydrocarbon spacer chains and
sidearms. The notion was that the two covalently attached chains would
define the channel’s overall length while the flexible sidearms
organized along the lipid axis to provide the other ‘wall’ of
the channel. A C–C bond is about 1 Å in the linear sense. The
dodecyl chain is therefore just under 14 Å. This provides a span of
≡28 Å plus the thickness of the macrocycle. Such a span was
expected to cover the insulator regime of the bilayer. The arbitrary
decision was made to locate the channel’s headgroup in the
bilayer’s midpolar regime rather than on the membrane surface.
Diaza-18-crown-6 groups were chosen as the macrocycles because
invertable nitrogen imparts flexibility to the system and obviates the
problem of stereoisomerism. The protonation state of the nitrogen atoms
within the bilayer is unknown at the time of this writing. It is worth
noting that the schematic of the channel and four phospholipid monomers
shows the latter with headgroups proportional to the lipid chain
lengths.
Alternative designs
The development of synthetic channel models has been considered in a
number of groups.21 In some cases,
compounds were designed de novo and in others the structures of
the products of certain reactions suggested that they might possess channel
activity. On occasion, the transport of Na+, K+ or
Ca2+ was not studied but the assessment of transport efficacy
was limited to H+ or Co2+. Thus, the references cited
represent a great range of approaches, designs, structural types and
success.Synthesis of channel 1
The first channel in the tris(macrocycle) family, designated
C12<N18N>C12<N18N>C12<N18N
>C12 in a shorthand we developed some years ago,22 presented the interesting problem of being
nearly, but not quite, symmetrical. Of course, there is two-fold symmetry
through the central macrocycle. The distal crowns, however, are attached to
dodecyl chains on either side but are not symmetrical. Our best current
synthetic approach23 is accomplished as
follows.In the first step, diaza-18-crown-6 is monoalkylated by 1-bromododecane
to give C12<N18N> (Scheme
1). This, in turn, is treated with 1,12-dibromododecane to give
C12<N18N>C12Br. Use of the latter to dialkylate
diaza-18-crown-6 affords channel 1 (1). This approach was the
model for the more than 30 members of this novel structural family now in
hand.
|
| Scheme 1 | |
Assessment of ionophoretic activity
Three methods were used to assess the efficacy of the synthetic cation
transporters: fluorescence, 23Na NMR and planar bilayer
conductance. The fluorescence technique24
was used for determining proton flux in a few compounds and only at an
early stage of the study. Planar bilayer methods are discussed below. The
bulk of the quantitative measurements were accomplished by using a dynamic
NMR method. In short, phospholipid liposomes (vesicles) are created in the
presence of NaCl. 23Na NMR shows a single line for
Na+(inside) and Na+ (outside). When Dy3+,
a shift reagent, is added to the external solution, the chemical shift of
the external Na+ changes. When an ionophore is added to the
bilayer, internal and external Na+ may equilibrate and the
exchange rate constant may be determined from the concentration dependence
of the linewidth change: K = 1/τ =
π(Δν1−Δν0).25Multiple experiments at concentration ranges from 0–20 μM are
required to determine the rate constant for a single transporter. The
experiments were therefore done in tandem with a standard of known
activity. In the early work, this standard was the naturally occurring,
channel-forming peptide gramicidin.26
Gramicidin is an excellent pore-former that sometimes functions even when
experimental conditions are not properly maintained. Thus, the failure of a
synthetic channel-former to transport Na+ might occur due to
poor experimental conditions rather than lack of efficacy and gramicidin
might function despite the experimental problems. We have thus adopted
Dn<N18N>C12<N18N>C12<N18N>Dn (Dn =
dimethylaminonaphthylsulfonyl or dansyl) as our experimental standard. The
‘dansyl channel’ transports cations very reproducibly but fails
when experimental conditions are not properly maintained. We have also
modified the [Na+] from 100 to 250 mM which gives better
entrapment and more reproducible experimental results.
Using the 23Na NMR method, we found channel 1
transported Na+ across a phospholipid bilayer at a rate about
27% of that observed for gramicidin. The exchange rate observed for
gramicidin is ≡175 s−1 so channel 1 is
transporting cations on the millisecond time scale. We were also able to
correlate the transport rate with a number of structural variations
although the details are beyond the scope of this review. It was
interesting to note, however, that when terminal macrocycles were altered
from C12<N18N>≡ to <18N>≡
(aza-18-crown-6), Na+ transport activity was lost. Replacement
of the sidearm by benzyl, substituted benzyl, naphthyl, dansyl and others
led to differences in transport rates but most sidearm changes afforded
functional channels.
Naming the family of compounds
Many of the early channels were tris(macrocycle)s and we referred to
these compounds as such. As structural variations led to the removal of one
or more macrocycles, the name was no longer appropriate. We considered the
name ‘hydraphile’ as a possibility because of its association
with the two-headed monster slain by Hercules. The dictionary27 provided additional inspiration in two other
definitions. A hydra is ‘any of several small freshwater polyps of
the genus Hydra and related genera, having a naked cylindrical
body and an oral opening surrounded by tentacles’. Clearly the shape
and tentacles were highly suggestive. An additional definition added to the
appropriateness of the name: ‘A persistent or multifaceted problem
that cannot be eradicated by a single effort’.Control experiments
The fact that sodium flux was observed in the presence of 1 was
very encouraging but not conclusive. It could mean that all of the design
concepts were as originally conceived. It is always nice to have
one’s ideas proven successful. Still, the fact of sodium transport
was permissive rather than conclusive. It was possible, for example, that
the tris(macrocycle) functioned simply by detergent action. To assess this
possibility, the tris(macrocycle) ionophore was replaced by either Triton
X-100, a neutral detergent, or sodium dodecyl sulfate, an anionic
detergent. The concentration range in the 23Na NMR experiment
was expanded from the typical 0–20 μM by ten-fold to 0–200
μM but no cation flux (line broadening) could be detected in either
case.21It was possible that the tris(macrocycle)s were unusually active carrier
molecules rather than pore-formers. A conventional concentric tube
apparatus was used to assess carrier transport through a bulk
CHCl3 membrane in a group of 10 compounds.28 In this experiment, a beaker is charged with
CHCl3 and water. A glass tube is then suspended in the beaker
through the upper water layer and into the CHCl3. The outer,
upper aqueous ring is thus separated from the inner core of water. A NaX
salt can be carried through the CHCl3 bulk membrane from inner
core to outer ring. On the atomic scale, a distance of
≡107
Å must be traversed so the channel mechanism is
precluded. The transport rates observed in this experiment (relative to
valinomycin) were compared to those obtained for the same compounds in the
23Na NMR/bilayer experiment (relative to gramicidin). In short,
the data showed no discernible correlation. This does not prove the channel
mechanism but clearly discounts carrier transport within the
bilayer.29 These findings comport with the
observation that fragments of the channel such as
C12<N18N>C12<N18N>C12 or
<18N>C12<N18N>C12<N18> and known
carriers such as PhCH2<N18N>CH2Ph were not
sufficiently active to show transport when assessed by the NMR method.
It is interesting to note that addition of the tris(macrocycle)s to the
CHCl3 concentric tube system led to a dramatic increase in
hydration of that solvent.27 No further
work was undertaken to resolve this issue because it was tangential to the
main thrust of the effort.
It was possible that the rate differences observed for structurally
related channels might be due only to variations in the extent of membrane
penetration. Octanol–water partition coefficients30 were determined for several substituted crown
ethers and the experimentally determined values were compared to data
calculated by the Hint module of Sybyl.27
Agreement between experiment and calculation was good. The data showed that
the tris(macrocycle)s favored octanol (i.e. the membrane) by
>1010 up to as much as 1030. Although the rate differential was not due to
differences in partitioning, a minor kinetic effect was observed. When the
ionophore was added to the preformed suspension of liposomes and then
analyzed immediately, the plots of 1/τ
vs. [ionophore] showed
curvature. If NMR analysis was delayed for an hour, the lines were
essentially straight. Likewise, if the vesicles were formed in the presence
of the ionophore (direct incorporation) linear data were obtained. Care was
thus taken to permit equilibration of the sample system.
The channel’s conformation
Changing the size of the central macrocycle diminished the transport
rate but did not preclude it. Substituting the central macrocycle by an
O(CH2CH2O)3 chain again impeded but did
not prohibit sodium transport. We concluded that the central macrocycle in
R<N18N>C12<N18N>C12<N18N>R was
parallel to the lipid axis rather than parallel to the other two
macrorings. Thus, we inferred that the cation passed by but not through the
central macrocycle. This conformation is illustrated in Fig. 3. |
| Fig. 3 | |
Assessment of optimal distances
The tris(macrocyclic) channels were designed to function in a
phospholipid bilayer but membrane dimensions and the placement of a channel
within it are elusive. An attempt was therefore made to experimentally
determine the optimal length of the channel. This was done by varying the
length of the covalent, hydrophobic spacers in
PhCH2<N18N>C12<N18N>C12<N1
8N>CH2Ph. It was assumed that the overall conformation would
remain similar as the chain length was varied by 2 methylenes on either
side of the central macrocycle. The starting point for this exercise is
indicated on the graph (Fig. 4) by an arrow.
It was anticipated that incremental lengthening of a flexible assembly
would lead to some reduction in efficacy as less favorable conformations
were adopted. As chain length diminished, it was expected that a point
would be reached at which the structure was simply too small to span the
bilayer. Note that each change of 2 methylene units in the spacer is an
overall change of 4 methylenes or ≡4 Å in span. Shortening the
chain by 4 Å or lengthening by 8 Å drops the transport rate to
about half. Shortening by 8 Å leads to an inactive ionophore. Note
that by ‘inactive’ we mean that no transport activity can be
detected by the 23Na NMR method. |
| Fig. 4 | |
Assessment of the conformation and location of the channel
within the bilayer
The synthetic tris(macrocycle) channel compounds can readily be modified
to incorporate various structural probes. In particular, fluorescent
headgroups can be included as an integral part of the structure. In
biochemical studies, for example, the indolyl residue of tryptophan is
often used as a fluorescent probe. Fluorescent dansyl residues were
incorporated into the channel as headgroups:
Dn<N18N>C12<N18N>C12<N18N>Dn,
2. The fluorescence spectrum was determined in a variety of
solvents from nonpolar to polar as well as in a phospholipid bilayer. Note
that lipids were carefully screened to be sure that any fluorescent
impurities were absent. The fluorescence maxima (λmax)
are shown in Fig. 5, plotted as a function
of solvent polarity (the Reichardt parameter,
ET).31 A dashed line
indicates the fluorescence maximum determined for the dansyl channel. The
polarity experienced by the dansyl group is between that of ethanol and
methanol—about what would be expected for the glyceryl ester regime
of a phospholipid. In any event, the dansyl environment of the channel is
significantly more polar than would be expected were it embedded in the
membrane’s ‘hydrocarbon slab. |
| Fig. 5 | |
Fluorescence depth quenching
The heterocyclic ‘doxyl’ group quenches fluorescence by
virtue of its unpaired electron spin. By using doxyl-substituted
phospholipids, it is possible to estimate how far from the bilayer’s
midplane is the dansyl ‘headgroup’. 7-Doxyl- and
12-doxyl-palmitoyl-substituted phosphatidylcholines were used along with
the dansyl channel to estimate headgroup separation.32 Application of the appropriate equations33 gives a value for the headgroup separation of 28
Å (i.e. the distance of the headgroup from the
bilayer’s midplane is 14 Å). If the hydrocarbon slab is
approximately 30 Å thick and the dansyl groups are ≡6 Å,
whether measured laterally or transversely, one concludes that the
channel’s termini are in or near the midpolar region created by the
glyceryl ester residues. This comports with the position estimated from
dansyl fluorescence.Experiments designed to address headgroup issues
It was assumed that the conformation illustrated in Fig. 1 required the distal macrocycles to function
both as headgroups and entry portals. An effort was made to confirm
experimentally the ability of diaza-macrocycles to serve as amphiphile
headgroups. It was found that a range of 2-armed diaza-18-crown-6
derivatives could form stable liposomes when sonicated in aqueous
suspension.34 In a molecule such as
C18<N18N>C18, the octadecyl chains can function
only as hydrophobic tails so the macrocycles must comprise the headgroups.
Successful formation of stable liposomes from R<N18N>R clearly
implies the efficacy of the crown as a headgroup. Evidence on the
interaction of the headgroup with cations is discussed below.The frequent observation of the rare amino acid tryptophan at the
boundary margins of putative transmembrane segments of proteins suggested
that it might play some important role in channel formation. In separate
work, we demonstrated that indole, the sidechain of tryptophan, could
function as a headgroup for the formation of stable liposomes.35 Clearly, tryptophan cannot function as an entry
portal for cations in the same sense that crowns do. We prepared
3, which incorporated the essential channel elements shown in
Fig. 1, but lacked the cation entry portal.
Compound 3 showed no cation transport ability as judged by any of
the analytical methods attempted.
H-bond-induced blockage of the headgroup
Because of our35 and
others’33,36 speculation that
tryptophan and/or its indole residue could play an anchoring role in
phospholipid bilayer membranes, we prepared a tris(macrocycle) terminated
in the indolylmethyl residue, i.e.
InCH2CH2<N18N>C12<N18N>C
12<N18N>CH2CH2In 4.
Although structurally similar to the highly active benzyl chan- nel
(PhCH2<N18N>C12<N18N>C12<N
18N>CH2Ph), the indolyl channel showed no ability to
transport Na+.37 Both CPK models
and Monte Carlo simulations showed that a hydrogen bond between the indole
NH and a macroring oxygen atom could form. An infrared band, attributable
to H-bond formation, did not alter its position during 100-fold dilution.
This suggests that the H-bond is intramolecular.This result, although inferential, is clearly important. It
implies that weak H-bond interactions can block the channel. This, in turn,
implies that the conformation in Fig. 2 is
correct or why would occlusion of the entry portal block the channel?
Replacement of the indolyl residue by a methylindolyl, i.e.
replacement of the NH group by NCH3, gave a compound,
5, that was fully active as a channel.
Application of the Hammett equation
If a cation enters the channel by going through or passing by the distal
macrocycle(s), it should experience the stereoelectronics of that group. We
prepared three channel compounds of the type
PhCH2<N18N>C12<N18N>C12<N1
8N>CH2Ph in which the aromatic ring of the benzyl group was
para-substituted. The substituents were H (shown), 4-methoxy and
4-nitro. A straight-line relationship was observed.38 Admittedly, the graph (Fig. 6) involves only three points but the
difficulty of synthesis and analysis will be apparent. |
| Fig. 6 | |
The critical results are as follows. First, r2 for
the three-point line is 0.95—a respectable value. Second, the slope
of the line is negative as expected for the interaction of a cation with a
neutral host. The slope is shallower than observed for complexation of
cations by dibenzyldiaza-18-crown-6 derivatives.39 This is expected for a transient interaction. A
third point, not apparent from the graph, is that when relative transport
rates obtained in the concentric tube experiment (see above) were plotted
vs. Hammett ς0, the straight-line
‘correlation’ had a zero slope and r2 =
0.4.
Changes in headgroup size
Only limited work has thus far been completed to assess the influence of
headgroup size. Two channels were prepared for this study. In both cases,
the terminal residue (‘flexible sidearm’ in Fig. 1) was fluorobenzyl. The distal macrocycles
were either 15- or 18-membered. When cation transport was assessed by using
the NMR method, transport of Na+ by the 15-membered ring channel
was found to be about 60% of that determined for the compound having
18-membered distal macrocycles.40The aggregation state of the channel
The availability of both the dansyl (2) and
N-methylindolyl (5) channels provided an opportunity to
assess the aggregation state of the channel. We found that the
N-methylindolyl-sidearmed channel absorbed light at 283 nm and
fluoresced at 343 nm—the wavelength at which the dansyl-terminated
channel absorbs. An experiment was undertaken in which the amount of
channel was held constant and the mole fractions of 2 and
5 were varied from 0→1 and 1→0, respectively. A
logarithmic plot of the fluorescence ratio as a function of mole fraction
gave a line with a slope of 1.12. The slope of this line has been
interpreted to be the aggregation state. Thus, at least for these two
compounds, the channel operates, within experimental error, as a
monomer.4123Na NMR transport results
The 23Na NMR experiment as it is used to assess transport in
a bilayer membrane was described above. A number of structural variations
have been incorporated into channel 1 and the relative transport
abilities of these compounds have been measured. Selected results are shown
in Table 1 using the shorthand described
above in which <N18N> represents 4,13-diaza-18-crown-6.
Table 1 Sodium ion transport by hydraphiles
Sidearm | Headgroup | Spacer | Center | krel |
---|
Dodecyl
(C12H25) | <N18N> | C12H24 | <N18N> | 27 |
Benzyl
(CH2C6H5) | <N18N> | C12H24 | O–2,6–C10H6–O | <2 |
Benzyl | <N18N> | C12H24 | <N18N> | 39 |
Dansyl | <N18N> | C12H24 | <N18N> | 24 |
Dansyl | <N18N> | C12H24 | <N15N> | 19 |
4-Fluorobenzyl | <N18N> | C12H24 | <N18N> | 26 |
2-(3-Indolyl)ethyl | <N18N> | C12H24 | <N18N> | <2 |
2-(N-Methyl-3-indolyl)-
ethyl | <N18N> | C12H24 | <N18N> | 23 |
None | <18N> | C12H24 | <N18N> | <2 |
Patch clamping results
The ability of various synthetic and peptidic compounds to function as
cation-conducting channels may be demonstrated by a technique called
‘planar bilayer conductance’, or PBC. In this technique, a
phospholipid bilayer is formed in a pinhole separating two salt phases.
When the membrane is formed over the pinhole, it turns dark and the system
is sometimes referred to as a black lipid membrane. Using electrodes and a
patch clamp amplifier, the electrical response of a bilayer membrane imbued
with a transporter may be observed. A membrane is normally insulating so
increases in the electrical response are interpreted as the passage of ions
through it.We conducted a number of experiments that confirmed ion transport. Our
most recent PBC results are for a calixarene-based channel.42 The patch clamp or PBC technique is wonderfully
sensitive but it is a difficult method to master and to reproduce. In some
cases, supposedly identical samples show high and no channel activity. In
other cases, the membrane itself collapses and terminates the study before
sufficient data have been acquired. We have thus preferred the NMR method;
it is cumbersome but, so far at least, it has proved to be reliable.
Conclusions
Clearly many channel models are possible. Our basic design is effective
and its modular design is proving useful to assess individual structural
issues. In work that is yet to be published, we have explored covalent
attachment of the sidearms and variations in the central relay unit.
Cation/anion selectivity, ion selectivity in general, and rectification all
remain important challenges in this emerging area.Acknowledgements
I warmly thank the many co-workers whose names are on the cited
references who have been intimately involved in this project. I appreciate
their hard work, their clever ideas, and their keen insight and criticism.
Support of this work by grants from NIH (GM 36262), NATO and NSF
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