Thermodynamic and kinetic stability of synthetic multifunctional rigid-rod β-barrel pores: Evidence for supramolecular catalysis

Article abstract of DOI:10.1021/ja0481878

The lessons learned from p-octiphenyl β-barrel pores are applied to the rational design of synthetic multifunctional pore 1 that is unstable but inert, two characteristics proposed to be ideal for practical applications. Nonlinear dependence on monomer co

Full text of DOI:10.1021/ja0481878

Published on Web 07/27/2004
Thermodynamic and Kinetic Stability of Synthetic
Multifunctional Rigid-Rod â-Barrel Pores: Evidence for
Supramolecular Catalysis
Svetlana Litvinchuk,† Guillaume Bollot,† Jiri Mareda,† Abhigyan Som,†
Dawn Ronan,† Muhammad Raza Shah,†,§ Philippe Perrottet,‡ Naomi Sakai,† and
Stefan Matile*,†
Contribution from the Department of Organic Chemistry and Laboratoire de Spectrome´trie de
Masse, UniVersity of GeneVa, GeneVa, Switzerland
Received March 30, 2004; E-mail: stefan.matile@chiorg.unige.ch
Abstract: The lessons learned from p-octiphenyl â-barrel pores are applied to the rational design of synthetic
multifunctional pore 1 that is unstable but inert, two characteristics proposed to be ideal for practical
applications. Nonlinear dependence on monomer concentration provided direct evidence that pore 1 is
tetrameric (n ) 4.0), unstable, and “invisible,” i.e., incompatible with structural studies by conventional
methods. The long lifetime of high-conductance single pores in planar bilayers demonstrated that rigid-rod
â-barrel 1 is inert and large (d ≈ 12 Å). Multifunctionality of rigid-rod â-barrel 1 was confirmed by adaptable
blockage of pore host 1 with representative guests in planar (8-hydroxy-1,3,6-pyrenetrisulfonate, KD ) 190
µM, n ) 4.9) and spherical bilayers (poly-L-glutamate, KD e 105 nM, n ) 1.0; adenosine triphosphate, KD
) 240 µM, n ) 2.0) and saturation kinetics for the esterolysis of a representative substrate (8-acetoxy-
1,3,6-pyrenetrisulfonate, KM ) 0.6 µM). The thermodynamic instability of rigid-rod â-barrel 1 provided
unprecedented access to experimental evidence for supramolecular catalysis (n ) 3.7). Comparison of
the obtained kcat ) 0.03 min-1 with the kcat ≈ 0.18 min-1 for stable analogues gave a global KD ≈ 39 µM3
for supramolecular catalyst 1 with a monomer/barrel ratio ≈ 20 under experimental conditions. The
demonstrated “invisibility” of supramolecular multifunctionality identified molecular modeling as an attractive
method to secure otherwise elusive insights into structure. The first molecular mechanics modeling
(MacroModel, MMFF94) of multifunctional rigid-rod â-barrel pore hosts 1 with internal 1,3,6-pyrenetrisulfonate
guests is reported.
Introduction
needed to form an active supramoleculesis observed only if
the concentration cM of the inactive monomer exceeds the
The performance of synthetic ion channels and pores depends
critically on their thermodynamic and kinetic stability.1-6 In the
case of synthetic multifunctional pores,3,6 kinetic stability is
crucial for control and detection of chemical processes that take
place within their confined and oriented internal space. Kinetic
stabilities, on one hand, can be assessed from the lifetime of
ion channels in conductance measurements: inert pores have a
long lifetime; labile ones are short-lived. On the other hand, it
is less straightforward to correlate thermodynamic stability and
the performance of synthetic multifunctional pores. In general,
thermodynamics of self-assembly are reflected in the dependence
of fractional supramolecular activity Y on the monomer con-
centration cM as described in the Hill equation:3a,7-10
concentration cB of the active supramolecule clearly (Figure
1).3a,9,10 A cM profile following n > 1 is, therefore, indicative
for unstable active supramolecules. Structural insights beyond
n remain, therefore, elusive with (n > 1) pores because
conventional spectroscopic methods report on inactive mono-
mers. Exergonic self-assembly, on the other hand, can yield
functional supramolecules that appear as (n ) 1) monomers in
the Hill equation (1).3a,10,11 Functional supramolecules character-
ized by n < 1 are metastable intermediates that transform into
inactive supramolecular products at high cM.3a,10,12 In practice,
stable synthetic multifunctional (n e 1) pores are problematic
because they already self-assemble in the media into prepores
with hydrophobic outer surfaces that tend to precipitate before
interacting with bilayer membranes. Endergonic self-assembly
(n > 1) from a pool of hydrophilic monomers during incorpora-
tion into a bilayer membrane bypasses this solubility problem
and can yield pores with excellent characteristics as long as
they are inert. As with pore-forming toxins from pathogenic
bacteria,13 derivatives of antifungal natural products such as
amphotericin B of practical interest,5p or chloride channel
mimics for replacement therapy in diseases such as cystic
log Y ) n log cM - n log KD
(1)
Namely, the expected nonlinear dependence characterized by
the Hill coefficient nsindicative for the number of monomers
† Department of Organic Chemistry.
‡ Laboratoire de Spectrome´trie de Masse.
§ Current address: HEJ Research Institute of Chemistry, University of
Karachi, Karachi-75270, Pakistan.
9
10.1021/ja0481878 CCC: $27.50 © 2004 American Chemical Society
J. AM. CHEM. SOC. 2004, 126, 10067-10075
10067

A R T I C L E S
Litvinchuk et al.
Translation of the “secret” of bacterial toxins on how to form
perfect pores from hydrophilic precursors to the rational design
of “ideal” synthetic multifunctional pores was challenging.
Rigid-rod â-barrels 2 with hydrophobic leucines (L) at the outer
and catalytic histidines (H) at the inner barrel surface form
synthetic multifunctional “HH pores” that are labile (single pore
lifetime τ ) 12 ms)14 and stable (cM profile with n ) 1).15 The
introduction of internal arginine-histidine dyads gives inert
(τ > 1 min)16 but stable (n e 1)10 RH pores 3. Destabilization
by external LWV triads gives unstable (n ) 4) but labile (τ <
1 ms) RH pores 4.10 Counteranion-mediated stabilization by
internal arginines (R)16,17 and â-sheet destabilization by external
tryptophans10 have been evoked to rationalize these character-
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Figure 1. Multifunctional rigid-rod â-barrels as pores. Endergonic (n >
1) and exergonic (n e 1, eq 1) self-assembly of monomeric rigid-rod
molecules 1m-4m into aqueous or transmembrane, inert or labile â-barrel
prepores or pores 1-4 depends on the nature of the amino acid residues at
the outer (gold) and inner (blue) barrel surface (single-letter abbreviations;
G ) -OCH2CO-). We caution that suprastructures in Figures 1 and 2 are
in part speculative representations that are, however, consistent with
experimental data and molecular models (see below).
fibrosis,5e the ideal synthetic multifunctional pore sensor may,
therefore, be an unstable but inert hydrophobic supramolecule
that assembles from a pool of hydrophilic precursors in the
media. Here, we report the first multifunctional rigid-rod â-barrel
pore with these ideal characteristics (i.e., 1, Figure 1).
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10068 J. AM. CHEM. SOC. VOL. 126, NO. 32, 2004

Synthetic Multifunctional Rigid-Rod â-Barrel Pores
A R T I C L E S
lability of transmembrane rigid-rod â-barrel pores was lysine
(K).15,18,19 Intermediate internal charge repulsion (ICR)20 be-
tween partially protonated internal amines may, therefore,
provide general access to the confined and oriented nanospace
needed to implement chemistry within synthetic multifunctional
pores,3a,14,21 also on the single-molecule level.3a,5t-w,21
With regard to multifunctionality, however, inner surfaces
decorated only with partially protonated butylamines are not
ideal.19 Maximal multifunctionality has been identified for pores
2-4 with one internal cation to contribute to anion recognition
and one internal histidine for catalysis. Over the years, these
after all too stable or too labile â-barrel pores have served very
well as adaptable supramolecular hosts,19,22 (enzyme) sensors,23
and catalysts.14,21,24-26 For example, the catalysis of the hy-
drolysis of ester 5 into phenol 6 by HH pores 2 and RH pores
3 was characterized by a transition-state stabilization of more
than 50 kJ/mol and a ground-state stabilization of 30-35 kJ/
mol (Figure 2).24 Preorganized ion pairing between the three
negative charges of the planar substrate and internal cationic
residues was proposed as the supramolecular basis of this quite
remarkable example of substrate recognition.
Much indirect evidence in support of the proposed supra-
molecular recognition is available (independence of catalysis
on substrate hydrophobicity,14 little dependence on partial H/R
mutation (2 versus 3),24 strong dependence on ionic strength
and pH,14 and so on). Moreover, several attractive applications
such as remote control of catalysis in pore 3 by electrostatic
steering with membrane polarization24 or the introduction of
pyrene-1,3,6-trisulfonates as cofactors for a broad variety of
otherwise inaccessible substrates25 became possible assuming
Figure 2. Multifunctional rigid-rod â-barrels as catalysts. Pyrene-1,3,6-
trisulfonate substrates like 5 are thought to bind to three proximal cationic
amino acid residues (here H, R, or K) at the inner surface of â-barrels 1-3
to initiate catalysis at the reactive site consisting of a proximal nucleophilic/
basic amino acid residue (here H) and an electrophilic center in the substrate
(here an ester). Putative catalyst-substrate complexes 1 ⊃ 5, 2 ⊃ 5, and 3
⊃ 5 include an axial view of (pre)pores 1-3 shown in Figure 1.
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surface of a pore, increases with pH for acidic residues (e.g., aspartate),
decreases with pH for basic residues (e.g., lysine, histidine, and arginine).
ICR model: pore activity is maximal at intermediate ICR. See ref 15.
(21) Som, A.; Sakai, N.; Matile, S. Bioorg. Med. Chem. 2003, 11, 1363-1369.
(22) (a) Das, G.; Onouchi, H.; Yashima, E.; Sakai, N.; Matile, S. ChemBioChem
2002, 3, 1089-1096. (b) Sorde´, N.; Matile, S. J. Supramol. Chem. 2002,
2, 191-199.
(23) (a) Das, G.; Talukdar, P.; Matile, S. Science 2002, 298, 1600-1602. (b)
Sorde´, N.; Das, G.; Matile, S. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
11964-11969. (c) Sorde´, N.; Matile, S. Peptide Sci. 2004, 76, 55-65.
(24) Sakai, N.; Sorde´, N.; Matile, S. J. Am. Chem. Soc. 2003, 125, 7776-7777.
(25) Som, A.; Matile, S. Eur. J. Org. Chem. 2002, 3874-3883.
(26) Baumeister, B.; Matile, S. Macromolecules 2002, 35, 1549-1555.
9
J. AM. CHEM. SOC. VOL. 126, NO. 32, 2004 10069

A R T I C L E S
Litvinchuk et al.
Scheme 1a
Table 1. Selected Adducts in the ESI Mass Spectra of 1m and
3m a
entry
1m (M ) 6035)b
3m (M ) 6258)b,c
H
H PO d
TFA
+ d
d
3
4
1
2
100 (863)
90 (877)
70 (879)
60 (891)
65 (893)
30 (895)
45 (895)
50 (909)
7
0
1
0
2
1
0
3
2
1
0
4
3
2
1
0
0
1
0
2
1
0
3
2
1
0
4
3
2
1
0
5
4
3
2
1
0
0
0
1
0
1
2
0
1
2
3
0
1
2
3
4
0
0
1
0
1
2
0
1
2
3
0
1
2
3
4
0
1
2
3
4
5
7
7
7
7
7
7
7
7
7
7
7
7
7
7
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
3
4
45 (923)
5
6
7
40 (937)
8
40 (907)
45 (909)
20 (911)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
20 (951)
55 (1007)
45 (1023)
25 (1025)
40 (1039)
45 (1042)
20 (1045)
30 (1044)
100 (1060)
90 (1077)
70 (1094)
35 (1058)
20 (1061)
45 (1111)
20 (1128)
30 (1077)
40 (1080)
40 (1083)
35 (1095)
45 (1098)
25 (1101)
a Measured as TFA salts under denaturing conditions (1m, CH3CN/H2O/
AcOH ) 74:24:2; 3m, CH3OH/CH3CN/H2O/AcOH ) 50:37:12:1). b Rela-
tive intensity of observed peaks in % (m/z). Consistent with the dynamic
nature of counteranion scavenging, the reported adduct patterns were
strongly dependent on experimental conditions (such as solvent purity) and
fully reproducible as qualitative trends rather than quantitatively. c Data from
ref 16. d Number of adducts (H+, H3PO4, and/or TFA) per monomer.
synthesized from p-octiphenyl 1a and tripeptide 1b (Scheme
1). These two intermediates were prepared in an overall 13 steps
from commercial starting materials following previously re-
ported procedures.14 Elongation of tripeptide 1b into pentapep-
tide 1c was as straightforward as expected. The N-terminus of
pentapeptide 1c was reacted with the carboxylate handles along
the rigid scaffold of rod 1a using HATU as coupling reagent,
TEA as base, and dimethylformamide (DMF) as the solvent of
choice. Deprotection of the resulting conjugate 1d with TFA
gave the target monomer 1m, which was purified by semi-
preparative RP-HPLC. Whereas the electrospray ionization mass
spectrometry (ESI MS) of the protected conjugate 1d gave the
a Conditions: (a) HATU, TEA, 22%; (b) TFA, quantitative; (c) self-
assembly; see text.
the same supramolecular recognition mechanism. Although all
this indirect evidence provides overwhelming support for
supramolecular (n e 1) pores (2/3) as active hosts and catalysts,
there is no direct experimental evidence available that excludes
molecular rods (2m/3m) as active species. In other words,
evidence for supramolecular multifunctionality is missing.
The objective of this study was to secure experimental
evidence in support of the hypothesis that the “ideal” synthetic
multifunctional pore is inert and unstable. To do so, we made
and studied rigid-rod â-barrel 1 with internal KH dyads (Figure
1). We report that the rationally designed inertness and instability
of synthetic multifunctional pores formed by this new barrel-
stave supramolecule provide access to high-conductance pores
with long lifetimes without losses in catalytic activity and to
experimental evidence for supramolecular catalysis, respectively.
These insights on function were, for the first time, simulated
on the structural level by molecular modeling.
expected peaks corresponding to [M + 8H]8+, [M + 7H]7+
,
[M + 6H]6+, and [M + 5H]5+, that of the final conjugate 1m,
measured as TFA salt under denaturing conditions, contained
many more peaks. These peaks suggested that the multiply
charged [M + 8H]8+, [M + 7H]7+, [M + 6H]6+, and [M +
5H]5+ appeared as TFA and H3PO4 adducts of various composi-
tion (Table 1). This indicated that some TFA anions were
replaced by phosphate anions present as “impurities” in the
media. Phosphate scavenging by KH-rich rod 1m was, however,
incomplete. In all cases, counterion-free polycation appeared
with highest intensity (entries 1 and 16), and the higher adducts
exhibited a preference for TFA rather than for phosphate (entries
34-36 versus 31-33, 28-30 versus 26-27, 23-24 versus 22,
Results and Discussion
Synthesis of Octakis(Gla-Leu-Lys-Leu-His-Leu-NH2)-p-
Octiphenyl 1m. The peptide-p-octiphenyl conjugate 1m was
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10070 J. AM. CHEM. SOC. VOL. 126, NO. 32, 2004

Synthetic Multifunctional Rigid-Rod â-Barrel Pores
A R T I C L E S
ing by K-rich rod 1m could indeed result in multifunctional pores
with the increased internal charge repulsion required for the
desired (n > 1) behavior (Figures 1 and 2, eq 1).
Rigid-Rod â-Barrels with Internal KH Dyads as Pores.
The activity of rigid-rod â-barrels 1 with internal lysine-
histidine (KH) dyads as pores was determined in large unila-
mellar vesicles (LUVs) that were composed of egg yolk phos-
phatidylcholine (EYPC) and loaded with the fluorescent probe
8-amino-1,3,6-naphthalenetrisulfonate (ANTS) and the quencher
p-xylene-bis-pyridinium bromide (DPX). After addition of KH
rod 1m, the release of fluorophore or quencher from EYPC
LUVs ⊃ ANTS/DPX was readily detectable as an increase of
ANTS emission.10,15 Despite clear disadvantages such as poor
sensitivity, the ANTS/DPX assay is, in our hands, ideal for
determining cM and pH profiles of synthetic multifunctional
pores because of its minimal dependence on pH and anion/cation
selectivity.10,11,15 According to the ANTS/DPX assay, the
activity of KH pore 1 depended strongly on pH. The bell-shaped
pH profile showed a sharp maximum at pH 5 (Figure 3A). At
pH > 5.0, the activity of KH pore 1 decreased like that of other
H-rich pores 2-4.10,15 At pH < 5.0, the activity of KH pore 1
decreased like that of K-rich rigid-rod â-barrel pores.15
Figure 3. (A) pH profile and (B) cM profile of rigid-rod â-barrel pore 1.
Fractional activity Y was measured following the increase in ANTS emission
during ANTS/DPX efflux from EYPC LUVs after addition of monomer
1m (≈250 µM EYPC, 100 mM KCl, 10 mM MES; (A) cM ) 800 nM, (B)
pH 5.0). (B) Curve fit to the Hill equation (1), n ) 4.0 ( 0.3, solid line.
The observed pH profile of pore 1, therefore, suggested that
the contributions of internal lysines and histidines to pore func-
tion are roughly additive. The ICR model (i.e., maximal pore
activity at intermediate internal charge repulsion)15,20 provided
a reasonable explanation for this additivity: pore 1 “implodes”
at pH >5.0 because of insufficient ICR from spacially remote
lysine residues (intrinsic pKa ≈ 10.5) and lacking ICR from
histidines (intrinsic pKa ≈ 6.0), whereas “explosion” of KH pore
1 at pH <5.0 isscompared to RH pore 3 or HH pore 2s
facilitated by contributions from fully protonated but counter-
anion-deficient internal lysines to increasing ICR from internal
histidines.
Around optimal pH, the cM profile of KH pore 1 in EYPC
LUVs ⊃ ANTS/DPX was nonlinear (Figure 3B). Fit to the Hill
equation (1) gave a Hill coefficient n ) 4.0 ( 0.3. As discussed
in the Introduction, this (n > 1) behavior had three significant
implications: (1) the active pore is a supramolecule (i.e., a
tetramersor, less likely, an octamer self-assembling from a
stable dimer, a dodecamer self-assembling from a stable trimer,
and so on), (2) this active tetramer is unstable (Figure 1), and
(3) structural studies by conventional methods are not applicable
to pore 1 because they will report on the excess inactive
monomer 1m in the system.
In planar EYPC bilayers with an applied voltage of V ) +25
mV, a high-conductance pore with long lifetime was observed
together with bursts of nearly identical conductance. As in
previous reports,10,18c we speculated that the labile pores are
intermediate to the formation of the inert pores. The mean
lifetime of the long-lived single KH pore 1 was τ ) 14.1 s
(Figure 4). The lifetime of high-conductance pore 1 was,
therefore, beyond that of many classical, biological low-
conductance ion channels such as gramicidin A. Clearly, the
unstable pores formed by rigid-rod â-barrels were as inert as
expected (Figure 1). In excellent agreement with â-sheet
destabilization at high polarization, where strong dipole-potential
repulsion may disturb the antiparallel alignment of the backbone
amides,27 the mean lifetime of single pore 1 decreased with
Figure 4. (top) Planar EYPC bilayer conductance in the presence of
p-octiphenyl 1m (4 µM cis, trans at ground) at +25 mV in 2 M KCl. (bottom)
Single-channel I-V profile of barrel 1.
and 8-10 versus 7). This suggested that the phosphate-enriched
lower adducts (entries 2, 4, 17, and 19) originate from TFA-
rich higher adducts by preferred TFA release rather than from
preferred phosphate binding.
This situation with K-rich rod 1m was different from that with
R-rich rod 3m, measured as a TFA salt under similar condi-
tions.16 With oligoarginines, less charged [entry 1 (1m) versus
entry 17 (3m)] and fully “phosphorylated” adducts were observed
(entries 2, 4, 7, 11, 17, 19, 22, 26, and 31). Moreover, the
phosphate counteranions of R-rich rod 3m were not as easily
released as with K-rich rod 1m [e.g., entry 16 versus entry 17].
This suggested that overall reduced anion (phosphate) scaveng-
(27) Bainbridge, G.; Gokce, I.; Lakey, J. H. FEBS Lett. 1998, 431, 305-308.
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J. AM. CHEM. SOC. VOL. 126, NO. 32, 2004 10071

A R T I C L E S
Litvinchuk et al.
circles). The obtained Hill coefficient n ) 2.0 ( 0.4 could
indicate that binding of two ATP molecules was required to
block ANTS/DPX translocation across tetrameric pore host 1.29
KH pore 1 could not be blocked with the nonfluorescent pyrene-
1,3,6,8-tetrasulfonate (PTS) at detectable concentrations (KD >
10 µM). Nevertheless, the adaptability required for applications
of synthetic multifunctional pores as enzyme sensors23 was
confirmed for KH pore 1 by the differences found in the
molecular recognition of poly-L-glutamate, ATP, and PTS.
Molecular recognition by KH pore hosts 1 not only blocked
the efflux of large organic anions (ANTS) and/or cations (DPX)
through KH pore hosts 1, it also hindered the translocation of
small inorganic cations (potassium) and anions (chloride). The
macroscopic conductance of planar EYPC bilayers doped with
pore hosts 1 was strongly reduced in the presence of increasing
concentrations of HPTS 6 (Figure 5B). The high KD ) 190 (
10 µM obtained from Hill analysis of the dose response curve
at V ) +100 mV was consistent with the poor blockage
observed with PTS in spherical bilayers. This comparably poor
host-guest interaction with KH pore 1 was clearly different
from HPTS blockage of HH pore 2 (KD ) 0.2 µM), RH pore 3
(KD ) 3.0 µM), and RH pore 4 (KD ) 3.0 µM) as well as the
KM ) 0.6 µM obtained for the esterolysis of AcPTS 5 by KH
catalyst 1 (see below). The high Hill coefficient n ) 4.9 ( 1.0
found for HPTS blockage of pore 1 supported the view that
multiple guest binding may be required to block the unusually
large KH pore 1, whereas high-affinity binding of one pyrene-
1,3,6-trisulfonate substrate at the inner barrel surface may suffice
for catalysis.
Rigid-Rod â-Barrels with Internal KH Dyads as Catalysts.
The catalytic activity of rigid-rod â-barrel 1 was evaluated using
AcPTS 5 as established model substrate (Figure 2).14,21,24
Esterolysis of substrate 5 was continuously detectable by
monitoring the increase in the fluorescence emission of the
product HPTS with time.14,21 The dependence of the esterolytic
activity of rigid-rod â-barrel 1 on pH was comparable to the
pH profile of rigid-rod â-barrel 1 as pore (Figure 6A, filled
circles, versus Figure 3A). The pH profile of KH catalyst 1 was
similar to that of HH catalyst 2.14 For complete comparison,
the pH profile of RH catalyst 3 was determined (Figure 6A,
empty circles). Different from KH and HH catalysts 1 and 2,
the catalytic activity of RH catalyst 3 did not decrease between
pH 5.5 and pH 6.8 (above pH 6.8, precipitation was observed).
This suggested that the loss in catalytic activity with increasing
pH may originate from neutralization of internal, proximal
histidines (2, intrinsic pKa ) 6.0) and lysines (1, intrinsic pKa
) 10.5) but not arginines (3, intrinsic pKa ) 12.5). This
reduction of the number of cations at the inner barrel surface
with increasing pH may either reduce the interaction with the
anionic substrate 5 orsaccording to the ICR model20sdestabilize
the active suprastructure.
Figure 5. Fractional activity Y of rigid-rod â-barrel pore hosts 1 as a
function of concentration cG of guests such as (A) poly-L-glutamate (filled
circles) and ATP (empty circles) in spherical EYPC LUVs ⊃ ANTS/DPX
(V ) 0 mV) and (B) HPTS 6 in planar EYPC bilayers (multichannel current
I at V ) +100 mV, 5 mM MES, 100 mM KCl, pH 5.0) with curve fit to
the Hill equation (1).
increasing voltage to a still quite remarkable τ ) 0.36 s at V )
+125 mV.
The current flowing across single pores showed linear voltage
dependence within (100 mV (Figure 4). Such ohmic behavior
is characteristic for symmetric â-sheet secondary structures as
in biological and rigid-rod â-barrel pores, where the opposing
orientation of the backbone amides results in no macrodipole.3a
A conductance g ) 4.98 ( 0.05 nS for the dominant single
pore resulted from the slope of this ohmic I-V profile.
Elimination of contributions from the resistivity of the recording
solution from g ≈ 5 nS using Hille’s equation7 gave a pore
diameter dHille ) 12 Å. Different from the underestimates
obtained with low-conductance pores, Hille diameters are
expected to reflect the internal space of high-conductance pores
like 1 quite accurately.21,28 dHille ) 12 Å is the largest Hille
diameter found so far for tetrameric rigid-rod â-barrel pores.3a
It was consistent with minimal counteranion scavenging ex-
pected with internal lysine residues and, consequently, maximal
internal charge repulsion to stabilize the large, oriented and
multifunctional “nanospace” within pore 1.
Rigid-Rod â-Barrel Pores with Internal KH Dyads as
Hosts. To combine molecular translocation with molecular
recognition, the activity of synthetic multifunctional pore 1 in
spherical EYPC LUVs ⊃ ANTS/DPX was assessed in the
presence of increasing concentrations of representative guests.
As with RH pore 3 (KD ) 150 nM, pH 4.5, KD ) 45 nM, pH
5.5),22b ANTS/DPX translocation across KH pore host 1 could
be efficiently blocked with partially R-helical poly-L-glutamate
as a representative guest (KD ) 105 ( 27 nM, pH 5.0, Figure
5A, filled circles). The obtained nanomolar apparent KD
suggested that nearly stoichiometric association obscured an
actual KD < 150 nM. The Hill coefficient n ) 1.0 ( 0.3 may
be indicative for the binding of one polypeptide guest per
tetrameric pore host 1.29 As with RH pore 4 (KD ) 82 µM, pH
6.5),23c about 3 orders-of-magnitude higher guest concentrations
were required to block KH pore host 1 with ATP (adenosine
triphosphate; KD ) 240 ( 25 µM, pH 5.0, Figure 5A, empty
Direct experimental evidence that the active catalysts are
tetrameric barrel-stave supramolecules (rather than monomeric
peptide-p-octiphenyl conjugates) was obtained, for the first
time, with the cM profile of catalyst 1 (Figure 6B, filled circles).
The nonlinear dependence of the initial velocity of product
formation on the concentration of monomeric peptide-p-
octiphenyl conjugate 1m gave a Hill coefficient n ) 3.7 ( 0.2
that was indicative for unstable tetramers as active supramo-
lecular catalysts (Figure 6B, solid). The stability of rigid-rod
â-barrel 2 known from pore characterization (n ) 1.0, Figure
1) was reflected in the cM profile for catalysis (n ) 0.9 ( 0.1,
(28) (a) Smart, O. S.; Breed, J.; Smith, G. R.; Sansom, M. S. P. Biophys. J.
1997, 72, 1109-1126. (b) Cruickshank, C. C.; Minchin, R. F.; Le Dain,
A. C.; Martinac, B. Biophys. J. 1997, 73, 1925-1931.
(29) Applied to host-guest chemistry, the meaning of the Hill coefficient n (or
sometimes h) can be more complex as well and include cooperativity
beyond the number n of guests bound per host. Compare, e.g.: Connors,
K. A. Binding Constants; John Wiley & Sons: New York, 1987; pp 59-
101.
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10072 J. AM. CHEM. SOC. VOL. 126, NO. 32, 2004

Synthetic Multifunctional Rigid-Rod â-Barrel Pores
A R T I C L E S
for the active supramolecule 1 was compatible with endergonic
self-assembly at the concentrations relevant for multifunction-
ality.
The KM ) 0.6 µM for esterolysis of AcPTS 5 by KH catalyst
1 was more than 2 orders of magnitude lower than the KD
)
190 µM for the blockage of KH pore 1 in planar bilayers by
HPTS. This is the first time that such a difference between
molecular recognition and catalysis was observed with multi-
functional rigid-rod â-barrels. Although the origin of this
difference is unknown, its coincidence with an exceptionally
large inner pore diameter and an exceptionally high Hill
coefficient for HPTS blockage (n ) 4.9) may be more than
accidental. Indeed, molecular simulations supported the view
that the origin of the high conductance of pore 1 may be not
only the absence of immobilized internal counteranions but also
a swelling of the rigid-rod â-barrel tetramer in response to high
internal charge repulsion. Above results suggested that the
structure of the inclusion complex accounting for pore blockage
may be different from that accounting for catalysis. The
possibility of different orientations for included pyrene-1,3,6-
trisulfonates is illustrated in modeling studies below (Figure
7B,C). Alternatively, it was conceivable that multiple pyrene-
1,3,6-trisulfonate binding may be needed for pore blockage,
whereas single AcPTS binding may suffice for catalysis. This
explanation was supported by n ) 4.9 for HPTS blockage.29
Molecular Modeling of Multifunctional Rigid-Rod â-Bar-
rel with Internal KH Dyads. The demonstrated experimental
inaccessibility to structural information on rigid-rod â-barrel
pore 1 identified molecular modeling as a unique resource for
insights on supramolecular multifunctionality at the molecular
level. Therefore, rigid-rod â-barrel 1 was constructed using
Maestro modeling software.30 Imitating the synthesis of barrel
1 (Scheme 1), four peptide-p-octiphenyl conjugates 1m were
constructed first and then assembled to give the supramolecular
tetramer. Taking counteranion-mediated charge neutralization
and the ICR model into account, only every second internal
lysine was protonated before preliminary geometry optimiza-
tion using the MMFF94 force field31 as implemented in the
MacroModel package.32
Figure 6. Dependence of supramolecular catalysis by rigid-rod â-KH barrel
1 on pH (A), cM (B), and cS (C) (filled circles) in comparison with HH
catalyst 2 (empty squares) and RH catalyst 3 (empty circles). The initial
velocity V(rel) of product formation was measured following the increase in
HPTS emission after addition of monomers 1m (filled circles: (A) 5.0 µM,
1.0 µM AcPTS 5; (B) pH 5.5, 1.0 µM 5; (C) 1.5 µM, pH 5.5), 3m (empty
circles: (A) 0.25 µM, 7.0 µM 5), or 2m (empty squares: (B) pH 5.5, 1.0
µM 5) in 10 mM MES, 100 mM KCl. Curves were fitted to the Hill ((B)
solid, n ) 3.7 ( 0.2; dotted, n ) 0.9 ( 0.1) and Michaelis-Menten
equations (KM ) 0.6 ( 0.1 µM, VMAX ) 181 ( 11 pM/s).
Figure 6B, dotted). Interpretation of the tetrameric, unstable and
“invisible” KH catalysts 1 as aqueous rigid-rod â-barrel prepores
with a suprastructure similar to that of the KH pores 1 in the
bilayer was supported by identical stoichiometry and thermo-
dynamic instability (Figure 1). Supramolecular catalyst and
supramolecular pore were, therefore, in all likelihood the same
multifunctional rigid-rod â-barrel 1. The remarkable consistency
throughout the characteristics of multifunctional pores 1-3
suggested that this conclusion applies for the more stable pores
2 and 3 as well, although direct evidence for supramolecular
function from cM profiles was not available in these cases
because of higher thermodynamic stability.
The energy-minimized rigid-rod â-barrel 1 had a height of
32.4 Å, a mean backbone-to-backbone diameter of 35 Å, and a
minimal inner van der Waals diameter of 16.2 Å (Figure 7A).
The inner pore diameter was in excellent agreement with the
experimental Hille diameter dHille ≈ 12 Å. â-Barrel 1 had an
M-helical twist of 20°. In a hypothetical rigid-rod â-barrel of
infinite length, this nearly negligible helicity would give rise
to a helix with 144 â-strands per turn, i.e., a helical pitch of
about 58 nm corresponding to 18 barrels on top of each other.
The preferred biphenyl torsion angles ω ≈ 123° in the
p-octiphenyl staves accounted for the circular, truly barrel-like
appearance of tetramer 1 viewed from the top. Preliminary
results indicated that uniform isomerization to energetically
conceivable biphenyl torsions ω ≈ 57°sin response to, e.g.,
reduced internal charge repulsionswould produce contracted
conformers with drastically reduced inner diameter. Possible
Variation of substrate concentration at constant catalyst
concentration and pH revealed the Michaelis-Menten constant
KM ) 630 ( 97 nM (Figure 6C). This remarkable substrate
recognition by KH catalyst 1 was as for HH catalyst 2 (KM
700 nM)14 and 10 times better than for RH catalyst 3 (KM
)
)
6.1 µM).24 The kcat ) 0.03 min-1 of KH catalyst 1 was, however,
clearly below that of HH catalyst 2 (kcat ) 0.13 min-1) and RH
catalyst 3 (kcat ) 0.24 min-1). This unusually low kcat was
consistent with the endergonic self-assembly of the supra-
molecular catalyst 1 identified with a nonlinear cM profile
(Figure 6B). Specifically, the assumption of a kcat′ ≈ 0.18 min-1
for an active supramolecule like that of HH and RH catalysts
suggested cB′ ≈ VMAX/kcat′ ) (kcatcM/4)/kcat′ ) 62.5 nM as the
effective concentration of KH catalyst 1 at cM ) 1.5 µM. This
result implied cM′ ) cM - 4cB′ ≈ 1250 nM as excess monomer
under these conditions. These effective monomer and tetramer
concentrations corresponded to an approximate monomer/barrel
ratio of cM′/cB′ ) 20. The resulting KD ) (cM′)4/cB′ ) 39 µM3
(30) Maestro 4.1; Schro¨dinger Inc.: Portland OR, 2001.
(31) (a) Halgren, T. A. J. Comput. Chem. 1996, 17, 490-641. (b) Halgren, T.
A. J. Comput. Chem. 1999, 20, 720-748.
(32) (a) MacroModel 7.0; Schro¨dinger, Inc.: Portland OR, 1999. (b) Mohamadi,
F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Caufield,
C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11,
440-467.
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J. AM. CHEM. SOC. VOL. 126, NO. 32, 2004 10073

A R T I C L E S
Litvinchuk et al.
Figure 7. Three-dimensional structure optimized by MacroModel (MMFF94) of rigid-rod â-barrel 1 (lysine protonation 50%, histidine protonation 0%) in
side view (left) and axial view (right) without guests (A) and with AcPTS (sulfonate protonation 0%) bound perpendicular (B, 1 ⊃ 5 ) and parallel to the
long barrel axis (C, 1 ⊃ 5)). Side views are wire presentations (O, red; N, blue; C, green; H, gray) with p-octiphenyls (A, black) and AcPTS highlighted
in ball-and-stick (B, C: O, red; S, yellow; C, green; H, gray). Axial views show electrostatic potentials mapped onto solvent-accessible surfaces (blue,
electron-poor; red, electron-rich) with (B, C) semitransparent surface to reveal barrel and AcPTS in wire and ball-and-stick presentation, respectively.
strain from the suppressed, intrinsic â-sheet helicity33 may
account for some local asymmetries in barrel 1; refined
simulations will be needed to evaluate their relevance.
Possible inclusion complexes of AcPTS 5 within supramo-
lecular catalyst 1 were simulated by docking of the substrate
between the first and second â-sheet hoops with the pyrene plane
either perpendicular (i.e., inclusion complex 1 ⊃ 5 , Figure 7B)
or parallel to the long barrel axis (1 ⊃ 5), Figure 7C). Before
docking, AcPTS was optimized with the density functional
(33) (a) Richardson, J. S. Nature 1977, 268, 495-500. (b) Flower, D. R. FEBS
Lett. 1994, 344, 247-250. (c) Nagano, N.; Hutchinson, E. G.; Thornton,
J. M. Protein Sci. 1999, 8, 2072-2084 (see also ref 13).
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Synthetic Multifunctional Rigid-Rod â-Barrel Pores
A R T I C L E S
theory (DFT)34 and B3LYP/6-31G* method.35 After this initial
optimization, substrate 5 was frozen in the DFT geometry. In
other words, substrate 5 was considered as the rigid body during
the optimizations of complex 1 ⊃ 5 by MMFF94 force field.
To obtain complex 1 ⊃ 5 , DFT-optimized AcPTS was docked
at the barrel entrance, spanning the interior to interact with
charged residues from opposing â-sheets. During optimization,
the substrate 5 moved into the pore to end up 10.8 Å from the
channel entrance (Figure 7B). Asymmetric barrel contraction
reduced the minimal internal diameter on the substrate side from
16.2 to 10.4 Å. This asymmetric barrel contraction into a cone-
shaped conformation originated from complete inward extension
of the internal lysine residues to reach the substrate for ion
pairing and multiple, bilateral H-bonding between two am-
monium cations per sulfonate anion (average N-H‚‚‚O distance
) 2.6 Å). These changes provided a compelling illustration for
likely contributions from supramolecular adaptability and guest
templation to pore blockage.
During optimization of 1 ⊃ 5), vertical movement of B3LYP/
6-31G* optimized substrate 5 from the barrel entrance toward
the barrel center as with 1 ⊃ 5 coincided with a lateral
displacement from one â-sheet surface to the interface between
adjacent â-sheets (Figure 7C). Inclusion complex 1 ⊃ 5)
exhibited complementary characteristics compared to 1 ⊃ 5
with regard to negligible barrel deformation and poor blockage.
This complementarity confirmed that molecular recognition
within synthetic multifunctional pores may occur with or without
a strong influence on molecular translocation (i.e., blockage),
whereas catalysis appeared possible in both cases (i.e., histi-
dine-carbonyl distances around 5 Å compatible with nucleo-
phile or base catalysis with possible electrophile activation and
transition-state stabilization by ammonium cations near the
carbonyl oxygen). Much room left by these initial simulations
is currently explored with emphasis on the impact of biphenyl
torsions, internal charge repulsion, guest templation, guest
diversity, solvents, bilayer membranes, and molecular dynamics
on barrel structure. Particular emphasis is on multiple guest/
substrate binding to ultimately simulate synthetic catalytic pores
(i.e., substrate transformation during transmembrane transloca-
tion),17 including the recent concept that blocker efflux through
blocked pores may translate the mechanism of selectivity of
biological ion channels36 to organic chemistry within synthetic
multifunctional pores.37
Figure 8. Notional energy diagrams summarizing the thermodynamic and
kinetic stabilities achieved with synthetic multifunctional pores 1-4.
Unstable and inert pores such as 1 are suggested to be “ideal” in practice.
Conclusions
Rigid-rod â-barrels 1 with internal lysine-histidine dyads
complete a comprehensive collection of synthetic multifunctional
pores that now covers all possible combinations of thermody-
namic and kinetic stabilities (Figure 8). The power of bacterial
pore-forming toxins underscores that endergonic self-assembly
from hydrophilic monomers into inert synthetic multifunctional
pores 1 is ideal for practical sensing applications because too
stable pores (such as 2 and 3) tend to precipitate rather than to
partition into bilayer membranes, whereas too labile pores (such
as 2 and 4) are useless as single-molecule sensors.
One attractive characteristic resulting from the rationally
designed inertness of rigid-rod â-barrels 1, on one hand, is their
long lifetime as single, multifunctional and large, i.e., high-
conductance, pores. Attractive characteristics resulting from the
instability of rigid-rod â-barrels 1, on the other hand, include
access to unprecedented experimental evidence for supramo-
lecular multifunctionality, expressed in n ) 4.0 for pore 1 and
n ) 3.7 for catalyst 1. Experimental support is provided for
minimal internal counterion immobilization and, as a conse-
quence, maximal internal charge repulsion to account for the
large inner diameter of pore 1 (d ≈ 12 Å). Multiple binding of
several small (HPTS, KD ) 190 µM, n ) 4.9) but not single
large blockers (polyglutamate, KD e 105 nM, n ) 1.0) may be
required to “fill” this large, swollen internal space of high-
conductance KH pore 1, whereas molecular recognition of single
small substrates may suffice for catalysis (AcPTS, KM ) 0.6
µM). Supported by preliminary results from molecular modeling,
we conclude that the multifunctional rigid-rod â-barrel pores 1
reported herein offer a superb platform to study chemical
processes that take place within their confined, oriented, inert,
and functionalized internal nanospace.
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Acknowledgment. We thank B. Baumeister for experimental
assistance, D. Jeannerat, A. Pinto, and J.-P. Saulnier for NMR
measurements, F. Gu¨lac¸ar for assistance with ESI MS, H. Eder
for elemental analyses, two reviewers for helpful suggestions,
and the Swiss NSF for financial support (200020-101486 and
National Research Program “Supramolecular Functional Materi-
als” 4047-057496).
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Supporting Information Available: Experimental Section
(PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
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