Single-Molecule Observation of the Intermediates in a Catalytic Cycle

Article abstract of DOI:10.1021/jacs.8b09282

The development of catalysts benefits from knowledge of the intermediate steps that accelerate the transformations of substrates into products. However, key transient species are often hidden in ensemble measurements. Here, we show that a protein nanoreac

Full text of DOI:10.1021/jacs.8b09282

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Article
Single-Molecule Observation of the Intermediates in a Catalytic Cycle
William J Ramsay, Nicholas A W Bell, Yujia Qing, and Hagan Bayley
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09282 • Publication Date (Web): 26 Nov 2018
Downloaded from http://pubs.acs.org on November 26, 2018
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Single-Molecule Observation of the Intermediates in a Catalytic Cycle
William J. Ramsay, Nicholas A. W. Bell, Yujia Qing, Hagan Bayley*
University of Oxford, Chemistry Research Laboratory, Oxford, OX1 3TA, UK
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ABSTRACT: The development of catalysts benefits from knowledge of the intermediate steps
that accelerate the transformations of substrates into products. However, key transient species are
often hidden in ensemble measurements. Here, we show that a protein nanoreactor can sample the
intermediate steps in a catalytic cycle by the continuous single-molecule observation of a
stoichiometric reaction in solution. By monitoring changes in the flow of ionic current through an
α-hemolysin protein pore, we observed three intermediate metal-ligand complexes in a gold(I)-
catalysed reaction that converts an acetylenic acid to an enol lactone, revealing a transitional
coordination complex that had been previously unobserved. A kinetic isotope effect helped assign
the various metal-ligand species. Measurements of the lifetimes of the intermediates allowed a full
kinetic analysis of the metal-catalysed reaction cycle.
INTRODUCTION
1
Chemical reactions catalysed by metals provide access to valuable pharmaceuticals and fine
2
chemicals. The control over bond-breaking and bond-forming processes during metal-catalysed
3
4-5
reactions requires awareness of the intermediate metal-ligand species
that mediate the
6
conversion of substrates into products. This critical information is restricted by the limited
number of techniques to monitor transient species continuously with molecular resolution.7-11
Although single-molecule fluorescence microscopy has enabled the investigation of the initiation
dynamics of a surface-supported palladium catalyst,12-14 the resolution of the multiple kinetic steps
involved in a catalytic cycle remains elusive. The nanoreactor approach used here is a versatile
technique to observe the kinetics of transient intermediates at the single-molecule level.
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5
The staphylococcal α-hemolysin (αHL) transmembrane pore (Figure 1a) has been used as a
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nanoreactor to monitor a variety of chemistries at the single-molecule level, including metal ion
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18
19-20
chelation, the detection of metal-coordinating neurotransmitters, a kinetic isotope effect,
21
Cu(I)-catalyzed azide-alkyne cycloaddition, and the continuous, stochastic movement of a small-
2
2
molecule walker along a five-foothold track. Here, we use the αHL nanoreactor to examine a
metal-catalysed reaction (Figure 1b). The lumen of an αHL pore was genetically engineered to
I
accommodate the binding of a single, catalytically active Au metal ion, which binds to alkyne
substrates (Figure 1c), and can catalytically cyclise acetylenic acids to enol lactones (Figure 1d).23-
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Figure 1. The P
H
αHL pore: a nanoreactor for the investigation of Au catalysis. (a) WT αHL (PDB code 7AHL) is a
mushroom-shaped transmembrane protein pore comprising seven identical subunits. A side view of the ribbon
structure shown with one of the subunits highlighted in green. (b) A sagittal section of the engineered αHL pore, P
embedded in a planar lipid bilayer (orange) for single-channel electrical recording. P contains one subunit with a
histidine substitution at position 145 (red in stick representation). The side chain of His-145 points into the lumen of
the transmembrane β barrel and can coordinate metal-ligand complexes from solution, entering from the trans
H
,
H
I
compartment. (c) An expanded view around His-145, with bound 4-pentynoic acid: Au (yellow sphere), 4-pentynoic
acid (stick representation: carbon, grey; hydrogen, white; oxygen, red). (d) The proposed catalytic cycle for
transformation of an acetylenic acid to an enol lactone.
In our study, the modified αHL pore serves as a sensing platform; the catalytic transformation
of acetylenic acids predominantly occurs in solution, while the pore samples the intermediate
metal-ligand species that are involved in the reaction and records the interconversions of each
intermediate. Continuous monitoring over the course of the reaction allows the assembly of
information connecting catalytic steps and thereby the construction of a catalytic cycle in accord
with our single-molecule observations. Our approach suggests a general method to follow metal-
catalysed aqueous reactions.
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RESULTS & DISCUSSION
Gold(I) binding to the synthetic pore.
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I
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Before investigating the Au catalysed reaction, we examined the binding of Au and Au -
alkyne complexes to the nanopore. We used a histidine-containing αHL pore to determine the
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binding kinetics of Au with an imidazole group at 21 ± 1 °C (Figure 2a). The same pore was
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subsequently used to study Au catalysis. A mutant αHL monomer, T145H, was assembled
together with wild-type (WT) subunits, and the heteroheptameric pore (WT) (T145H) (referred to
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1
as P ) was purified by SDS-polyacrylamide gel electrophoresis (Supplementary Section 1.2,
H
Figure S1). The histidine substitution at position 145 in P orientates the imidazole side-chain into
H
I
the lumen of the β barrel, where it can coordinate to a single Au cation (none of the natural His
residues in the protein are in the conductive pathway, Figure S2). The amplitude of the metal-
binding events was enhanced by positioning the coordinating ligand near the pore constriction.26
PH was characterised by current recording in planar lipid bilayers and carried a single-channel
current of −76.6 ± 2.1 pA at −50 mV in 2 M KCl, 10 mM 2-(N-morpholino)ethanesulfonic acid
(MES), pH 6.1 (n = 25; Table S1). The addition of gold(I) chloride to the trans compartment
resulted in the fluctuation of the ionic current between two discrete levels separated by ΔI = 1.10 ±
0
.07 pA (Figure 2b), where ΔI is the difference between the current carried by the unoccupied pore
I
I
(
P ) and that of the pore with bound Au (P ·Au ), i.e. metal binding results in an increase in the
H
H
unitary conductance of the pore.18 Although metal binding to modified αHL pores has been
observed to both induce and block the flow of current, the subtle reasons behind this phenomenon
have yet to be elucidated. The dominant gold species in 2 M KCl at pH 6.1 and 21 ± 1 °C in the
I
−
binding experiments is the linear molecule Au Cl , as the gold concentrations (≤5 µM) are well
2
I
−
III
−
III
below the isoactivity boundary between Au Cl and Au Cl and disproportionation to Au and
2
4
Au is negligible.27-28 The events associated with Au binding had a mean lifetime (τ ) of 2.1 ± 0.2
0
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off
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−
s and were not seen with WT pores (Figure S11). By assuming that the Au Cl concentration
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2
inside the pore equals that in solution,16 we performed a kinetic analysis of the events, and
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deduced that the histidine residue and Au reversibly form a binary complex (Figure 2c, top). The
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−
reciprocal of the mean inter-event interval (τ ) is proportional to the Au Cl concentration, which
on
2
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−
is consistent with a bimolecular interaction for which 1/τ = k [Au Cl ]. In contrast, a plot of the
on
on
2
I
−
reciprocal of the mean lifetime of the complex (τ ) versus the Au Cl concentration has a near
off
2
zero slope (Figure 2c, bottom), which is consistent with a unimolecular dissociation mechanism
1/τ = k ). The values of τ and τ were determined by fitting dwell-time histograms for each
(
off
off
on
off
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−
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-1 -1
Au Cl concentration to single exponential functions. The forward (k = 1.52 ± 0.05 × 10 M s )
2
on
-1
and reverse (koff = 0.49 ± 0.05 s ) rate constants derived from the τ values yield an equilibrium
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association constant of Au to imidazole of K = 3.1 ± 0.3 × 10 M (n = 4, independent
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experiments).
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Figure 2. The P
H
pore binds Au . (a) Ribbon representation of the P
single histidine side chain (red) binds Au (yellow sphere) reversibly. (b) Single-channel recordings at −50 mV with 2
M KCl, 10 mM MES (pH 6.1) in both compartments, and 0 to 5 µM Au Cl in the trans compartment. The two current
levels correspond to the unoccupied pore (P
Proposed kinetic scheme describing Au binding to the P pore and plots of the reciprocals of the mean inter-event
2
intervals (τon) and the dwell times of Au (τoff) versus the Au Cl concentration.
H
pore viewed from the bottom of the β barrel. The
I
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−
I
H
) and to the pore with Au Cl
2 H
bound to the histidine residue (P ·Au ). (c)
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H
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−
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We
also
prepared
the
phosphine-coordinated
Au -complex
chloro(tris(2-
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carboxyethyl)phosphine) gold(I) (Au (tcep)Cl) in situ by mixing chloro(tetrahydrothiophene)
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gold(I) (Au (tht)Cl) with TCEP (tris(2-carboxyethyl)phosphine) (Supplementary Section 1.4).
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Phosphine ligated Au -complexes are commonly used as catalysts to activate alkenes and
alkynes,29-31 where the phosphine ligand engenders solubility to a discrete Au complex. In order to
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follow the Au -catalysed cyclisation of acetylenic acids at the single-molecule level, we found that
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the use of Au (tcep)Cl was a necessity (see below). Although we observed TCEP coordination to
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−
31
1
Au Cl in 2 M KCl solutions by P{ H} NMR spectroscopy (Figure S4), in single-molecule
2
experiments, we did not observe blocking events consistent with the coordination of TCEP (or
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THT) to Au bound to His-145 when Au (tcep)Cl was added to the trans compartment (Figures
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−
S12 and S13). Rather, we observed fluctuations in the ionic current consistent with Au Cl
2
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binding that occurs from the ligand exchange of Au (tcep)Cl in 2 M KCl solution (Table S2 and
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Supplementary Section 2.3). Thus, although Au (tcep)Cl is present in solution, we did not have to
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consider events associated with TCEP binding to P -bound Au when we used Au (tcep)Cl in our
H
alkyne binding and catalytic investigations. We postulate that the dwell time, τ , of TCEP bound
off
I
to a Au ion bound to P is beyond the resolution of the experiment, perhaps exacerbated by the
H
trans influence of the imidazole coordinating group.32
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Alkyne Binding to gold(I)
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A key, first step in homogeneous Au -catalysed reactions of alkynes is the formation of a Au -
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33-36
alkyne π-complex, in which the alkyne binds side-on to a single Au centre (Figure 1d).
Before
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investigating a Au catalysed reaction, we sought to detect this binding behaviour at the single-
molecule level. We prepared a series of alkyne esters (1 – 3; Figure 3, a to c), which can
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coordinate to but not react on Au , and investigated their binding behaviour to Au and the
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histidine pore P . Alkyne-ester 1 and Au (tcep)Cl were mixed in a 1:1 ratio and added to the trans
H
compartment of the recording apparatus at −50 mV in 2 M KCl, 10 mM MES, pH 6.1. NMR
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spectroscopy confirmed that these components do not react (Figures S14 and S15) and that the
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38
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−
binding of the alkyne-ester to Au (tcep)Cl is weak. In addition to the binding of Au Cl to
2
histidine, two further reversible current blockades were observed: at 4.8 ± 0.2 pA (with respect to
PH) with an average lifetime of 95 ± 4 ms (level 3), and at 2.2 ± 0.2 pA with an average lifetime of
2
41 ± 30 ms (level 2) (Figure 3a). As we anticipated observing only one reversible current
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blockade from the coordination of a linear, Au -1 π-complex, the observation of two reversible
current blockades prompted further investigation. These current levels were not observed with the
WT pore.
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Figure 3. Detection of Au -alkyne complexes using the P
with an idealised trace overlaid and color coded for each level) and noise analyses for the bound states of Au -
coordinated alkyne-esters 1 – 3 to P (N = number of events). Inset in (c): expanded view of level 3. Concentrations
H
pore. (a – c) Electrical current recordings (shown in grey
I
H
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(Au (tcep)Cl and alkyne-ester added in a 1:1 stoichiometry: 1, 50 µM; 2, 50 µM; 3, 50 µM. All traces are displayed on
the same scale. Conditions: 2 M KCl, 10 mM MES, pH 6.1, 21 ± 1 °C, at an applied potential of −50 mV. (d) Kinetic
model used to determine the rate constants of the observed transitions. (e) Proposed kinetic scheme describing the
restricted rotation of alkyne esters 3 and 4 between the trans (Z) configuration and the cis (E) configuration. (f) All-
points histogram of the current amplitudes in level 3 observed with 3. Inset: idealised trace (QuB) of level 3 (red)
overlaid on the recorded current trace (black).
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Direct transitions from the unoccupied pore (level 1) to level 3 or level 2 and transitions
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between levels 3 and 2 were commonly observed, but transitions from the Au -occupied level
(
level 0) to levels 3 and 2 were rarely recorded (<1% of the total transitions) (Figure S16). We
created a four-state model to describe the observed transitions (Figure 3d). The rate constants of
the transitions between each state were determined by analysing idealised current traces with
Hidden Markov modelling (QUB 2.0, Buffalo University) (Table 1). The association rate from
level 1 to level 2 (corresponding to rate constant k ) or level 3 (rate constant k ) showed a first-
1
2
13
I
order dependence on the concentration of the Au -1 complex (Figure S17), while dissociation (rate
constants k21 and k ) was independent of the concentration, consistent with a bimolecular
31
I
interaction between Au -1 and P . The transitions between levels 3 and 2 (k and k ) were
H
23
32
concentration independent, suggesting that level 2 and level 3 arise from rearrangement of the
I
39
coordination complex. As Au is known to form three-coordinate complexes in addition to the
more commonly observed linear, two-coordinate complexes, we tentatively assign levels 2 and 3
I
I
to two different coordination states: a linear, Au -1 π-complex and a three-coordinate, Au complex
chelated by the alkyne and ester groups (Figure 3d). We propose the three-coordinate, AuI
complex in order to account for our single-molecule observation that the transitions between level
2
and 3 are concentration independent; although this is an atypical binding motif, similar
heterotopic and multidentate ligand binding to AuI (Refs. 39-40) and AuIII (Ref. 41) have been reported.
I
I
We did not observe binding of Au -ester (Figure S18) or Au -acid (Figure S19) complexes to P ,
H
indicating that coordination must be through the alkyne. Because the amplitude of the current
block for an organic molecule depends approximately on how far it extends into the pore lumen,18
the chelate state was assigned to the smaller current blockade (level 2). Although this argument is
the simplest and most chemically plausible, it encouraged us to investigate additional alkyne-ester
binding activity to PH.
I
The addition of a 1:1 mixture of Au (tcep)Cl and alkyne-ester 2 to the trans side of P
H
I
produced current blockades and transitions similar to those produced by Au -1 under the same
experimental conditions (Figure 3b and Figure S21); 2 differs from 1 by only an additional carbon
atom in the chain between the alkyne and ester group. We found that the rate constant of the
I
transition from level 3 to level 2 (k ), which is proposed to describe the chelation of Au by 2 to
3
2
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-1
form a 7-membered ring, was slower (2.9 ± 0.2 s ) than the formation of a 6-membered ring with
I
-1
Au and 1 (4.4 ± 0.4 s ) (Figure S22); similar small differences in ring-closing rates have been
4
2
observed with platinum(II)-diamine chelate complexes. The opening of the 7-membered ring
-1
(
k ) formed with 2 was slightly faster (8.1 ± 0.4 s ) than the 6-membered ring with 1
23
-1
I
(
6.4 ± 0.5 s ). These results support our initial level 3 assignment as the Au -alkyne π-complex.
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Further, we determined the binding kinetics of Au -3 to P (Figure 3c) (Figures S24 and S25). The
H
-1
dimethyl substituents of alkyne-ester 3 did not affect the rate of ring-opening (k = 6.4 ± 0.3 s ),
2
3
as compared with alkyne-ester 1 (which forms the same chelate ring size), but slightly retarded the
-1
rate of ring formation (k = 3.0 ± 0.3 s ) as expected from the steric influence. Although the noise
3
2
I
that arises from level 2, which is assigned as the Au -alkyne chelate complex, was similar among
I
alkyne-esters 1 – 3, we observed considerable noise in the amplitude of level 3 with Au -3,
I
I
compared to Au -1 and Au -2 (Figure 3, a to c, bottom). We attribute this observation to the
4
3
restricted rotation of the σ-bond within the ester group, as it is the slowest rotating bond within
the molecule (Figure 3e). By using Hidden Markov modelling (QUB 2.0, Buffalo University) to
I
analyse the fluctuations of level 3 from Au -3 as a two state system with transitions from the trans
-1
(
Z) configuration to the cis (E) isomer (Figure 3f), we determined k (701 ± 24 s ) and k
(2193
cis
trans
-1
-1
±75 s ) and calculated an E – Z free energy difference (ΔG°) of 0.52 kcal mol (ΔG° =
RTln(k /k )), consistent with literature values.44 We also prepared the ethyl ester of 4-
−
cis trans
pentynoic acid (4), which omits the dimethyl substituents of 3, and observed a similar fluctuation
-1
in the amplitude of level 3 (Figure S26), and found faster kcis (1748 ± 131 s ) and k
trans
-1
-1
(
3274 ± 237 s ) values, yielding a smaller E – Z free energy difference of 0.37 kcal mol . This
result suggests that the observed restricted rotation was a result of the ethyl ester group. The
I
barrier to ester rotation is consistent with a linearly-coordinated, Au -alkyne π-complex to P , in
H
support of our assignment of level 3. We also investigated the binding of tert-butyl propargyl
I
carbonate to Au in the presence of P , in which the tert-butyl group sterically prevents chelation.
H
We observed only a single current blockade, consistent with the formation of a simple π-complex
and not a chelate (Figure S27). We could thus observe two different coordination modes of alkyne-
I
esters 1 – 3 to Au with P and provide plausible assignments of the coordinating species.
H
I
Pore[a]
Table 1. Rate constants for the interaction of Au -alkyne-esters 1 – 3 with the P
H
1
2
3
Transition
Rate Constant
n[b]
32
40
164
163
64
Rate Constant
2.5 ± 0.1 × 10
4.0 ± 0.6
1.15 ± 0.09 × 10
6.4 ± 0.6
8.1 ± 0.4
n[b]
35
42
193
190
99
Rate Constant
n[b]
21
27
136
139
47
-1 -1
2
2
2
k
k
k
k
k
k
12 (M s )
2.3 ± 0.2 × 10
4.2 ± 0.5
1.2 ± 0.1 × 10
10.5 ± 0.5
6.4 ± 0.5
1.6 ± 0.2 × 10
4.1 ± 0.2
6.2 ± 1.2 × 10
11.1 ± 0.8
6.4 ± 0.3
-1
21 (s )
-
1 -1
3
3
2
13 (M s )
-
1
31 (s )
-
1
23 (s )
-
1
32 (s )
4.4 ± 0.4
72
2.9 ± 0.2
104
3.0 ± 0.3
19
[
a] Conditions: 2 M KCl, 10 mM MES, Chelex, pH 6.1, recorded at −50 mV and 21 ± 1 °C (n = 3)
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
I
[
b] n = number of events recorded in a selected experiment when 150 µM of both Au (tcep)Cl and alkyne ester were
added to the trans compartment (refer to Figure 3)
Gold(I) catalysed cyclisation of acetylenic acids
The intramolecular cyclisation of acetylenic acids to produce enol lactones is catalysed by AuI
salts in organic solvent (Figure 1d).23-25 It has been proposed that the Au catalyst first coordinates
I
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to and activates the alkyne to promote the addition of the nucleophilic carboxylic acid. The
I
corresponding vinyl-Au species, which has been isolated and visualised with X-ray
crystallography,45-46 protodemetallates to yield the product and returns Au to the catalytic cycle.
I
I
Accordingly, we found that the treatment of 4-pentynoic acid with Au (tcep)Cl at room
temperature in water (buffered at pH 6.0, with and without 2M KCl) cleanly produced γ-
methylene-γ-butyrolactone (Figures S28 and S29); these conditions were used because they were
similar to the conditions employed in the nanoreactor experiments. The selective exo-dig
cyclisation was also observed in organic media (Figure S30), consistent with literature
observations. An exo-lactone was also produced catalytically from the similar acetylenic acid, 2,2-
dimethyl-4-pentynoic acid, under the same aqueous conditions (Figure S31). As we were able to
I
observe Au -alkyne binding to the P pore, we examined the intermediates in the catalytic
H
cyclisation of acetylenic acids at the single-molecule level by using the nanoreactor approach.
I
When Au (tcep)Cl (300 µM) was added to the trans compartment of the bilayer apparatus
containing 4-pentynoic acid (300 µM; stoichiometric conditions) in 2 M KCl, 10 mM MES, pH
6
.1, at an applied potential of −50 mV (Figure 4a), we observed three reversible blockades of P in
H
I
addition to the open pore current (level 1) and the level with Au bound (level 0) (Figure 4b and
Figures S32 and S33). Because the catalytic reaction proceeds in the trans compartment, PH
I
samples and records the intermediate Au -ligand species present in solution during the
transformation. Transitions linking intermediates are observed as partial reaction cycles that occur
on P , permitting construction of a reaction cycle and the associated kinetics. Performing the
H
I
reaction under catalytic conditions (2 µM Au (tcep)Cl and 500 µM 4-pentynoic acid) did not yield
I
sufficient binding events of Au -ligand intermediate species for statistical analysis. Further, when
I
−
30 µM (10 mol%) of Au Cl was used as the catalyst with 300 µM 4-pentynoic acid, although the
2
I
same reversible blockades were initially observed as seen with Au (tcep)Cl, the ionic current
through the nanopore quickly became blocked (Figure S34). We postulate that in the absence of a
I
I
phosphine-coordinating ligand, aggregated Au and Au -ligand complexes form in solution and
1
7
I
block the nanopore. The three current blockades suggested that there are three different Au -
ligand species involved in the catalytic cycle (n = 5, independent experiments). We also recorded
only three current blockades with the acetylenic acid 2,2-dimethyl-4-pentynoic acid (n = 3)
(
Figures S35 and S36). The largest blockade observed with 4-pentynoic acid (6.9 ± 0.3 pA) was
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designated level 4 and had a life-time of 26 ± 1 ms. Smaller blockades of 5.2 ± 0.2 pA (level 3)
and 3.8 ± 0.2 pA (level 2) had longer life-times of 152 ± 8 ms and 205 ± 8 ms, respectively (Figure
4
b). The number of events at each observed level decreased over the course of the reaction as 4-
pentynoic acid was converted to the enol lactone. The cumulative plots of the events fitted well to
exponential functions, as expected for a decay with a first-order dependence on each intermediate
(Figure 4c). As the time-dependence of the derivative of a cumulative events plot reflects the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
7
change in concentration of an analyte with time (Supplementary Section 4.1), and the initial
concentration of 4-pentynoic acid was known, we were able to determine a rate constant of 5.7 ±
-4
-1
0
.8 × 10 s (the three intermediates gave the same rate constant and was averaged over five
experiments to determine the overall rate constant). The similar calculated rate constant for the
conversion of all of the observed intermediates (levels 4, 3 and 2) suggest that they interconvert at
rates considerably faster than the rate of product formation (see below). When the reaction was
1
followed by H NMR under the same conditions in buffered D O (2 M KCl, 10 mM MES, p(DH)
2
-4
-1
-4
6
.1), a similar rate constant of 4.3 ± 0.7 × 10 s was obtained. A rate constant of 5.5 ± 0.5 × 10
-1
s was determined with 2,2-dimethyl-4-pentynoic acid (Figure S37).
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1
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2
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2
2
2
2
2
2
2
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
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0
1
2
3
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5
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9
0
1
2
3
4
5
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8
9
0
1
2
3
4
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
Figure 4. Single-molecule detection of catalytic intermediates. (a) A single P pore was incorporated into a planar
lipid bilayer. An acetylenic acid was introduced to the trans compartment and the catalyst, Au (tcep)Cl, was added to
initiate the reaction and catalytically produce the enol lactone. Conditions: 2 M KCl, 10 mM MES, pH 6.1, 21 ± 1 °C.
I
(
b) At an applied potential of −50 mV, analysis of the electrical recording during the reaction (grey) identified three
I
current blocking events accessed from either the Au -bound state (level 0; red) or the unoccupied pore (level 1; green),
which were assigned as level 4 (black), level 3 (purple), and level 2 (blue). (c) Cumulative plots of the reaction
I
progress. The number of recorded events is proportional to the concentration of the intermediate Au -species and
decreases over time as the substrate is converted to the product. (d) The transitions from each level observed in the
electrical recordings were analysed to determine accessible transitions. This information was combined to create a
kinetic model that describes the transitions between current levels. (e) Proposed kinetic scheme describing the proton-
I
mediated demetallation step (k20). (f) Our proposed catalytic cycle that describes the Au -catalysed cyclisation of
acetylenic acids. (g) A recording of a complete catalytic cycle for the transformation of 4-pentynoic acid within P
that transitions from levels 1→4→3→2→0. The chemical structure proposed for each level is shown.
H
We analysed the transitions from each level during the reaction of either 4-pentynoic acid or
I
2
,2-dimethyl-4-pentynoic acid with Au (Figure 4d, top). We observed reversible transitions from
1
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the Au -bound state (level 0) to level 1 (open pore), level 3 and 4. Similarly, reversible transitions
were observed from the open pore (level 1) to levels 0, 3 and 4. We also found that several
pathways were rarely observed (≤ 2% of the total transitions from that level): transitions from
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
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4
4
4
4
4
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5
5
5
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5
5
6
I
either the open pore (level 1) or the Au -bound state (level 0) to level 2, and transitions between
levels 2 and 4. This revealed that level 2 was accessed predominantly from level 3. We collated
this information to construct a five-state model that describes these transitions (Figure 4d, bottom).
The rate constants associated with the transitions were determined by Hidden Markov modelling
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
QUB 2.0, Buffalo University) (Table 2). Control experiments involving the addition of
I
Au (tcep)Cl and excess enol lactone to the trans compartment under the same conditions revealed
I
that the enol lactone product of the catalytic reaction did not coordinate to Au (Figure S38). As we
also observed that level 2 does not occur from the coordination of a ligand, this suggested that
I
level 2 may be assigned to the vinyl-Au complex, which is the last intermediate in the proposed
I
catalytic cycle (Figure 1d). To better support this assignment, and as the vinyl-Au species
I
protodemetallates to release the enol lactone and reintroduce Au into the catalytic cycle (Figure
I
4
e), we repeated the reaction with 4-pentynoic and Au in D O solution (n = 3, independent
2
-4 -1
experiments) (Figures S39 and S40). We determined a rate constant of 3.8 ± 1.3 × 10 s (using
I
300 µM of both 4-pentynoic acid and Au (tcep)Cl; Figure S41), slower than that observed in H O
2
-4
-1
1
(
5.7 ± 0.8 × 10 s , performed with the same reactant concentrations) and consistent with H
-4 -1
NMR spectroscopy experiments in D O (4.3 ± 0.7 × 10 s ); we also extrapolated a product
2
-4 -1
formation rate constant of ~7 × 10 s from individual rate constants (Supplementary Section 4.3),
which agrees (roughly) with the bulk-observed rate constant. We found a small solvent kinetic
hydrogen-deuterium isotope effect48-50 in the transition from level 2 to level 0, k20H / k20D = 1.7
(
Table 2), and determined statistically insignificant (P < 0.05) isotope effects in the transitions
from level 4 and level 3 to level 0 (k40H / k40D = 1.2 and k30H / k30D = 1.1). This unambiguously
establishes that the transition from level 2 to level 0 is a proton mediated step, consistent with our
I
assignment of level 2 to the vinyl-Au species, which requires protonation for the product to
I
dissociate from the Au center. We also observed solvent kinetic isotope effects of < 1 for
transitions to the open pore (k , k , k , and k ), coincident with isotope effects of > 1 for
0
1
21
31
41
transitions from the open pore (k , k and k ) (Table 2). We postulate that these solvent isotope
1
0
13
14
effects result from the difference in hydrogen bond strength between H O and D O to the His-145
2
2
coordinating group.51
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
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5
5
5
6
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Table 2. Rate constants for the steps involved in the Au -catalysed conversion of of acetylenic acids to enol lactones
as determined with the P pore
H
2
,2-dimethyl-4-pentynoic
acid
4
-pentynoic acid
O[c] (n = 3)
O[a] (n = 5)
n[b]
4137
190
120
4127
154
825
22
52
34
156
199
25
D
n[b]
1899
99
k /k
H
D
H
2
O[a] (n = 3)
1.2 ± 0.2
n[b]
Transition
H
2
2
-1
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
01 (s )
03 (M s )
1.5 ± 0.3
3.9 ± 0.4
0.40 ± 0.09
0.83 ± 0.31
1.33 ± 0.66
3.25 ± 1.11
1.55 ± 0.71
1.08 ± 0.47
1.73 ± 0.55
0.46 ± 0.17
0.85 ± 0.25
1.09 ± 0.41
0.64 ± 0.12
1.08 ± 0.40
0.83 ± 0.47
1.17 ± 0.44
0.74 ± 0.16
0.86 ± 0.43
4260
296
130
4276
174
798
24
-1 -1
3
2
3
2
3
3
2
2
2
3
3
2
3
3
3
1.0 ± 0.2 × 10
5.8 ± 1.7 × 10
2.8 ± 0.8 × 10
6.4 ± 2.5 × 10
1.6 ± 0.5 × 10
0.8 ± 0.2
1.9 ± 0.7
1.0 ± 0.3
1.6 ± 0.4
3.0 ± 0.5
1.6 ± 0.5
0.5 ± 0.3
1.1 ± 0.2
26.4 ± 5.6
1.1 ± 0.4
1.3 ± 0.4 × 10
4.3 ± 1.8 × 10
8.7 ± 1.2 × 10
4.1 ± 0.9 × 10
1.5 ± 0.5 × 10
0.5 ± 0.1
1.2 ± 0.2 × 10
7.4 ± 0.3 × 10
2.9 ± 0.1 × 10
7.6 ± 1.7 × 10
1.8 ± 0.6 × 10
0.7 ± 0.1
1.8 ± 0.2
1.4 ± 0.2
1.7 ± 0.2
2.6 ± 0.3
1.8 ± 0.2
1.4 ± 0.2
1.9 ± 0.7
25.2 ± 0.4
3.5 ± 0.3
-
1 -1
04 (M s )
31
-
1 -1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10 (M s )
1899
225
1022
10
55
13
94
230
20
27
80
-
1 -1
13 (M s )
-
1 -1
14 (M s )
-
1
20 (s )
-
1
21 (s )
4.2 ± 0.4
1.1 ± 0.1
1.5 ± 0.4
4.7 ± 0.3
1.5 ± 0.2
0.59 ± 0.09
0.9 ± 0.3
35.8 ± 2.4
1.2 ± 0.4
29
51
-
1
23 (s )
-
1
30 (s )
281
182
41
124
131
812
109
-
1
31 (s )
-
1
32 (s )
-
1
34 (s )
31
-
1
40 (s )
130
817
31
-
1
41 (s )
1025
29
-
1
43 (s )
[
[
a] Conditions: 2 M KCl, 10 mM MES, Chelex, pH 6.1, recorded at −50 mV and 21 ± 1 °C
b] n = number of events recorded in a selected experiment when 300 µM of both Au (tcep)Cl and acetylenic acid
were added to the trans compartment (refer to Figure 4)
c] Conditions: 2 M KCl, 10 mM MES, Chelex, p(DH) 6.1, recorded at −50 mV and 21 ± 1 °C
I
[
The life-times (τ ) and amplitudes of levels 4 and 3 observed during the catalytic cycle were
off
similar to the life-times of the binding events established with the unreactive alkyne-esters. We
I
therefore assigned level 4 in the catalytic cycle to the Au -alkyne π-complex and level 3 to the
formation of the alkyne-acid chelate (Figure 4d). We propose a catalytic cycle for the cyclisation
of acetylenic acids in accord with our observations at the single-molecule level (Figure 4f). In the
I
first step, the substrate coordinates to an open site on Au through the alkyne group to form a linear
I
complex, activating the alkyne for nucleophilic attack. The Au center must then position the
I
carboxylic acid to attack the activated alkyne. The resultant vinyl-Au species is then protonated to
I
release the enol lactone irreversibly and allow Au to continue in the catalytic cycle. Although the
identity of the catalytic intermediates cannot be proven definitively with our nanopore sensing
approach, our catalytic model is the simplest and most chemically plausible scenario to describe
the single-molecule experiments. Our strategy for the observation of catalysis at the single-
molecule level relies on sampling the reaction as it proceeds in solution, observing the rates of
transitions within the pore, and compiling this information to construct a catalytic cycle. We
observed only a single event during a two hour electrical recording with 4-pentynoic acid that
showed a sequence corresponding to a complete catalytic cycle without dissociation from the
nanoreactor (i.e. levels 4 to 3 to 2 to 0) (Figure 4g). This is consistent with an expectation based on
the probabilities of the different transitions (Supplementary Section 4.4). Furthermore, the
I
observation of the transitions from levels 4 and 3 (Au -alkyne intermediate species) to level 0 (the
1
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Au -bound state) indicate that a substrate can dissociate from the catalytic Au center before
irreversible production of the enol lactone.
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CONCLUSIONS
I
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In this study, we have monitored the Au -catalysed transformation of acetylenic acids into enol
lactones in real time, observing transitions at the single-molecule level and thereby establishing the
intermediates in the catalytic cycle. The nanoreactor approach has allowed direct observation of
the individual reaction intermediates and an analysis of the lifetimes and the allowed transitions of
each species. Therefore, the approach has advantages over ensemble measurements, which are
often limited to monitoring the rate-determining step. Although our approach is limited to the
investigation of reactions that occur in water, aqueous metal-catalysed reactions are of increasing
importance in bio-orthogonal and green chemistry. As we have been able to discriminate between
4
7
enantiomeric amino acids by binding to a copper(II) complex within a nanopore, we believe our
approach may be adapted to identify the handedness of a chiral intermediate involved in a metal-
catalysed reaction. We anticipate that the observation of the reactions of ligands on catalytically
active metal centres bound within a nanopore will shed light on additional multistep reaction
mechanisms.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information for this paper is available free of charge on the ACS Publications
website.
Synthetic details, characterisation data, and single-molecule analyses.
AUTHOR INFORMATION
Corresponding Author
*
E-mail: hagan.bayley@chem.ox.ac.uk
ORCID
Hagan Bayley : 0000-0003-2499-6116
Notes
The authors declare no competing financial interest.
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1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
4
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4
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4
4
4
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6
ACKNOWLEDGEMENTS
This work was supported by a European Research Council Advanced Grant (COSIMO). Y.Q. was
the recipient of China Scholarship Council-University of Oxford Scholarship.
0
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REFERENCES
1
. Magano, J.; Dunetz, J. R., Large-scale applications of transition metal-catalyzed couplings for
the synthesis of pharmaceuticals. Chem. Rev. 2011, 111 (3), 2177-2250.
. Beller, M.; Blaser, H. U.; Eds., Organometallics as Catalysts in the Fine Chemical Industry,
vol 42 of Topics in Organometallic Chemistry. Springer, Berlin, Heidelberg: 2012.
. Topczewski, J. J.; Cabrera, P. J.; Saper, N. I.; Sanford, M. S., Palladium-catalysed
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3
transannular C–H functionalization of alicyclic amines. Nature 2016, 531 (7593), 220-224.
4. Thomas, A. A.; Denmark, S. E., Pre-transmetalation intermediates in the Suzuki-Miyaura
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Figure 1. The PH αHL pore: a nanoreactor for the investigation of AuI catalysis. (a) WT αHL (PDB code 7AHL)
is a mushroom-shaped transmembrane protein pore comprising seven identical subunits. A side view of the
ribbon structure shown with one of the subunits highlighted in green. (b) A sagittal section of the engineered
αHL pore, PH, embedded in a planar lipid bilayer (orange) for single-channel electrical recording. PH
contains one subunit with a histidine substitution at position 145 (red in stick representation). The side chain
of His-145 points into the lumen of the transmembrane β barrel and can coordinate metal-ligand complexes
from solution, entering from the trans compartment. (c) An expanded view around His-145, with bound 4-
pentynoic acid: AuI (yellow sphere), 4-pentynoic acid (stick representation: carbon, grey; hydrogen, white;
oxygen, red). (d) The proposed catalytic cycle for transformation of an acetylenic acid to an enol lactone.
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Figure 2. The PH pore binds AuI. (a) Ribbon representation of the PH pore viewed from the bottom of the β
barrel. The single histidine side chain (red) binds AuI (yellow sphere) reversibly. (b) Single-channel
recordings at −50 mV with 2 M KCl, 10 mM MES (pH 6.1) in both compartments, and 0 to 5 µM AuICl in the
trans compartment. The two current levels correspond to the unoccupied pore (PH) and to the pore with
AuICl2− bound to the histidine residue (PH·AuI). (c) Proposed kinetic scheme describing AuI binding to the
PH pore and plots of the reciprocals of the mean inter-event intervals (τon) and the dwell times of AuI (τoff)
versus the AuICl2− concentration.
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Figure 3. Detection of AuI-alkyne complexes using the PH pore. (a – c) Electrical current recordings (shown
in grey with an idealised trace overlaid and color coded for each level) and noise analyses for the bound
states of AuI-coordinated alkyne-esters 1 – 3 to PH (N = number of events). Inset in (c): expanded view of
level 3. Concentrations (AuI(tcep)Cl and alkyne-ester added in a 1:1 stoichiometry: 1, 50 µM; 2, 50 µM; 3,
5
0 µM. All traces are displayed on the same scale. Conditions: 2 M KCl, 10 mM MES, pH 6.1, 21 ± 1 °C, at
an applied potential of −50 mV. (d) Kinetic model used to determine the rate constants of the observed
transitions. (e) Proposed kinetic scheme describing the restricted rotation of alkyne esters 3 and 4 between
the trans (Z) configuration and the cis (E) configuration. (f) All-points histogram of the current amplitudes in
level 3 observed with 3. Inset: idealised trace (QuB) of level 3 (red) overlaid on the recorded current trace
(black).
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Figure 4. Single-molecule detection of catalytic intermediates. (a) A single PH pore was incorporated into a
planar lipid bilayer. An acetylenic acid was introduced to the trans compartment and the catalyst,
AuI(tcep)Cl, was added to initiate the reaction and catalytically produce the enol lactone. Conditions: 2 M
KCl, 10 mM MES, pH 6.1, 21 ± 1 °C. (b) At an applied potential of −50 mV, analysis of the electrical
recording during the reaction (grey) identified three current blocking events accessed from either the AuI-
bound state (level 0; red) or the unoccupied pore (level 1; green), which were assigned as level 4 (black),
level 3 (purple), and level 2 (blue). (c) Cumulative plots of the reaction progress. The number of recorded
events is proportional to the concentration of the intermediate AuI-species and decreases over time as the
substrate is converted to the product. (d) The transitions from each level observed in the electrical
recordings were analysed to determine accessible transitions. This information was combined to create a
kinetic model that describes the transitions between current levels. (e) Proposed kinetic scheme describing
the proton-mediated demetallation step (k20). (f) Our proposed catalytic cycle that describes the AuI-
catalysed cyclisation of acetylenic acids. (g) A recording of a complete catalytic cycle for the transformation
of 4-pentynoic acid within PH that transitions from levels 1→4→3→2→0. The chemical structure proposed
for each level is shown.
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