ARTICLES PUBLISHED ONLINE: 18 MAY 2014 | DOI: 10.1038/NCHEM.1949
Single-molecule analysis of chirality in a multicomponent reaction network Mackay B. Steffensen†‡, Dvir Rotem†‡ and Hagan Bayley* Single-molecule approaches to chemical reaction analysis can provide information that is not accessible by studying ensemble systems. Changes in the molecular structures of compounds tethered to the inner wall of a protein pore are known to affect the current carried through the pore by aqueous ions under a fixed applied potential. Here, we use this approach to study the substitution reactions of arsenic(III) compounds with thiols, stretching the limits of the protein pore technology to track the interconversion of seven reaction components in a network that comprises interconnected Walden cycles. Single-molecule pathway analysis of ‘allowed’ and ‘forbidden’ reactions reveals that sulfur–sulfur substitution occurs with stereochemical inversion at the arsenic centre. Hence, we demonstrate that the nanoreactor approach can be a valuable technique for the analysis of dynamic reaction systems of relevance to biology.
I
n the present study, substitution at arsenic(III) by thiols (RSH) has been observed at the single-molecule level within a protein nanoreactor by ionic current recording1. In the nanoreactor approach, reactants are tethered to the wall of an engineered protein pore in a lipid bilayer1. The current carried through the pore by aqueous ions (such as Kþ and Cl2) under an applied potential is monitored by electrical recording. Changes in current flow arising from individual bond-making and bond-breaking events are tracked, often with a submillisecond time resolution. No bulky tags are required to observe the chemistry and it is possible to use high concentrations of reagents1, which is challenging in single-molecule fluorescence experiments. In this way, it has been possible to observe a variety of covalent reactions of small molecules, including the photodeprotection of 2-nitrobenzyl carbamates2, the formation and cleavage of disulfide bonds3, a step-by-step polymerization4, photochemical5 and thermal6 isomerizations and an addition–elimination displaying a hydrogen–deuterium isotope effect7. The potential of the approach for sensing chemically reactive analytes has also been evaluated8. The nanoreactor approach has also proved useful for examining the making and breaking of As–S bonds in organoarsenic(III) molecules6,9. Reactions of arsenic(III) compounds with thiols10 occur in medicine11, toxicology12 and enzymology13 and in the application of probes for cellular imaging14. Examples in pharmacology include the original ‘magic bullet’ (salvarsan15), treatments for trypanosomiasis16 and the use of arsenic trioxide to manage cancer11,17. Further, arsenic isotopes are under consideration for nuclear medicine and imaging18. As well as their real and fictional roles in homicide and assassination, arsenic(III) reagents have been used in chemical warfare19 and for crowd control20. Arsenate reductases, which convert arsenate to arsenite, rely on As–S chemistry13. Arsenic(III) reagents have also been applied extensively in biochemistry and cell biology, notably the bisarsenicals used for the sitespecific labelling of proteins containing tetracysteine motifs14. Although trivalent arsenic molecules with alkyl and aryl substituents are relatively inert to substitution and undergo slow inversion21,22, arsenite, arsenous acids and other organoarsenic(III) compounds with As–O and As–S bonds are more labile6,9,13,23,24. In the present work we examine a reaction network that allows us to determine the stereochemistry of thiol substitution at
arsenic(III). First, an arylarsonous acid makes an As–S bond on the wall of the protein nanoreactor to form PSAs(OH)Ar (P ¼ protein). The hydroxyl of this adduct is then substituted with one of two free thiols (R1SH or R2SH) to form PSAs(SR1)Ar or PSAs(SR2)Ar. The interconversion of PSAs(SR1)Ar and PSAs(SR2)Ar by substitution with R2SH and R1SH is also monitored. The identification of ‘allowed’ and ‘forbidden’ reactions allows the stereochemistry of thiol interchange to be assigned. a
H2N cis
SH
O
O
SH
O N H
HO OH
H2N
L-Penicillamine
H N
OH O
O
Glutathione
b As
PSH
PSH
OH
R PSH
PSH
As
HO
R
HO trans
As
SO3–
HO 4-sulfophenyl arsonous acid (SPAA)
Figure 1 | The aHL nanoreactor. a, Protein nanoreactor PSH is a heteroheptameric staphylococcal aHL pore with a single cysteine residue at position 117 (space-filling spheres) in one of the seven subunits. The side chain of the cysteine contains a thiol group (yellow), which projects into the lumen of the pore. The structures of the reagents are shown at the side of the pore from which they are added. b, Reactions of SPAA within the PSH pore6.
Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK, † Present addresses: Southern Utah University, Physical Science Department, 351 West Center, Cedar City, Utah 84720, USA (M.B.S.), Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel (D.R.), † These authors contributed equally to this work. * e-mail: [email protected] NATURE CHEMISTRY | VOL 6 | JULY 2014 | www.nature.com/naturechemistry
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S C
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H2N O
R = SO3
OH L-Penicillamine
c
d
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1
OH
H N
N H
HO
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O
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Figure 2 | Assignment of current levels. a, Part of the current recording for a partial reaction containing the PSH pore, SPAA (trans) and L-penicillamine (cis). It is known from reactions with PSH and SPAA alone that levels A and B correspond to the two enantiomers of the arsonous acid adduct, PSAs(OH)Ar. Therefore, C and D correspond to adducts in which the hydroxyl of PSAs(OH)Ar has been substituted with the sulfur of L-penicillamine. b, One stereochemical interpretation of the recording in a. To avoid confusion, we have used the relative stereochemical configurations that were ultimately shown to participate in the reactions (Figs 3 and 5). c, Part of the current recording for a partial reaction containing the PSH pore, SPAA (trans) and glutathione (cis). E and F correspond to adducts in which the hydroxyl of PSAs(OH)Ar has been substituted with the sulfur of glutathione. d, One stereochemical interpretation of the recording in c. e, Part of the current recording for the full reaction containing the PSH pore, SPAA (trans), L-penicillamine (cis) and glutathione (cis). Current levels are assigned from a and c. An extended segment of a recording is provided in Supplementary Fig. 5. f, One stereochemical interpretation of the recording in e. Measurements were made in 2 M KCl, 20 mM bis–tris propane, 100 mM EDTA, pH 8.5, in the presence of 200 mM SPAA (trans) and 250 mM L-penicillamine or 250 mM glutathione, or both, in the cis compartment. 604
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P
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P
A
B
AR* BS*
AR* BS*
AR* BS*
AR* BS*
D
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DR* CS*
DS* CR*
DR* CS*
DS* CR*
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ER* FS*
ES* FR*
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ER* FS*
Case 1 r r
Case 2 i r
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S
S
S
P
P
P
was converted only to D (and vice versa) and B only to C (and vice versa). A similar experiment with SPAA and glutathione (g-Lglutamyl-L-cysteinylglycine, a single diastereomer) identified levels E and F (Fig. 2c,d), and we saw that A was converted only to F (and vice versa) and B only to E (and vice versa). In the complete reaction, with both L-penicillamine and glutathione, transitions between all seven components (P, A–F) could be observed (Fig. 2e,f).
P
A
B
A
B
A
B
A
B
D
C
D
C
D
C
D
C
E
F
E
F
E
F
E
F
Figure 3 | Analysis of the reaction network. Left: the two principal branches of the network. Right: Cases 1–4, four possible stereochemical pathways, based on retention (r) or inversion (i) at arsenic(III). The diastereomer A of the initial adduct PSAs(OH)Ar is given the relative stereochemistry R*, so diastereomer B is S*. These relative designations are used because the absolute configurations around arsenic(III) are unknown. O ↔ S, O ligand (–OH) substitution by an S nucleophile (or the reverse reaction); S ↔ S, S ligand substitution by an S nucleophile (thiol interchange). , the step is expected according to the proposed stereochemical pathway, but it is not observed by experiment; , not expected, but observed; , expected and observed; , not expected and not observed. Cases 1 and 2 are not permissible based, in part, on the finding that reactions A E and B F are not observed. Cases 3 and 4 are both permissible and both require inversion at arsenic(III) during thiol interchange (S ↔ S). Case 4 requires inversion and case 3 requires retention during OH substitution by an S nucleophile (O ↔ S). We favour the pathway depicted in Case 4, in which both O ligand and S ligand substitutions proceed with inversion.
Results Reaction of PSH with 4-sulfophenylarsonous acid. We have previously examined the reaction between a single thiol group (the side chain of a Cys residue) on the internal wall of an ahaemolysin (aHL) pore (PSH , Fig. 1a) and 4-sulfophenylarsonous acid (SPAA) to form an As–S bond (PSAs(OH)Ar)9. We were also able to observe stereochemical inversion at arsenic(III) in the bound adduct, which has a mean lifetime of 1 s at pH 8.4 (Fig. 1b)6. Because the protein is chiral, the adducts are diastereomers and physically distinguishable by ionic current recording. In the present work, we observe a far more complex reaction network within the nanoreactor that involves seven current levels. We examine the displacement of the hydroxyl group in PSAs(OH)Ar (where PSH contains a single cysteine at position 117) by a sulfur nucleophile (for example, R1SH) to form PSAs(SR1)Ar. We have also been able to observe the displacement of R1S in this adduct by a second sulfur nucleophile (for example, R2SH) to form PSAs(SR2)Ar and show by single-molecule reaction pathway analysis that this displacement occurs with inversion of configuration at the As atom. These reactions were examined by adding SPAA to the trans side of the lipid bilayer and the thiols to the cis side (Fig. 1a), so that they would not react with one another but instead migrate to the reaction site within the pore1. Assignment of the seven current levels. So that the entire reaction network could be examined, it was first necessary to assign the seven observed current levels to the different reactants. In support of our assignments, we show here short sections of far longer traces from various partial reactions. Reaction with SPAA (trans) alone identified the levels A and B arising from the two enantiomers of PSAs(OH)Ar (ref. 6). Reaction with SPAA (trans) and L-penicillamine (cis) yielded two additional levels, C and D, which must correspond to the two enantiomers of PSAs(SR1)Ar (Fig. 2a,b). At this point, the basis of our stereochemical analysis could be seen as it was apparent from long current traces that A
Examination of the full reaction network. We collected data from reactions that included all three soluble reactants—SPAA (trans), L-penicillamine (cis) and glutathione (cis)—involving thousands of transitions (Fig. 4a, Supplementary Table 1). Because it was not practicable to carry out a detailed analysis of the reaction pathway by manual techniques, we used the software packages Clampfit and QuB, which are normally used for the analysis of singlechannel recordings from ion channels. In addition to the allowed transitions identified in the partial reactions, we find that transitions D ↔ E (and not D ↔ F) and C ↔ F (and not C ↔ E) occur (Figs 3 and 4a, Supplementary Table 1). To check the validity of the computer analysis, some of the data were evaluated by hand, and a strong correspondence was found between the manual and computational methods (Supplementary section ‘Results’, Supplementary Table 2, Supplementary Figs 1, 2). Stereochemistry of the allowed reactions. Examination of the data in Fig. 4a suggests two pathways for PSH ↔ PSAs(OH)Ar ↔ PSAs(SR1)Ar ↔ PSAs(SR2)Ar: P ↔ A ↔ D ↔ E and P ↔ B ↔ C ↔ F, which must reflect stereochemically allowed transformations. Because P is a branch-point, the two pathways can be assembled (Fig. 3, left) and we can then examine how proposed stereochemistries for each step fit in with the remaining observed transitions that are not simple epimerizations at As (A ↔ B and so on). The absolute configurations of the reactants (for example, A versus B) are not known, so we have used relative configurations (R*, S*, Fig. 3). There are four possibilities (Fig. 3, Cases 1 to 4) and we focus on steps A E and A F. In two of the possibilities (Cases 1 and 4), all substitutions at As occur with the same stereochemistry; that is, both O ↔ S and S ↔ S substitutions go with retention of configuration (Case 1) or both go with inversion (Case 4). However, in Case 1, transition A E is expected according to the designated stereochemical pathway, but not observed, while A F is not expected, but is observed experimentally (Fig. 4a). By contrast, in Case 4, A E is not expected and not observed, while A F is expected and experimentally observed. Therefore, Case 1 is not viable. In the second pair of the four cases (Cases 2 and 3), Case 2 imparts inversion during O ↔ S substitution and retention during S ↔ S substitution, while in Case 3, O ↔ S takes place with retention and S ↔ S with inversion. In this pair, the experimental data fit only Case 3 (Figs 3 and 4a). We can therefore conclude that S ↔ S occurs with inversion (the viable Cases 3 and 4), but that the situation for O ↔ S is unresolved. We favour Case 4, in which both O ↔ S and S ↔ S occur with inversion. A matrix analysis can also be used to describe experimentally observed transitions in the reaction network (Supplementary Table 3, Supplementary Fig. 3), and this also rules out Cases 1 and 2 (see Supplementary section ‘Additional results’). The data also rule out the possibility that the substitution reactions occur with racemization at As. Single-molecule pathway analysis, as used here, suggests a reaction scheme (Fig. 5) based on Case 4 (Fig. 3), which requires that both O ↔ S and S ↔ S substitutions go with inversion. All 42 possible rates of interconversion for the seven species were computed with the QuB software package from transition frequencies obtained by single-channel recording (Fig. 4b). The rates of the inversions C ↔ D and E ↔ F for the species with penicillamine and glutathione ligands, respectively, are of the same order of magnitude as the rates
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0.033 ± 0.009 0.072 ± 0.018 0.026 ± 0.003
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0.011 ± 0.002 0.019 ± 0.001
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0.11 ± 0.01
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6 99
0.12 ± 0.01 0.072 ± 0.042
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F
0.20 ± 0.04 0.20 ± 0.07
0.37 ± 0.11 0.20 ± 0.02
0.33 ± 0.02 0.094 ± 0.016
0.030 ± 0.013
0.12 ± 0.04 0.11 ± 0.01
0.14 ± 0.04 0.075 ± 0.008
Figure 4 | Transition counts and computed reaction rates. The reaction components are 200 mM SPAA (trans) and 250 mM L-penicillamine and 250 mM glutathione (both cis) in 2 M KCl, 20 mM bis-tris propane, 100 mM EDTA, pH 8.5. Red and blue letters represent the different stereoisomers. Allowed (green) and forbidden (red) transitions are indicated based on the observed frequencies and their compatibility with the Case 4 reaction network. a, Transitions between current levels were counted by computer (Clampfit 10 software). The results from a typical experiment are shown. The 3,221 transitions are represented in a row to column fashion, that is, row D, column C is the number of transitions for D C. The transitions for two additional traces are presented in Supplementary Table 1. Of the 73 forbidden transitions assigned by computer, 49 were judged to be allowed after careful visual inspection of the individual transitions, and 20 were found to involve substates other than P or A to F ( Supplementary section ‘Additional results’). b, The transition rates of the allowed transitions (green) were computed with the QuB software package. Values are means of three trials+s.d. The transitions are represented in row to column fashion, that is, row D, column C is the rate for D C under the experimental conditions. P
HO As
SO3 SH
HO
R S
A
OH
As
B
S As R1 =
OH
H2N O
R = SO3
OH
R
S
As
L-Penicillamine
R
R1 S
R2 =
D
S
C
As S
R1
R
O
O N H
HO H2N
H N
OH O
O
Glutathione
S R E
S
As S
R2
R2
As
S F
R
Figure 5 | The complete reaction network. The reactions occur within an aHL protein nanopore (PSH), which contains a single cysteine residue that projects into the lumen. The reactants are SPAA and the thiols L-penicillamine (R1, maroon) and glutathione (R2, orange). Double green arrows indicate reversible reactions that are allowed (Figs 4a and 2) and occur with inversion according to the scheme in Fig. 3, Case 4. Double red arrows indicate reactions that are not observed and would occur with retention according to Fig. 3, Case 4. Red-green arrow combinations include the observed loss of As from PSH and the unlikely direct ‘termolecular’ formation of C, D, E or F, which is not observed (Figs 4a and 2). The double green arrows from P ↔ A and P ↔ B indicate the observed formation and breakdown of the initial PSAs(OH)Ar adduct. The absolute stereochemical configurations of the various adducts are unknown. However, if A is assigned the relative configuration R*, the relative configurations in Case 4 are: R*: A, C, E; S*: B, D, F (Fig. 3). 606
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of O ↔ S substitutions (transitions A ↔ D, B ↔ C for penicillamine and A ↔ F, B ↔ E for glutathione) and the S ↔ S thiol interchanges (C ↔ F and D ↔ E). In addition, the inversion A ↔ B, which does not involve a thiol nucleophile, is far slower. This suggests that, at the concentrations of penicillamine and glutathione used, the inversions C ↔ D and E ↔ F occur through thiol interchange and not by a unimolecular mechanism. Rate constants for the network reactions. The rate constants for the interconversions were calculated from current recordings carried out in the presence of all three reagents (SPAA, penicillamine and glutathione) or in the absence of one of the two thiols (Supplementary Table 4). All combinations of reactants gave similar values. It was assumed that the concentrations of reactants within the nanoreactor were the same as the concentrations in bulk solution1. Examination of the rate constants revealed several interesting points. For example, the rate constants for hydrolysis of arsenic(III) adducts of PSH with two S substituents (kDA , kDP, kCB , kCP, kFA , kFP, kEB , kEP) are much smaller than the hydrolysis rate constants of arsenic(III) adducts of PSH with a single S substituent (kAP and kBP) (Supplementary Table 4). This suggests that an arsenic(III) reagent can be temporarily ‘locked’ onto a cysteine residue by substitution of the hydroxyl group with a thiol ligand. We also note that the rate constants associated with enantiomeric adducts differ significantly. For example, kCP ¼ 0.033 s21 while kDP ¼ 0.072 s21 (Supplementary Table 4). This is because C and D are actually diastereomers (the protein provides a chiral environment6), so the rates are expected to diverge. Finally, in a reaction network, the products of the rate constants in any circular pathway in the clockwise and anticlockwise directions must be equal6,25. We have shown that this is valid for 13 different pathways (Supplementary Fig. 4).
Discussion The Walden inversion, described in 1896, demonstrated a simple conversion of one enantiomer (at a carbon centre) to the other, and offered critical experimental support for van’t Hoff’s concept of tetrahedral stereochemistry at carbon. Our work on arsenic stereochemistry is based on interwoven Walden cycles visualized at the single-molecule level. The tracking of seven reaction components by electrical recording showed that S ↔ S substitution at an arsenic(III) centre occurs with inversion of the relative stereochemical configuration. Such measurements made in a protein nanoreactor are not possible using ensemble approaches, because of the numerous species present. The nanoreactor approach is therefore a valuable means for the detailed analysis of complex dynamic chemical systems26.
Methods For full details see Supplementary Methods. Heteroheptameric staphylococcal aHL pores (PSH) were prepared with a single cysteine at position 117 in one of the seven subunits9. SPAA was prepared as described in ref. 9. Solutions of thiols were made fresh each day. Single-channel current recordings were performed in a planar bilayer apparatus. The bilayer was formed from 1,2-diphytanoyl-sn-glycero-3phosphocholine. Steps in the ionic current reflecting reversible chemistry were recorded at þ50 mV with an Axopatch 200B patch-clamp amplifier (Axon Instruments). The signal was filtered with a low-pass Bessel filter (80 dB/dec) with a corner frequency of 2 kHz and sampled at 10 kHz with a DigiData 1200 A/D converter (Axon Instruments). To analyse the current traces we used Clampfit (version 10.0, Axon Instruments) and QuB software (http://www.qub.buffalo.edu).
4. Shin, S-H. & Bayley, H. Stepwise growth of a single polymer chain. J. Am. Chem. Soc. 127, 10462–10463 (2005). 5. Loudwig, S. & Bayley, H. Photoisomerization of an individual azobenzene molecule in water: an on–off switch triggered by light at a fixed wavelength. J. Am. Chem. Soc. 128, 12404–12405 (2006). 6. Shin, S-H., Steffensen, M. B., Claridge, T. D. W. & Bayley, H. Formation of a chiral center and pyramidal inversion at the single-molecule level. Angew. Chem. Int. Ed. 46, 7412–7416 (2007). 7. Lu, S., Li, W-W., Rotem, D., Mikhailova, E. & Bayley, H. A primary hydrogen– deuterium isotope effect observed at the single molecule level. Nature Chem. 2, 921–928 (2010). 8. Wu, H. C. & Bayley, H. Single-molecule detection of nitrogen mustards by covalent reaction within a protein nanopore. J. Am. Chem. Soc. 130, 6813–6819 (2008). 9. Shin, S-H., Luchian, T., Cheley, S., Braha, O. & Bayley, H. Kinetics of a reversible covalent-bond forming reaction observed at the single molecule level. Angew. Chem. Int. Ed. 41, 3707–3709 (2002). 10. Shen, S., Li, X-F., Cullen, W. R., Weinfeld, M. & Le, X. C. Arsenic binding to proteins. Chem. Rev. 113, 7769–7792 (2013). 11. Liu, J. X., Zhou, G. B., Chen, S. J. & Chen, Z. Arsenic compounds: revived ancient remedies in the fight against human malignancies. Curr. Opin. Chem. Biol. 16, 92–98 (2012). 12. Argos, M., Ahsan, H. & Graziano, J. H. Arsenic and human health: epidemiologic progress and public health implications. Rev. Environ. Health 27, 191–195 (2012). 13. Messens, J. & Silver, S. Arsenate reduction: thiol cascade chemistry with convergent evolution. J. Mol. Biol. 362, 1–17 (2006). 14. Adams, S. R. & Tsien, R. Y. Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins. Nature Protoc. 3, 1527–1534 (2008). 15. Lloyd, N. C., Morgan, H. W., Nicholson, B. K. & Ronimus, R. S. The composition of Ehrlich’s salvarsan: resolution of a century-old debate. Angew. Chem. Int. Ed. 44, 941–944 (2005). 16. Burchmore, R. J., Ogbunude, P. O., Enanga, B. & Barrett, M. P. Chemotherapy of human African trypanosomiasis. Curr. Pharm. Des. 8, 256–267 (2002). 17. Zhang, X. W. et al. Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML. Science 328, 240–243 (2010). 18. Jennewein, M. et al. Vascular imaging of solid tumors in rats with a radioactive arsenic-labeled antibody that binds exposed phosphatidylserine. Clin. Cancer Res. 14, 1377–1385 (2008). 19. Goldman, M. & Dacre, J. C. Lewisite: its chemistry, toxicology, and biological effects. Rev. Environ. Contam. Toxicol. 110, 75–115 (1989). 20. Henriksson, J., Johannisson, A., Bergqvist, P. A. & Norrgren, L. The toxicity of organoarsenic-based warfare agents: in vitro and in vivo studies. Arch. Environ. Contam. Toxicol. 30, 213–219 (1996). 21. Senkler, G. H. & Mislow, K. The barrier to pyramidal inversion in ethylmethylphenylarsine. J. Am. Chem. Soc. 94, 291 (1972). 22. Baechler, R. D., Casey, J. P., Cook, R. J., Senkler, G. H. & Mislow, K. Effect of ligand electronegativity on the inversion barriers of arsines. J. Am. Chem. Soc. 94, 2859–2861 (1972). 23. Dill, K., Huang, L., Bearden, D. W., McGown, E. L. & O’Connor, R. J. Activation energies and formation rate constants for arsenical-antidote adducts as determined by dynamic NMR spectroscopy. Chem. Res. Toxicol. 4, 295–299 (1991). 24. Serves, S. V., Charalambidis, Y. C., Sotiropoulos, D. N. & Ioannou, P. V. Reaction of arsenic(III) oxide, arsenous and arsenic acids with thiols. Phosphorus Sulfur Silicon 105, 109–116 (1995). 25. Richard, E. A. & Miller, C. Steady-state coupling of ion-channel conformations to a transmembrane ion gradient. Science 247, 1208–1210 (1990). 26. Hunt, R. A. & Otto, S. Dynamic combinatorial libraries: new opportunities in systems chemistry. Chem. Commun. 47, 847–858 (2011).
Acknowledgements This work was supported by the Medical Research Council and a European Research Council advanced grant. H.B. was the holder of a Royal Society–Wolfson Research Merit Award. M.B.S. was the holder of a Ruth L. Kirschstein NIH Postdoctoral Fellowship (F32L078236).
Author contributions
Received 25 October 2013; accepted 9 April 2014; published online 18 May 2014
M.B.S. and D.R. contributed equally to this work. M.B.S. and H.B. designed the research. M.B.S. performed the experimental work. M.B.S., D.R. and H.B. analysed data and wrote the manuscript.
References
Additional information
1. Bayley, H., Luchian, T., Shin, S-H. & Steffensen, M. B. in Single Molecules and Nanotechnology (eds Rigler, R. & Vogel, H.) Ch. 10, 251–277 (Springer, 2008). 2. Luchian, T., Shin, S-H. & Bayley, H. Kinetics of a three-step reaction observed at the single molecule level. Angew. Chem. Int. Ed. 42, 1926–1929 (2003). 3. Luchian, T., Shin, S-H. & Bayley, H. Single-molecule covalent chemistry with spatially separated reactants. Angew. Chem. Int. Ed. 42, 3766–3771 (2003).
Supplementary information is available in the online version of the paper. Reprints and permissions information is available online at www.nature.com/reprints. Correspondence and requests for materials should be addressed to H.B.
Competing financial interests The authors declare no competing financial interests.
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Single-molecule analysis of chirality in a multicomponent reaction network Mackay B. Steffensen†‡, Dvir Rotem†‡ and Hagan Bayley* Single-molecule approaches to chemical reaction analysis can provide information that is not accessible by studying ensemble systems. Changes in the molecular structures of compounds tethered to the inner wall of a protein pore are known to affect the current carried through the pore by aqueous ions under a fixed applied potential. Here, we use this approach to study the substitution reactions of arsenic(III) compounds with thiols, stretching the limits of the protein pore technology to track the interconversion of seven reaction components in a network that comprises interconnected Walden cycles. Single-molecule pathway analysis of ‘allowed’ and ‘forbidden’ reactions reveals that sulfur–sulfur substitution occurs with stereochemical inversion at the arsenic centre. Hence, we demonstrate that the nanoreactor approach can be a valuable technique for the analysis of dynamic reaction systems of relevance to biology.
I
n the present study, substitution at arsenic(III) by thiols (RSH) has been observed at the single-molecule level within a protein nanoreactor by ionic current recording1. In the nanoreactor approach, reactants are tethered to the wall of an engineered protein pore in a lipid bilayer1. The current carried through the pore by aqueous ions (such as Kþ and Cl2) under an applied potential is monitored by electrical recording. Changes in current flow arising from individual bond-making and bond-breaking events are tracked, often with a submillisecond time resolution. No bulky tags are required to observe the chemistry and it is possible to use high concentrations of reagents1, which is challenging in single-molecule fluorescence experiments. In this way, it has been possible to observe a variety of covalent reactions of small molecules, including the photodeprotection of 2-nitrobenzyl carbamates2, the formation and cleavage of disulfide bonds3, a step-by-step polymerization4, photochemical5 and thermal6 isomerizations and an addition–elimination displaying a hydrogen–deuterium isotope effect7. The potential of the approach for sensing chemically reactive analytes has also been evaluated8. The nanoreactor approach has also proved useful for examining the making and breaking of As–S bonds in organoarsenic(III) molecules6,9. Reactions of arsenic(III) compounds with thiols10 occur in medicine11, toxicology12 and enzymology13 and in the application of probes for cellular imaging14. Examples in pharmacology include the original ‘magic bullet’ (salvarsan15), treatments for trypanosomiasis16 and the use of arsenic trioxide to manage cancer11,17. Further, arsenic isotopes are under consideration for nuclear medicine and imaging18. As well as their real and fictional roles in homicide and assassination, arsenic(III) reagents have been used in chemical warfare19 and for crowd control20. Arsenate reductases, which convert arsenate to arsenite, rely on As–S chemistry13. Arsenic(III) reagents have also been applied extensively in biochemistry and cell biology, notably the bisarsenicals used for the sitespecific labelling of proteins containing tetracysteine motifs14. Although trivalent arsenic molecules with alkyl and aryl substituents are relatively inert to substitution and undergo slow inversion21,22, arsenite, arsenous acids and other organoarsenic(III) compounds with As–O and As–S bonds are more labile6,9,13,23,24. In the present work we examine a reaction network that allows us to determine the stereochemistry of thiol substitution at
arsenic(III). First, an arylarsonous acid makes an As–S bond on the wall of the protein nanoreactor to form PSAs(OH)Ar (P ¼ protein). The hydroxyl of this adduct is then substituted with one of two free thiols (R1SH or R2SH) to form PSAs(SR1)Ar or PSAs(SR2)Ar. The interconversion of PSAs(SR1)Ar and PSAs(SR2)Ar by substitution with R2SH and R1SH is also monitored. The identification of ‘allowed’ and ‘forbidden’ reactions allows the stereochemistry of thiol interchange to be assigned. a
H2N cis
SH
O
O
SH
O N H
HO OH
H2N
L-Penicillamine
H N
OH O
O
Glutathione
b As
PSH
PSH
OH
R PSH
PSH
As
HO
R
HO trans
As
SO3–
HO 4-sulfophenyl arsonous acid (SPAA)
Figure 1 | The aHL nanoreactor. a, Protein nanoreactor PSH is a heteroheptameric staphylococcal aHL pore with a single cysteine residue at position 117 (space-filling spheres) in one of the seven subunits. The side chain of the cysteine contains a thiol group (yellow), which projects into the lumen of the pore. The structures of the reagents are shown at the side of the pore from which they are added. b, Reactions of SPAA within the PSH pore6.
Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK, † Present addresses: Southern Utah University, Physical Science Department, 351 West Center, Cedar City, Utah 84720, USA (M.B.S.), Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel (D.R.), † These authors contributed equally to this work. * e-mail: [email protected] NATURE CHEMISTRY | VOL 6 | JULY 2014 | www.nature.com/naturechemistry
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OH R 2
4 R1
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S
3 D
S C
R
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H2N O
R = SO3
OH L-Penicillamine
c
d
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P
SH
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A B
1
OH
H N
N H
HO
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O
O
O
O
H2N Glutathione OH
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S As A
E F
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9 15
7 11 12 13
10 s 1
R1 R
S As
S
8 14
2
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As S
OH A
R S
D
R
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E
Figure 2 | Assignment of current levels. a, Part of the current recording for a partial reaction containing the PSH pore, SPAA (trans) and L-penicillamine (cis). It is known from reactions with PSH and SPAA alone that levels A and B correspond to the two enantiomers of the arsonous acid adduct, PSAs(OH)Ar. Therefore, C and D correspond to adducts in which the hydroxyl of PSAs(OH)Ar has been substituted with the sulfur of L-penicillamine. b, One stereochemical interpretation of the recording in a. To avoid confusion, we have used the relative stereochemical configurations that were ultimately shown to participate in the reactions (Figs 3 and 5). c, Part of the current recording for a partial reaction containing the PSH pore, SPAA (trans) and glutathione (cis). E and F correspond to adducts in which the hydroxyl of PSAs(OH)Ar has been substituted with the sulfur of glutathione. d, One stereochemical interpretation of the recording in c. e, Part of the current recording for the full reaction containing the PSH pore, SPAA (trans), L-penicillamine (cis) and glutathione (cis). Current levels are assigned from a and c. An extended segment of a recording is provided in Supplementary Fig. 5. f, One stereochemical interpretation of the recording in e. Measurements were made in 2 M KCl, 20 mM bis–tris propane, 100 mM EDTA, pH 8.5, in the presence of 200 mM SPAA (trans) and 250 mM L-penicillamine or 250 mM glutathione, or both, in the cis compartment. 604
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P
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P
P
A
B
AR* BS*
AR* BS*
AR* BS*
AR* BS*
D
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DR* CS*
DS* CR*
DR* CS*
DS* CR*
F
ER* FS*
ES* FR*
ES* FR*
ER* FS*
Case 1 r r
Case 2 i r
Case 3 r i
Case 4 i i
E
O
S
S
S
P
P
P
was converted only to D (and vice versa) and B only to C (and vice versa). A similar experiment with SPAA and glutathione (g-Lglutamyl-L-cysteinylglycine, a single diastereomer) identified levels E and F (Fig. 2c,d), and we saw that A was converted only to F (and vice versa) and B only to E (and vice versa). In the complete reaction, with both L-penicillamine and glutathione, transitions between all seven components (P, A–F) could be observed (Fig. 2e,f).
P
A
B
A
B
A
B
A
B
D
C
D
C
D
C
D
C
E
F
E
F
E
F
E
F
Figure 3 | Analysis of the reaction network. Left: the two principal branches of the network. Right: Cases 1–4, four possible stereochemical pathways, based on retention (r) or inversion (i) at arsenic(III). The diastereomer A of the initial adduct PSAs(OH)Ar is given the relative stereochemistry R*, so diastereomer B is S*. These relative designations are used because the absolute configurations around arsenic(III) are unknown. O ↔ S, O ligand (–OH) substitution by an S nucleophile (or the reverse reaction); S ↔ S, S ligand substitution by an S nucleophile (thiol interchange). , the step is expected according to the proposed stereochemical pathway, but it is not observed by experiment; , not expected, but observed; , expected and observed; , not expected and not observed. Cases 1 and 2 are not permissible based, in part, on the finding that reactions A E and B F are not observed. Cases 3 and 4 are both permissible and both require inversion at arsenic(III) during thiol interchange (S ↔ S). Case 4 requires inversion and case 3 requires retention during OH substitution by an S nucleophile (O ↔ S). We favour the pathway depicted in Case 4, in which both O ligand and S ligand substitutions proceed with inversion.
Results Reaction of PSH with 4-sulfophenylarsonous acid. We have previously examined the reaction between a single thiol group (the side chain of a Cys residue) on the internal wall of an ahaemolysin (aHL) pore (PSH , Fig. 1a) and 4-sulfophenylarsonous acid (SPAA) to form an As–S bond (PSAs(OH)Ar)9. We were also able to observe stereochemical inversion at arsenic(III) in the bound adduct, which has a mean lifetime of 1 s at pH 8.4 (Fig. 1b)6. Because the protein is chiral, the adducts are diastereomers and physically distinguishable by ionic current recording. In the present work, we observe a far more complex reaction network within the nanoreactor that involves seven current levels. We examine the displacement of the hydroxyl group in PSAs(OH)Ar (where PSH contains a single cysteine at position 117) by a sulfur nucleophile (for example, R1SH) to form PSAs(SR1)Ar. We have also been able to observe the displacement of R1S in this adduct by a second sulfur nucleophile (for example, R2SH) to form PSAs(SR2)Ar and show by single-molecule reaction pathway analysis that this displacement occurs with inversion of configuration at the As atom. These reactions were examined by adding SPAA to the trans side of the lipid bilayer and the thiols to the cis side (Fig. 1a), so that they would not react with one another but instead migrate to the reaction site within the pore1. Assignment of the seven current levels. So that the entire reaction network could be examined, it was first necessary to assign the seven observed current levels to the different reactants. In support of our assignments, we show here short sections of far longer traces from various partial reactions. Reaction with SPAA (trans) alone identified the levels A and B arising from the two enantiomers of PSAs(OH)Ar (ref. 6). Reaction with SPAA (trans) and L-penicillamine (cis) yielded two additional levels, C and D, which must correspond to the two enantiomers of PSAs(SR1)Ar (Fig. 2a,b). At this point, the basis of our stereochemical analysis could be seen as it was apparent from long current traces that A
Examination of the full reaction network. We collected data from reactions that included all three soluble reactants—SPAA (trans), L-penicillamine (cis) and glutathione (cis)—involving thousands of transitions (Fig. 4a, Supplementary Table 1). Because it was not practicable to carry out a detailed analysis of the reaction pathway by manual techniques, we used the software packages Clampfit and QuB, which are normally used for the analysis of singlechannel recordings from ion channels. In addition to the allowed transitions identified in the partial reactions, we find that transitions D ↔ E (and not D ↔ F) and C ↔ F (and not C ↔ E) occur (Figs 3 and 4a, Supplementary Table 1). To check the validity of the computer analysis, some of the data were evaluated by hand, and a strong correspondence was found between the manual and computational methods (Supplementary section ‘Results’, Supplementary Table 2, Supplementary Figs 1, 2). Stereochemistry of the allowed reactions. Examination of the data in Fig. 4a suggests two pathways for PSH ↔ PSAs(OH)Ar ↔ PSAs(SR1)Ar ↔ PSAs(SR2)Ar: P ↔ A ↔ D ↔ E and P ↔ B ↔ C ↔ F, which must reflect stereochemically allowed transformations. Because P is a branch-point, the two pathways can be assembled (Fig. 3, left) and we can then examine how proposed stereochemistries for each step fit in with the remaining observed transitions that are not simple epimerizations at As (A ↔ B and so on). The absolute configurations of the reactants (for example, A versus B) are not known, so we have used relative configurations (R*, S*, Fig. 3). There are four possibilities (Fig. 3, Cases 1 to 4) and we focus on steps A E and A F. In two of the possibilities (Cases 1 and 4), all substitutions at As occur with the same stereochemistry; that is, both O ↔ S and S ↔ S substitutions go with retention of configuration (Case 1) or both go with inversion (Case 4). However, in Case 1, transition A E is expected according to the designated stereochemical pathway, but not observed, while A F is not expected, but is observed experimentally (Fig. 4a). By contrast, in Case 4, A E is not expected and not observed, while A F is expected and experimentally observed. Therefore, Case 1 is not viable. In the second pair of the four cases (Cases 2 and 3), Case 2 imparts inversion during O ↔ S substitution and retention during S ↔ S substitution, while in Case 3, O ↔ S takes place with retention and S ↔ S with inversion. In this pair, the experimental data fit only Case 3 (Figs 3 and 4a). We can therefore conclude that S ↔ S occurs with inversion (the viable Cases 3 and 4), but that the situation for O ↔ S is unresolved. We favour Case 4, in which both O ↔ S and S ↔ S occur with inversion. A matrix analysis can also be used to describe experimentally observed transitions in the reaction network (Supplementary Table 3, Supplementary Fig. 3), and this also rules out Cases 1 and 2 (see Supplementary section ‘Additional results’). The data also rule out the possibility that the substitution reactions occur with racemization at As. Single-molecule pathway analysis, as used here, suggests a reaction scheme (Fig. 5) based on Case 4 (Fig. 3), which requires that both O ↔ S and S ↔ S substitutions go with inversion. All 42 possible rates of interconversion for the seven species were computed with the QuB software package from transition frequencies obtained by single-channel recording (Fig. 4b). The rates of the inversions C ↔ D and E ↔ F for the species with penicillamine and glutathione ligands, respectively, are of the same order of magnitude as the rates
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0.033 ± 0.009 0.072 ± 0.018 0.026 ± 0.003
0.15 ± 0.02 0.12 ± 0.04
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0.024 ± 0.003
F
0.011 ± 0.002 0.019 ± 0.001
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0.11 ± 0.01
0.058 ± 0.035
C
6 99
0.12 ± 0.01 0.072 ± 0.042
D
F
0.20 ± 0.04 0.20 ± 0.07
0.37 ± 0.11 0.20 ± 0.02
0.33 ± 0.02 0.094 ± 0.016
0.030 ± 0.013
0.12 ± 0.04 0.11 ± 0.01
0.14 ± 0.04 0.075 ± 0.008
Figure 4 | Transition counts and computed reaction rates. The reaction components are 200 mM SPAA (trans) and 250 mM L-penicillamine and 250 mM glutathione (both cis) in 2 M KCl, 20 mM bis-tris propane, 100 mM EDTA, pH 8.5. Red and blue letters represent the different stereoisomers. Allowed (green) and forbidden (red) transitions are indicated based on the observed frequencies and their compatibility with the Case 4 reaction network. a, Transitions between current levels were counted by computer (Clampfit 10 software). The results from a typical experiment are shown. The 3,221 transitions are represented in a row to column fashion, that is, row D, column C is the number of transitions for D C. The transitions for two additional traces are presented in Supplementary Table 1. Of the 73 forbidden transitions assigned by computer, 49 were judged to be allowed after careful visual inspection of the individual transitions, and 20 were found to involve substates other than P or A to F ( Supplementary section ‘Additional results’). b, The transition rates of the allowed transitions (green) were computed with the QuB software package. Values are means of three trials+s.d. The transitions are represented in row to column fashion, that is, row D, column C is the rate for D C under the experimental conditions. P
HO As
SO3 SH
HO
R S
A
OH
As
B
S As R1 =
OH
H2N O
R = SO3
OH
R
S
As
L-Penicillamine
R
R1 S
R2 =
D
S
C
As S
R1
R
O
O N H
HO H2N
H N
OH O
O
Glutathione
S R E
S
As S
R2
R2
As
S F
R
Figure 5 | The complete reaction network. The reactions occur within an aHL protein nanopore (PSH), which contains a single cysteine residue that projects into the lumen. The reactants are SPAA and the thiols L-penicillamine (R1, maroon) and glutathione (R2, orange). Double green arrows indicate reversible reactions that are allowed (Figs 4a and 2) and occur with inversion according to the scheme in Fig. 3, Case 4. Double red arrows indicate reactions that are not observed and would occur with retention according to Fig. 3, Case 4. Red-green arrow combinations include the observed loss of As from PSH and the unlikely direct ‘termolecular’ formation of C, D, E or F, which is not observed (Figs 4a and 2). The double green arrows from P ↔ A and P ↔ B indicate the observed formation and breakdown of the initial PSAs(OH)Ar adduct. The absolute stereochemical configurations of the various adducts are unknown. However, if A is assigned the relative configuration R*, the relative configurations in Case 4 are: R*: A, C, E; S*: B, D, F (Fig. 3). 606
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of O ↔ S substitutions (transitions A ↔ D, B ↔ C for penicillamine and A ↔ F, B ↔ E for glutathione) and the S ↔ S thiol interchanges (C ↔ F and D ↔ E). In addition, the inversion A ↔ B, which does not involve a thiol nucleophile, is far slower. This suggests that, at the concentrations of penicillamine and glutathione used, the inversions C ↔ D and E ↔ F occur through thiol interchange and not by a unimolecular mechanism. Rate constants for the network reactions. The rate constants for the interconversions were calculated from current recordings carried out in the presence of all three reagents (SPAA, penicillamine and glutathione) or in the absence of one of the two thiols (Supplementary Table 4). All combinations of reactants gave similar values. It was assumed that the concentrations of reactants within the nanoreactor were the same as the concentrations in bulk solution1. Examination of the rate constants revealed several interesting points. For example, the rate constants for hydrolysis of arsenic(III) adducts of PSH with two S substituents (kDA , kDP, kCB , kCP, kFA , kFP, kEB , kEP) are much smaller than the hydrolysis rate constants of arsenic(III) adducts of PSH with a single S substituent (kAP and kBP) (Supplementary Table 4). This suggests that an arsenic(III) reagent can be temporarily ‘locked’ onto a cysteine residue by substitution of the hydroxyl group with a thiol ligand. We also note that the rate constants associated with enantiomeric adducts differ significantly. For example, kCP ¼ 0.033 s21 while kDP ¼ 0.072 s21 (Supplementary Table 4). This is because C and D are actually diastereomers (the protein provides a chiral environment6), so the rates are expected to diverge. Finally, in a reaction network, the products of the rate constants in any circular pathway in the clockwise and anticlockwise directions must be equal6,25. We have shown that this is valid for 13 different pathways (Supplementary Fig. 4).
Discussion The Walden inversion, described in 1896, demonstrated a simple conversion of one enantiomer (at a carbon centre) to the other, and offered critical experimental support for van’t Hoff’s concept of tetrahedral stereochemistry at carbon. Our work on arsenic stereochemistry is based on interwoven Walden cycles visualized at the single-molecule level. The tracking of seven reaction components by electrical recording showed that S ↔ S substitution at an arsenic(III) centre occurs with inversion of the relative stereochemical configuration. Such measurements made in a protein nanoreactor are not possible using ensemble approaches, because of the numerous species present. The nanoreactor approach is therefore a valuable means for the detailed analysis of complex dynamic chemical systems26.
Methods For full details see Supplementary Methods. Heteroheptameric staphylococcal aHL pores (PSH) were prepared with a single cysteine at position 117 in one of the seven subunits9. SPAA was prepared as described in ref. 9. Solutions of thiols were made fresh each day. Single-channel current recordings were performed in a planar bilayer apparatus. The bilayer was formed from 1,2-diphytanoyl-sn-glycero-3phosphocholine. Steps in the ionic current reflecting reversible chemistry were recorded at þ50 mV with an Axopatch 200B patch-clamp amplifier (Axon Instruments). The signal was filtered with a low-pass Bessel filter (80 dB/dec) with a corner frequency of 2 kHz and sampled at 10 kHz with a DigiData 1200 A/D converter (Axon Instruments). To analyse the current traces we used Clampfit (version 10.0, Axon Instruments) and QuB software (http://www.qub.buffalo.edu).
4. Shin, S-H. & Bayley, H. Stepwise growth of a single polymer chain. J. Am. Chem. Soc. 127, 10462–10463 (2005). 5. Loudwig, S. & Bayley, H. Photoisomerization of an individual azobenzene molecule in water: an on–off switch triggered by light at a fixed wavelength. J. Am. Chem. Soc. 128, 12404–12405 (2006). 6. Shin, S-H., Steffensen, M. B., Claridge, T. D. W. & Bayley, H. Formation of a chiral center and pyramidal inversion at the single-molecule level. Angew. Chem. Int. Ed. 46, 7412–7416 (2007). 7. Lu, S., Li, W-W., Rotem, D., Mikhailova, E. & Bayley, H. A primary hydrogen– deuterium isotope effect observed at the single molecule level. Nature Chem. 2, 921–928 (2010). 8. Wu, H. C. & Bayley, H. Single-molecule detection of nitrogen mustards by covalent reaction within a protein nanopore. J. Am. Chem. Soc. 130, 6813–6819 (2008). 9. Shin, S-H., Luchian, T., Cheley, S., Braha, O. & Bayley, H. Kinetics of a reversible covalent-bond forming reaction observed at the single molecule level. Angew. Chem. Int. Ed. 41, 3707–3709 (2002). 10. Shen, S., Li, X-F., Cullen, W. R., Weinfeld, M. & Le, X. C. Arsenic binding to proteins. Chem. Rev. 113, 7769–7792 (2013). 11. Liu, J. X., Zhou, G. B., Chen, S. J. & Chen, Z. Arsenic compounds: revived ancient remedies in the fight against human malignancies. Curr. Opin. Chem. Biol. 16, 92–98 (2012). 12. Argos, M., Ahsan, H. & Graziano, J. H. Arsenic and human health: epidemiologic progress and public health implications. Rev. Environ. Health 27, 191–195 (2012). 13. Messens, J. & Silver, S. Arsenate reduction: thiol cascade chemistry with convergent evolution. J. Mol. Biol. 362, 1–17 (2006). 14. Adams, S. R. & Tsien, R. Y. Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins. Nature Protoc. 3, 1527–1534 (2008). 15. Lloyd, N. C., Morgan, H. W., Nicholson, B. K. & Ronimus, R. S. The composition of Ehrlich’s salvarsan: resolution of a century-old debate. Angew. Chem. Int. Ed. 44, 941–944 (2005). 16. Burchmore, R. J., Ogbunude, P. O., Enanga, B. & Barrett, M. P. Chemotherapy of human African trypanosomiasis. Curr. Pharm. Des. 8, 256–267 (2002). 17. Zhang, X. W. et al. Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML. Science 328, 240–243 (2010). 18. Jennewein, M. et al. Vascular imaging of solid tumors in rats with a radioactive arsenic-labeled antibody that binds exposed phosphatidylserine. Clin. Cancer Res. 14, 1377–1385 (2008). 19. Goldman, M. & Dacre, J. C. Lewisite: its chemistry, toxicology, and biological effects. Rev. Environ. Contam. Toxicol. 110, 75–115 (1989). 20. Henriksson, J., Johannisson, A., Bergqvist, P. A. & Norrgren, L. The toxicity of organoarsenic-based warfare agents: in vitro and in vivo studies. Arch. Environ. Contam. Toxicol. 30, 213–219 (1996). 21. Senkler, G. H. & Mislow, K. The barrier to pyramidal inversion in ethylmethylphenylarsine. J. Am. Chem. Soc. 94, 291 (1972). 22. Baechler, R. D., Casey, J. P., Cook, R. J., Senkler, G. H. & Mislow, K. Effect of ligand electronegativity on the inversion barriers of arsines. J. Am. Chem. Soc. 94, 2859–2861 (1972). 23. Dill, K., Huang, L., Bearden, D. W., McGown, E. L. & O’Connor, R. J. Activation energies and formation rate constants for arsenical-antidote adducts as determined by dynamic NMR spectroscopy. Chem. Res. Toxicol. 4, 295–299 (1991). 24. Serves, S. V., Charalambidis, Y. C., Sotiropoulos, D. N. & Ioannou, P. V. Reaction of arsenic(III) oxide, arsenous and arsenic acids with thiols. Phosphorus Sulfur Silicon 105, 109–116 (1995). 25. Richard, E. A. & Miller, C. Steady-state coupling of ion-channel conformations to a transmembrane ion gradient. Science 247, 1208–1210 (1990). 26. Hunt, R. A. & Otto, S. Dynamic combinatorial libraries: new opportunities in systems chemistry. Chem. Commun. 47, 847–858 (2011).
Acknowledgements This work was supported by the Medical Research Council and a European Research Council advanced grant. H.B. was the holder of a Royal Society–Wolfson Research Merit Award. M.B.S. was the holder of a Ruth L. Kirschstein NIH Postdoctoral Fellowship (F32L078236).
Author contributions
Received 25 October 2013; accepted 9 April 2014; published online 18 May 2014
M.B.S. and D.R. contributed equally to this work. M.B.S. and H.B. designed the research. M.B.S. performed the experimental work. M.B.S., D.R. and H.B. analysed data and wrote the manuscript.
References
Additional information
1. Bayley, H., Luchian, T., Shin, S-H. & Steffensen, M. B. in Single Molecules and Nanotechnology (eds Rigler, R. & Vogel, H.) Ch. 10, 251–277 (Springer, 2008). 2. Luchian, T., Shin, S-H. & Bayley, H. Kinetics of a three-step reaction observed at the single molecule level. Angew. Chem. Int. Ed. 42, 1926–1929 (2003). 3. Luchian, T., Shin, S-H. & Bayley, H. Single-molecule covalent chemistry with spatially separated reactants. Angew. Chem. Int. Ed. 42, 3766–3771 (2003).
Supplementary information is available in the online version of the paper. Reprints and permissions information is available online at www.nature.com/reprints. Correspondence and requests for materials should be addressed to H.B.
Competing financial interests The authors declare no competing financial interests.
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