A primary hydrogen–deuterium isotope effect observed at the single-molecule level Siran Lu † , Wen-Wu Li † , Dvir Rotem, Ellina Mikhailova and Hagan Bayley * The covalent chemistry of reactants tethered within a single protein pore can be monitored by observing the time-dependence of ionic current flow through the pore, which responds to bond making and breaking in individual reactant molecules. Here we use this ‘nanoreactor’ approach to examine the reaction of a quinone with a thiol to form a substituted hydroquinone by reductive 1,4-Michael addition. Remarkably, a primary hydrogen–deuterium isotope effect is readily detected at the single- molecule level during prototropic rearrangement of an initial adduct. The observation of individual reaction intermediates allows the measurement of an isotope effect whether or not the step involved is rate limiting, which would not be the case in an ensemble measurement. S ingle-molecule studies in solution, usually using various forms of force measurement or fluorescence detection, continue to improve in sensitivity and time resolution and are providing a new outlook on many dynamic processes in chemistry and physics that cannot be attained through ensemble studies 1–4 . For example, single-molecule approaches can reveal rare reaction path- ways or illuminate fast reaction steps embedded between slow steps. In macromolecules, such as enzymes, dynamic and static disorder may be apparent. In biology, the output of single-molecule experiments over the last decade has been explosive, increasing our understanding of protein folding 5 , catalysis by ribozymes 6 and enzymes 3,7,8 , the mechanisms of molecular motors 9,10 and how molecules and assemblies function within cells 11,12 . In contrast to the abundance of work on biological macromolecules, far fewer single-molecule studies have been done on the chemistry of small molecules in solution. Under high vacuum, scanning tunnelling microscopy (STM) has continued to produce valuable findings 13–15 , but the tools required to observe bond making and breaking in solution— such as dyes, beads, nanoparticles and scanning probe tips—are highly perturbing. Nevertheless, significant progress has been made. For example, the breaking of Si–C and S–Au bonds subjected to large forces has been observed 16 . More recently, force spectroscopy has been used to examine details of the chemical cleavage of disulfide bonds 17 . Fluorogenic substrates have been used to monitor esterolysis and transesterification on a Li þ /Al 3þ layered double hydroxide catalyst 18 and a reduction catalysed by Au nanoparticles 19 . Our laboratory has developed an alternative approach for exam- ining the single-molecule chemistry of small molecules in aqueous solutions 20 . The reactants are tethered to the wall of a nanoreactor, which comprises an engineered protein pore. The pore is located in a planar bilayer apparatus so that the current carried through the pore by ions (such as K þ and Cl 2 ) in an applied potential can be monitored (Fig. 1a, Supplementary Fig. S1). Alterations in current flow, caused by individual bond-making and bond-breaking events within the pore, can be monitored often with sub-millisecond time resolution. Furthermore, no bulky tags are required to follow the chemistry and it is possible to use high concentrations of reagents, which is problematic in single-molecule fluorescence experiments. A wide variety of covalent reactions of small molecules has been observed by this approach including the making and breaking of S–As bonds 21 , the photodeprotection of 2-nitrobenzyl carbamates 22 , the cleavage of disulfide bonds 23 , a step-by-step polymerization 24 , and photochemical 25 and thermal isomeriza- tions 26 . The potential of the approach for sensing chemically reac- tive analytes has also been evaluated 27 . In the present work, we show that the nanoreactor approach can be used to measure a primary hydrogen–deuterium isotope effect during C–H(D) bond cleavage. Furthermore, we show that, because we are observing individual reactions, the isotope effect can be observed under conditions when the cleavage step is not rate limiting. A single-molecule isotope effect observed by fluor- escence has been reported when deuterated NADPH is used in the enzymatic reaction carried out by dihydrofolate reductase and may represent a kinetic isotope effect for hydride transfer 28 . The present work is the first example of a kinetic isotope effect for a small molecule examined in solution at the single- molecule level. We examined the reaction of a quinone with a thiol, in which the final product is a thioether hydroquinone 29,30 . The hydroquinone is formed by a reductive 1,4-Michael addition, which occurs through a tetrahedral intermediate 30 . Similar chemistry has been observed for thiosulfate and a quinone 31 and benzenesulfinic acid and a quinone 32,33 ; a kinetic isotope effect was observed for the benzene- sulfinic acid reaction in bulk solution 32,33 . Specifically, we have examined the reaction of 2,3-dimethoxy-5-methyl-1,4-benzoqui- none (ubiquinone-0, UQ, 1) in which there is one available position for reaction with a thiol. The thiol was the side chain of a Cys residue positioned within the lumen of an a-haemolysin (aHL) protein pore. Various quinones have been used previously to covalently modify proteins at Cys residues: for example, to construct artificial photosynthetic systems 34,35 . The nanoreactor used here was an aHL heteroheptamer consisting of one copy of the single-cysteine mutant T117C and six copies of the wild-type (WT) protein (WT) 6 (T117C) 1 (P SH , Fig. 1a) 21 . In separate experiments, the pore was reacted with UQ (1) and 2,3-dimethoxy-5-methyl-6-bromo-1,4-benzoquinone Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK; † These authors contributed equally to this work. *e-mail: hagan.bayley@chem.ox.ac.uk ARTICLES PUBLISHED ONLINE: 12 SEPTEMBER 2010 | DOI: 10.1038/NCHEM.821 NATURE CHEMISTRY | VOL 2 | NOVEMBER 2010 | www.nature.com/naturechemistry 921 © 2010 Macmillan Publishers Limited. All rights reserved.