Dynamic Interconversions of Single Molecules Probed by Recognition Tunneling at Cucurbit[7]uril‐Functionalized Supramolecular Junctions

Abstract We introduce a versatile recognition tunneling technique using doubly cucurbit[7]uril‐functionalized electrodes to form supramolecular junctions that capture analytes dynamically by host–guest complexation. This results in characteristic changes in their single‐molecule conductance. For structurally related drug molecules (camptothecin, sanguinarine, chelerythrine, and berberine) and mixtures thereof, we observed distinct current switching signals related to their intrinsic conductance properties as well as pH‐dependent effects which can be traced back to their different states (protonated versus neutral). The conductance variation of a single molecule with pH shows a sigmoidal distribution, allowing us to extract a pK a value for reversible protonation, which is consistent with the reported macroscopic results. The new electronic method allows the characterization of unmodified drug molecules and showcases the transfer of dynamic supramolecular chemistry principles to single molecules.


S1. Recognition tunneling method
Preparation of test solutions. Cucurbit [7]uril (CB7) was prepared according to the reported procedure 1 . Camptothecin (CPT), sanguinarine (SA), chelerythrine (CHE), and berberine (BE) were purchased from Sigma Aldrich and used without further purification. Owing to the moderate solubility of CPT, SA and CHE, stock solutions of 5 mM were prepared in DMSO in order to allow the titration experiments. 0.5 mL of the CPT stock solution was added to Milli-Q water at the desired pH (adjusted with HClaq and NaOHaq) and the solution was subsequently sonicated for 10 min to ensure rapid mixing. Test solutions of the other analytes were prepared accordingly. Tunneling measurements. The preparation and chemical modification of the STM tip and the substrate has been described in our previous work. 2 The conductance measurements were performed at ambient temperature on a Keysight 6500 instrument. Before the conductance experiment, the distance between STM tip and substrate was adjusted to maintain a gap of set-point = 4 pA and the instrument was warmed up for 2 hours to stabilize the tip. All tunneling traces were collected at a bias of 0.2 V with a sampling rate of 10 kHz. A blank control experiment (without CB7 reader molecules) was performed with the HDPE-coated tip for a leakage test. The low-noise level in the tunneling traces at GB = 50 pS (Noise = 2 pA) and 20 pS (Noise = 1 pA) indicated that the insulation probe is capable of single-molecule electronic detection ( Figure S1a). In addition, we carried out control experiments in the STM studies, as shown in Figure S1 b-d. No current switching signals are generated when the electrodes are not functionalized (b) or only one electrode is functionalized (c), or when no target molecules are present (d). The current jump signals only appear when both electrodes are functionalized with CB7 and the target molecule is present in solution. Statistical analysis of the current spikes was automated by using home-built Labview programs as described previously 2 . The conductance histograms comprised current spikes extracted from thousands of individual I(t) traces and the error bars were calculated from the full width at half-maximum (FWHM) of the fitting peaks.

S2. Conductance measurements in dependence on CPT concentration
As shown in Figure S2, the conductance peaks at GB = 50 pS remain unchanged, while the proportion of molecular junction events in GA (CB7---CPTH + ---CB7) and GW (CB7---CB7) increases with increasing concentration of CPT. The area of signal counts of GA and GW are integrated respectively and compared, the ratios of the two areas are listed in Table S1.   Figure S3. Typical current-time traces of CB7---CPT---CB7 junctions obtained from pH 2 to pH 12.

S4. Two-dimensional histograms of CB7---CPT---CB7 junctions at different pH values
The 2D histograms were constructed by overlaying the selected current spikes from hundreds of individual I(t) traces in each experiment over a wide pH range, using bins of 5 ms along the time axis and 10 0.05 G0 bins along the conductance axis. We plotted the 2D histogram by setting the origin of the time axis to the start point at which the current suddenly increased and the end until it came back to the set-point current. As can be seen in Figure S4, the counts of constructed histograms decrease with increasing pH value, indicating that the CB7---CPT---CB7 supramolecular junction depletes at high pH. As the solution became less acidic from pH 2 to pH 6, the initially dominant protonated lactone form of CPT gradually converted into the neutral lactone form which bound less strongly to the junction compared to the protonated form. Besides, the proportion of CPT carboxylate form (which interacted only to a negligible extent with CB7) increased concomitantly above pH 6, as observed from the UV-vis absorption and fluorescence spectra (see Section S6), which further minimized the formation probability of the supramolecular junctions. In contrast to the reduction of the events probability, the absolute conductance values increased gradually from pH 2.0 to 7.8, as marked by the blue lines in Figure S4 a-h.

S5. Conductance distribution of CB7---CPT---CB7 junctions at different pH values
The conductance values at different pH were extracted from the 2D histogram and further constructed 1D conductance histograms (logarithmic bin 200 bins along the conductance axis). Overlaid 1D conductance histograms for CB7---CPT---CB7 junction over pH range of 2.0-7.8 are shown in Figure S5. The conductance histograms suggest that the conductance increases slowly from pH 2.0 to 4.3 and rises up rapidly from pH 6.4 to 7.8.  shift the pKa of CPT to 6.8 and 6.2, respectively, at the excited and the ground state. 4 It was assumed that by forming a 1:2 complex with CB7, the protonation of the quinoline nitrogen became easier, possibly because the electron density is enriched by trapping between the portals of two CB7 hosts. 4 As the pH rises, the carboxylate form of CPT gradually takes over the lactone form, and, as a result, the pharmacological activity of CPT is diminished. Above pH 8, CPT exists mostly in its carboxylate form. 5 Dong et al. studied the binding behavior of CPT and CB7 at pH 2, 6 and confirmed the formation of a 1:2 complex, as had been observed by Hazra near neutral pH. However, experimental structural information on the 1:2 complex remains elusive. Nonetheless, the 1:1 complex prevails in solution if CB7 and CPT are employed in low concentrations. This allowed us to use a 1:1 binding model in the fitting, the obtained binding constant is 8.4 × 10 5 M -1 and 7.9 × 10 5 M -1 , respectively, from UV-vis and fluorescence titration. The increasing absorption at 407 nm as well as emission at 515 nm refers to the growing fraction of the protonated form, which is induced by encapsulation within CB7 ( Figure S6). 1 H NMR studies of CPT and CB7 in D2O revealed an upfield shift of the quinoline ring protons, while the protons on the other rings shifted downfield, indicating a preferential immersion of the quinoline ring inside the CB7 cavity while the other part of the drug molecule remains positioned outside the cavity 6 . This 1 H NMR shift pattern corresponds better to a partial 1:1 inclusion complex than to a 2:1 inclusion complex, such that the second CB7 macrocycle interacts likely very weakly, and more superficially with CPT. At basic pH values, we found no shift of the 1 H NMR peaks of CPT upon addition of 2 equiv. of CB7, suggesting the absence of detectable binding; this is in line with the spectroscopic titration results (see Figures S6 and S7). We were not able to measure the 1 H NMR in acidic D2O, because the solubility of CPT was too low in this medium.

S7. DFT calculations for CB7---CPT---CB7 junctions
Molecular geometry optimizations were carried out with the Gaussian 09 program by using dispersion-corrected density functional theory (DFT-D3) and the B3LYP functional along with the 6-31G(d,p) basis set. Grimme's D3 empirical dispersion correction with Becke-Johnson damping (GD3BJ) was used. Vibrational frequency analyses were performed for all geometry-optimized structures, confirming the absence of imaginary frequencies, and therefore identifying them as energy minima. The host-guest complex structures were optimized between two gold electrodes to form molecular devices in the Atomistix-Tool-Kit software. We applied DFT methods within the generalized gradient approximation (GGA) by using the Perdew-Burke-Ernzerhof (PBE) exchange-correlation function to simulate the optimized structures. The single zeta polarized level was used for Au atoms and the double zeta polarized level was adopted for C, H, O, N and Cl atoms based on the linear combination of atomic orbitals (LCAO) basis set approach.
The Au electrodes were settled to a 6×6×2 supercell. The isolated organic complexes were optimized and the complexes were inserted between the left and right Au electrodes, and the resulting supramolecular junctions were relaxed until the total free energy reached a minimum. During the relaxation, the force tolerance was set to 0.5 eV/Å. It is worth noting that we placed one adjacent chloride atom in the CB7---CPTH + ---CB7 system to simulate the effect of protonation (HCl) while maintaining an overall neutral assembly.
First principles calculations were carried out to investigate electronic transport properties. In the series of calculations, the exchange-correlation potential was approximated within the GGA-PBE functional for exchange and correlation effects. A mesh cutoff energy of 75 Hartree and a (1,1,89) k-point mesh within the Monkhorst-Pack scheme were utilized. The single-ζ polarization basis set was used for the gold atoms and the double-ζ basis set with polarization functions was used for all other atoms. According to the Landauer formalism, the conductance G of a supramolecular junction can be calculated, where e is the electron charge, h is Planck's constant, and Tn is the transmission coefficient of the individual transport channels which describes how effective a molecule performed in scattering an incoming electron from the right lead into the left lead. In this way, the conductance values of the molecule under zero bias voltage were obtained. As shown in Figure S9, the blue and red arrows show the forward and backward electron transport pathways in the molecular system with the thickness of the arrow representing the absolute transmission magnitudes. The main transmission pathways for both systems are along Au→left CB7→CPT→Au. There is almost no arrow pointing to or from the CB7 molecule that encapsulates the quinoline ring; instead, this macrocycle serves to facilitate direct CPT---Au transmission through the formation of inclusion complexes. Therefore, the computational modelling results account for the experimental observation that the recognition tunneling signals are "switched on" upon addition of CPT, because the CPT is (expectedly, due to its aromatic character) more conductive than the aliphatic macrocyclic host. Secondly, and again in agreement with the experiment, the electron transmission modelling predicts a higher conductance for the neutral junction than for the protonated one, as indicated by the arrows connecting the right Au surface to the junction. This effect is fully consistent with the deeper immersion of the neutral quinoline ring (see Figure 2c), which places it more closely to the Au surface (3.25 versus 4.41 Å, see Figure S9). Counterintuitively, even though the neutral quinoline ring is more deeply immersed into CB7, this does not increase the distance of the lactone group to the second Au surface, but rather decreases it (10.14 and 10.64 Å for the Au-O and Au-C distances for CPT, shorter than in the case of CPTH+ with 10.71 and 10.77 Å), as a consequence of different quinoline protonation-induced tilting angles (also see Figure 2c); this shorter bridging distance for the neutral form may be a second contributor to the increased conductivity.

S10. Repeated release and binding studies of CPT at the supramolecular junctions
We conducted repeated release/binding studies by alternatively adding CPT and salt in the STM measurement cell and monitoring the tunneling current changes, as shown in Fig. S18.
The STM gap was set to an initial current of 4 pA (GB= 20 pS) and both electrodes were functionalized with CB7. In the first 10 minutes, no current switching signals were observed in the absence of analyte. Once 0.5 mM CPT was added to the system, dramatic jumps occurred in the current and signal frequency (red points) that saturated after ca. 10 minutes.
Subsequently, 0.1 M Ca 2+ was added to the solution and an immediate reduction of the signal frequency (blue points) is seen in the time-resolved signal frequency change, suggesting the effective release of CPT from the supramolecular junction by competitive CB7 portal binding.
Subsequently, we rinsed the electrodes with DI water and repeated the measurements by adding CPT and salt alternatively for another two cycles. The repeated release-binding results demonstrate the reversibility of the processes and robustness of the supramolecular junctions.

S11. Summary of analyte affinities to CB7 and their molecular conductance values at the supramolecular junctions
The conductance values of all studied molecules at different pH are summarized in Table S3.
The GA values of SA and CHE are the same, within error, as might be expected from their closely related structures, but both display a slightly lower conductance than BE. In contrast to CPT, SA and CHE do not exhibit a change in conductance upon changing from acidic to neutral pH.