Semiquantitative Submolecular Barrier Height Measurements of 4-Aza

Jan 20, 2016 - Florida Institute of Technology, Department of Chemistry, 150 West University Boulevard, Melbourne, Florida 32901, United States. ‡. ...
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Semiquantitative Submolecular Barrier Height Measurements of 4‑Aza-8-fluorotryptanthrin Monolayers on HOPG: Orbital-Mediated Tunneling Krishnan Sriraman,§ Raymond J. Terryn, III,† Xixuan Guo,§ Mark J. Novak,‡ J. Clayton Baum,§ and Joel A. Olson*,§ §

Florida Institute of Technology, Department of Chemistry, 150 West University Boulevard, Melbourne, Florida 32901, United States Department of Chemistry and Applied Biological Sciences, South Dakota School of Mines and Technology, 501 E. Saint Joseph Street, Rapid City, South Dakota 57701, United States

‡

ABSTRACT: 4-Aza-8-fluoroindolo[2,1-b]quinazoline-6,12-dione (4-aza-8-fluorotryptanthrin) was imaged via scanning tunneling microscopy and barrier height tunneling spectroscopy at the graphite−solution interface. Submolecular resolution was achieved for the apparent barrier height (ϕap) images and revealed that ϕap displays significant variation over the area of the molecule. Topographic effects and electrostatic potential effects were found to be insufficient to explain the behavior of the measured ϕap variations. Specifically, where barrier height images were collected over regions of large molecular orbital (MO) wave function amplitudes, the measured ϕap did not agree with either affecting phenomenon. The authors attribute the disparity to the fact that electron tunneling is orbital-mediated for these systems. Thus, the ϕap is dominated by the spatial disposition of the mediating MO in locations of large wave function amplitude. This hypothesis is corroborated by excellent agreement with a computational treatment that takes into account only the spatial disposition of the MO wave function. It is possible that these measurements could be used to predict molecular behaviors, especially when a redox event or electron coordination is involved.

1. INTRODUCTION Tryptanthrin (indolo[2,1-b]quinazoline-6,12-dione 1, Figure 1) is a weakly basic alkaloid that occurs naturally in a number of

Insight into a molecule’s pharmacological and toxicological mechanisms of action may be gained by analyzing its electronic and geometric properties at the molecular and submolecular levels, and how these may affect interaction with the surroundings. More specifically, substrate−receptor interactions would require favorable interactions of the frontier molecular orbitals (HOMOs and LUMOs) as an initial trigger event. However, other important factors including sterics and the electronic charge density over the entire molecule cannot be excluded when elucidating a potential mechanism. A possible approach to gain insight into tryptanthrins’ mechanism(s) of action would be to generate detailed submolecular energy barrier maps of their frontier molecular orbitals using scanning tunneling spectroscopy (STS)12 as an ancillary to scanning tunneling microscopy (STM). Although scanning tunneling microscopy is commonly used to topographically image molecules,13,14 the data collected give very limited information about the electronic properties of the molecules. The STS technique aids in obtaining detailed information about energetic barriers of various surfaces. Local barrier height data can be measured by means of an ac synchronous detection of the tunneling current under feedback control of the STM system, so that barrier height data can be obtained

Figure 1. Structures of tryptanthrin (1) and 4-aza-8-fluorotryptanthrin (2).

different plant species1 whose extracts have been historically used as traditional remedies to treat various fungal infections2 and to reduce inflammation and fever.3 More recently, this compound and its analogues have been shown to possess significant activity against a variety of pathogenic bacteria,4−6 eukaryotic parasites such as Plasmodium falciparum,7,8 Leishmania. donovani,9 and Trypanosoma brucei10 and have generated considerable interest as potential therapeutic agents against these neglected tropical diseases along with tuberculosis11 due to their broad spectrum of activities, stability, and ease of synthesis. Although inhibitory concentration (IC50) values of some tryptanthrin analogues against the aforementioned parasites have been reported to be in the low nanogram/ milliliter range, very little is known about their mechanisms of action at the cellular and molecular levels. © 2016 American Chemical Society

Received: December 7, 2015 Revised: January 9, 2016 Published: January 20, 2016 3420

DOI: 10.1021/acs.jpcc.5b11959 J. Phys. Chem. C 2016, 120, 3420−3427

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topographic contribution to variations in the apparent barrier height. This equation is expanded upon and refined below, based on the results reported here. Although barrier height images of organic molecules have been collected, little has been reported about the actual values of the barrier height energies, following a trend in which most of the published data seemed to indicate anomalously low or high values of measured barrier height energies as compared to macroscopically measured surface work functions.25,27 One possible reason for these anomalous apparent barrier height energies is due to possible surface contaminants while scanning under ambient conditions. Also, as mentioned above, measured barrier heights depend on the local charge variations and the image potential,28,29 and due to these dependencies, the values measured are not true barrier heights but instead are defined as apparent barrier height (ϕap) energies. This is why the apparent barrier height (ϕap) term is used here to present our data. For many adlayer systems the electron tunneling is mediated by molecular orbitals (MOs) that exist in the adlayer itself, rather than exclusively with the substrate states, as for the case of stearic acid mentioned above.30−38 These sorts of adlayers typically consist of conjugated moieties, and in particular cyclic aromatic species, since one effect of conjugation is an energetic relaxation of the states such that they may fall within the energy ranges typically measured in STM tunneling. Additionally, adsorption onto HOPG further relaxes the energies of aromatic systems.39 Indeed, this is likely why good MO images are possible at STM bias voltages that seem anomalously too low to access these electron states.40−42 While the presence of tunneling MO states has been thoroughly investigated via I−V tunneling spectroscopy, the effect of MO-mediated tunneling on ϕap measurements is not well reported in the literature, especially for adlayers at the graphite−solution interface. As a continuation of efforts to image and study the electronic and geometric properties of tryptanthrin analogues utilizing STM,40−42 we present herein ϕap and topography data on 4aza-8-fluorotryptanthirn (2, Figure 1). Two reasons in particular led to compound 2 being chosen for this study. First, we are able to obtain good submolecular-resolution images of the frontier molecular orbitals. Submolecular resolution is critical since the topographic images must be utilized as a spatial reference for the direct comparative analysis with ϕap images that are simultaneously generated. Second, this compound shows a low IC50 (600 ng/mL) and a low LD50 (400 ng/mL) against E. coli,5 as well as a low IC50 versus the malaria pathogen P. falciparum, 1.53 and 0.84 ng/mL versus the D-6 and W-2 strains, respectively,7,8 but is a poor DNA intercalator compared to other tryptanthrin analogues.5,43 Ultimately, correlation of observed biological activities with barrier height energetics of a class of molecules would suggest a mechanism of action based on redox events and/or covalent modification. Potential mechanisms of action such as intercalation into DNA would likely not be dependent on the barrier heights associated with the individual lobes of a tryptanthrin’s frontier molecular orbitals.43,44 As such we chose a tryptanthrin analogue whose mechanism would most likely be redox in nature.

simultaneously (via a lock-in amplifier) with a topographic image.15 The spatial barrier height data can be considered to correspond to an energy barrier map and can be further quantified in terms of apparent barrier energy, ϕap, in electron volts, according to eq 1. ϕap =

⎛ dlnI ⎞2 ⎛ dlnI ⎞2 ℏ2 ⎟ = 0.95 × ⎜ ⎟ ×⎜ ⎝ dz ⎠ 8m ⎝ dz ⎠

(1)

In eq 1, ℏ is the reduced Planck’s constant and m is the electron mass; the coefficient thus reduces to the value of 0.95 if dZ is in Å, and the numerical result is in eV. The lock-in amplifier provides dI/dZ, from which the logarithm can be determined and inserted into eq 1. The result, ϕap, is termed the “apparent barrier energy” rather than just the barrier energy for reasons described below. Historically the STS technique has been used mainly to measure the barrier height energies of metals16−19 and semiconductor surfaces.20 These barrier height measurements can provide chemically specific information about a surface, local charge densities, work functions, and band bending effects on semiconductor surfaces.21 The STS technique has also been applied to measure barrier heights of organic molecules adsorbed onto various substrates such as highly oriented pyrolytic graphite (HOPG) and metal surfaces.22−24 However, reports in the literature are relatively sparse, particularly for systems at the graphite−solution interface. We have recently reported on detailed ϕap measurements of stearic acid adlayers from 1-phenyloctane onto HOPG.25 This study is noted here because it reported the observation of two mechanisms responsible for spatial variations of barrier height measurements over an adlayer surface. It should be mentioned that STM tunneling into or out of these types of adsorbates, e.g., alkane acids, is not orbital mediated since the molecular states fall well outside of the energy range containing the tunneling states. Thus, imaging of stearic acid monolayers involves tunneling only with the electron states of the HOPG substrate. The first observed mechanism is a result of charge separation within the adlayer. Specifically, where polar moieties are present, the resulting charge separation affects the substrate states such that a region of negative electrostatic potential (ESP) enhances the native surface dipole and displays a region of increased ϕap. Conversely, a region of positive ESP opposes the native surface dipole, causing a decrease in ϕap. These effects are typically manifested in the corresponding topographic images as well, since an increase in ϕap corresponds to a retraction of the substrate states toward the surface, which appears as a topographic depression, and vice versa. This effect is observed specifically for regions of a surface where tunneling is predominantly between the tip and the substrate states. The second mechanism is a manifestation of the topography itself and results in the appearance of the topography embedded into variations in the ϕap. The topographic contribution is universally present and is a factor in all such barrier energy maps.25,26 This is one reason for the difficulty in interpreting ϕap images. In our recent work, we have combined these phenomena into a simple descriptive mathematical relationship that takes these effects into account, eq 2:25 ϕap = Tϕ(qs)

2. EXPERIMENTAL SECTION 4-Aza-8-fluorotryptanthrin (2) was synthesized in a single step using commercially available 3-azaisatoic anhydride (Starks Associates, Buffalo, NY) and 5-fluoroisatin (Aldrich, Milwaukee, WI) according to the protocol of Mitscher et al.45 A

(2)

Here ϕ(qs) is the barrier height associated with the substrate states that is a function of the surface charge, and T is the 3421

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The Journal of Physical Chemistry C saturated solution for STM imaging was prepared by dissolving 20 mg of 2 in 2.0 mL of 1-phenyloctane solvent (98%, Aldrich, Milwaukee, WI). A drop of this solution was then placed on a freshly cleaved HOPG surface (ZYA grade, Momentive Performance Materials, New Smyrna Beach, FL) resulting in monolayer formation. The tip was prepared mechanically by cutting a 0.25 mm Pt−Ir wire (85%:15% Alfa Aeaser, Ward Hill, MA). Imaging was performed by immersing the tip in the solution under ambient conditions. An RHK SPM1000 instrument with a PicoSPM scan head was used for collecting the images. The conditions applied for imaging were a sample bias voltage of +0.80 V and a set point of 0.1 nA. Images were comprised of 128 × 128 data points collected over an imaging area of 7 × 7 nm. All images were corrected for drift by comparing sequential alternating slow-scan direction images and adjusting the vertical scale and horizontal skew accordingly. Barrier height images were collected simultaneously with the topographic images by applying a sine wave signal (Stanford Research systems DS360 function generator model) of 10 mV amplitude and 6 kHz frequency to the modulated input of the Z-piezo. Actual applied modulation was 0.1937 Å. The modulated tunneling current signal was measured using a lock-in amplifier (Stanford Research Systems model SR530). The lock-in amplifier output was then used to generate dI/dz images which differ from actual barrier height images in that the lock-in amplifier output has not been converted into an actual barrier energy numerical value (this is noted where appropriate); however, the dI/dz images correlate to actual barrier heights in that an increase in one corresponds to an increase in the other. The numerical values of apparent barrier height energies were calculated using eq 1. B3LYP density functional theory (DFT) calculations with a 6-31G* basis set46,47 were performed utilizing Spartan’14 software.48

Figure 2. Topography and corresponding dI/dz images. (a) A topographic STM image of an adlayer of 4-aza-8-fluorotryptanthrin on HOPG. Also, a single adlayer molecule is indicated with a box. (b) A dI/dz image of the same adlayer as the topographic image shown in (a); the two images correspond on a pixel-by-pixel basis. The box contains the same single molecule as that for the topographic image in (a). The images were collected at a sample bias of +0.80 V. The scale bars correspond to 2 nm distance.

adsorbed systems, with adlayers at the metal−vacuum interface typically displaying lower work function values than for bare surfaces.49,50 Early reports of ϕap measurements of adlayers adsorbed onto HOPG from organic solvent failed to report quantitative values for ϕap at all.23,24 More recently, the authors have reported ϕap values for adlayers of stearic acid in 1phenyloctane that were obviously anomalously elevated, reaching values as high as 7.2 eV, with significant variation between experiments.25 Therefore, in order to be able to draw any semiquantitative internal comparisons, the authors have typically utilized normalized ϕap values where the average apparent barrier energy is the normalization factor. Consistent with previous investigations of this class of molecules, for the given imaging conditions (sample bias = +0.80 V) it is expected that the images will correspond to the LUMO of the molecule, shown in Figure 3a. Each lobe of the LUMO is labeled according to the convention previously reported.42 It is important to note that the shapes of the LUMO lobes presented here are generally the same as those for the parent compound as previously reported; that is, each lobe represented in Figure 3a is also present in tryptanthrin itself, though specific geometric aspects of the lobes themselves, e.g., size, might be different.42 Figure 3b shows an expanded view of the boxed region depicted in Figure 2a; this region contains a single 4-aza-8-fluorotryptanthrin molecule. The features of the LUMO are not specifically discernible in this image, especially as compared to the excellent lobe-to-lobe topographic features previously reported by the authors for both occupied and unoccupied states for this class of compounds.41,42 This is most likely due to the imaging conditions that are necessary for collecting ϕap images. In particular, the modulation of the tip and the slower scan rates required to allow the lock-in amplifier sufficient response time

3. RESULTS An STM topographic image of 2 is shown in Figure 2a. The image is 7 nm in width. A scale bar corresponding to 2 nm is shown in the lower left of the image. As seen in Figure 2a, 4aza-8-fluorotryptanthrin spontaneously forms ordered monolayers, much like other tryptanthrin analogues that have been reported,40−42 and a repetitive pattern of features is easily discernible; each feature corresponds to an individual molecule, one of which is indicated by the boxed region, and the length of the molecule is found to be approximately 1.2 nm as expected. Figure 2b shows the dI/dz map associated with the topographic image in Figure 2a. Note that Figure 2b corresponds to Figure 2a on a pixel-for-pixel basis. What is immediately apparent is that the periodicity of the features present in the dI/dz map is identical to those in the corresponding topographic image. The agreement in periodicity indicates that the dI/dz map does result from the molecules in the adlayer. The same region that is boxed in Figure 2a is also boxed in Figure 2b and indicates the location of a single adsorbed molecule. An identical scale bar to that depicted in Figure 2a is also shown in Figure 2b. The numerical values of the data from Figure 2b can be used to calculate the ϕap directly via eq 1 (data not shown). The actual ϕap values from the image in Figure 2b range from 0.79 to 3.8 eV over the region of the molecule. These values are higher than what would typically be expected for bare HOPG in air (∼0.6 eV on average, though sometimes lower),27 which is the same value that the authors have observed for HOPG under the solvent, 1-phenyloctane.25 However, work functions, and presumably thus ϕap, can show significant variation for 3422

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Figure 3. LUMO, topographic image, and dI/dz image of a single 4-aza-8-fluorotryptanthrin molecule. The molecule selected for the images is in the boxed region shown in Figure 2. (a) A DFT-calculated LUMO for 4-aza-8-fluorotryptanthrin (provided for comparison). Individual lobes are labeled. (b) A topographic image of the single adlayer molecule is shown, allowing geometrical assignment of its location. The molecular structure and outlines of the LUMO lobes are overlaid. (c) The dI/dz image of the same single adlayer molecule is shown. As in (b), the molecular structure and LUMO outline are overlaid. For ease of comparison, individual lobes of the LUMO are labeled in all images.

cause a degradation of image quality as compared to purely topographic imaging experiments−especially those collected under ambient conditions where drift effects are pervasive. However, since the molecule is asymmetric upon adsorption, e.g., 4-aza-8-fluorotryptanthrin’s only plane of symmetry is broken upon adsorption, therefore the LUMO of the adsorbed species is asymmetric, and the topography data can be used to easily assign the positions of the molecules in the adlayer. Indeed the molecular position is depicted in Figure 3b with the overlay of a tube model on the image, as well as outlines of the individual lobes of the LUMO. Note that the bend in the polycyclic arrangement caused by the five-member ring of the indolo group as well as the fact that the γ3* lobe is present over the indolo carbonyl, whereas there is no corresponding lobe over the quinazoline carbonyl on the B ring, make the assignment of the position of the molecule straightforward. One interesting aspect of these images, as well as those previously reported by the authors, is that under the bias conditions used, the images were collected off-resonance from the LUMO state itself. However, as has been noted in our previous work, imaging at lower biases generates images that correspond very well to the individual frontier orbitals, suggesting that they alone are mediating the tunneling for these systems under these imaging conditions. Note that current−voltage spectroscopy was attempted at room temperatures under ambient conditions, but no MO peaks could be identified in the differential spectra. This lack of peaks was presumably due to the energetic broadening of the MO states at higher (room) temperatures. Figure 3c shows the single-molecule dI/dz map corresponding pixel-for-pixel to the topographic map in Figure 3b. Since the dI/dz map corresponds exactly, then the exact same molecular placement can be superimposed, as shown in Figure 3b. As discussed earlier, one well-known effect that can vary the ϕap on a surface is the augmentation or reduction of the quiescent surface dipole of the substrate electron states due to ESP variations in an adlayer.25,51 In particular, this is observed when the substrate states are primarily mediating the electron tunneling under the bias conditions utilized.25 A negative ESP augments the surface dipole, causing an increase in the ϕap, whereas a positive ESP opposes the surface dipole, causing a corresponding decrease in ϕap. Therefore, the authors compared the ESP map of the molecule as determined by DFT to the dI/dz image, shown in Figures 4a and 4b, respectively. The tube model of the molecule is superimposed on the ESP surface shown in Figure 4a. As can easily be seen by

Figure 4. Comparison of (a) the DFT-calculated electrostatic potential (ESP) of 4-aza-8-fluorotryptanthrin and (b) the dI/dz image of a single adlayer molecule (taken from Figure 3c). More negative ESP is shown in red, and more positive ESP is shown in blue (ranging from −199 to +126 kJ/mol as indicated by the scale bar). Generally, the dI/dz image does not correlate with the ESP distribution, suggesting that the tunneling is not primarily via substrate states, especially in regions where the MO amplitude is large; in these regions, the measured dI/dz is instead a function of the spatial decay of the MO itself.

a comparison of Figures 4a and 4b, the ϕap clearly does not show a direct correlation with the ESP distribution in the molecule; several features are in direct disagreement. For instance, the γ1* lobe is the brightest portion of the dI/dz image. However, this location of the molecule shows a very positive ESP, which should correspond to a low ϕap and thus a dark region in the dI/dz image; this is perfectly at odds with what would normally be expected. Furthermore, the portion of the molecule with the most negative ESP is along the lower edge, from the indolo-carbonyl to the 4-aza position; however, the dI/dz image displays a distinct dim feature along that portion of the molecule. Note that the above disagreements with conventional interpretations of these images are specifically over portions of the molecule where the mediating MO is present. However, there are regions where the MO is not present; here we see a more conventional response of the ϕap to the features of the 3423

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The Journal of Physical Chemistry C molecule. For instance, the quinazoline carbonyl does not have an MO lobe situated over it, so it is expected that the HOPG states contribute more substantially to tunneling at this location. A comparison of the dI/dz image in Figure 4a and the ESP in Figure 4b in this region reveals that there is a brighter feature in the image, correlated with a negative ESP at that location. This is perfectly in agreement since the negative ESP would augment the surface dipole and thus increase the ϕap. This particular location would correspond to a depressed region in the corresponding topographic region (Figure 3b). Again, this is in perfect agreement with tunneling that is mediated by substrate states; as the tunneling barrier increases, the states retract thus also corresponding to a depressed topography. Additionally, Figure 4b shows more positive ESPs predicted near the termini of the hydrogen atoms in the 1, 2, 3, 7, and 9 positions. And indeed the dI/dz image shows distinct dim features in these locations. This is consistent with the positive ESP reducing the surface dipole in those locations, resulting in a decreased ϕap. Finally, the fluorine in the 8position shows a slightly negative ESP in Figure 4b, but no lobe of the LUMO is present in that location (thus the tunneling would be mediated only by the substrate states). And as expected, the dI/dz image displays a moderately bright contrast, consistent with this arrangement. These results are consistent with two separate modes of tunneling, either mediated by the MO or mediated by the substrate states. Furthermore, these two modes can be present within an image of a single molecule, depending on the distribution of the mediating MOs. In regions where the tunneling is mediated by the substrate states, variations in the ϕap correspond closely to the ESP distribution of the adlayer, as per ϕ(qs) in eq 2. However, in regions where the tunneling is mediated by the MO, the ϕap depends on other phenomena. It is important to keep in mind that, for the measurement method described here, the barrier energy is not measured directly but is instead inferred from spatial measurements. The spatial aspect of these measurements is beneficial when attempting to collect high-resolution data, since it is not limited by the Heisenberg Uncertainty Principle, as is sometimes the case for spatial measurements of energy-correlated data; e.g., I−V tunneling spectroscopy.15 But since these measurements are fundamentally spatial, their values depend simply on the spatial distributions of the states that are measured. For substrate states, these spatial distributions fall neatly into understood theory. However, for the MO-mediated case, the picture is less clear; the ϕap is possibly not a true barrier energy at all, but instead simply a manifestation of the spatial distribution of the mediating states, although these phenomena are certainly interrelated. The implications of these phenomena regarding molecular behaviors are described more fully in the conclusions. Based on these results, we can then describe a more fully developed relationship between ϕap and the states present at the surface. Quite simply, an additional term can be added to eq 2: ϕap = T[ϕ(qs) + ϕMO]

ρs (z , E) =

1 ϵ

E

∑

|ψn(z)|2 (4)

En = E −ϵ

Here z is the distance from the surface plane of atoms, E is the energy, ϵ is the energy range over which the tunneling occurs (the difference between the sample and tip Fermi levels set by the bias voltage, and assumed to be relatively small), and ψn(z) is the nth surface wave function within the energy range measured as a function of distance from the surface plane. Assuming that the states of the substrate and the states of the adlayer are not coupled, and since the substrate states and the MO states are superimposed spatially, they can be considered to contribute separately to ρs and can be described by eq 5: ρs (z , E) =

1 ϵ

E

∑ [|ψSSn(z)|2 + |ψMOn(z)|2 ] (5)

En

Here ψSSn(z) are the states contributed by the substrate, and ψMOn(z) are the states contributed by the MOs, both as a function of distance from the surface plane of the substrate. Generally, surface states are considered to decay exponentially from the surface from the value at the surface plane (ψ(z) = ψ(0)e−κz). Indeed, eq 1 is derived exactly from this relationship, since the value of the decay constant, κ, is proportional to the root of the difference between the state energy and the potential barrier into which the state decay occurs. However, now a distinction must be made in that the exponential decay regime of the MO should originate at the plane of the adlayer atoms rather than the plane of the substrate atoms. In this case, z = 0 is still referenced to the plane of substrate atoms; however, now a spacing term, δ, is introduced as the distance between the substrate surface plane and the adlayer plane, typically ∼4 Å. Including these terms, and separating the sums, yields eq 6: ρs (z , E) =

E

1 ϵ

∑ |ψSSn(0)|2 e−2k

+

1 ϵ

SSz

En E MO(z − δ)

∑ |ψMOn(δ)|2 e−2κ

(6)

En

Here, ψMOn(δ) represents the exponential portion of the MO wave function at the adlayer plane. Note that, generally speaking, κ is dependent only on the energy difference of a particular state and the energy for the free electron, so the same decay constant might be included for both terms here. However, as the experiments indicate, the measured ϕap varies across the molecule. This is a direct result of differences in the state decay within the same energy range. Thus, different terms are used, in particular κSS for the substrate states and κMO for the MO states. Since the tunneling current is proportional to ρs, it then follows that ∂ρ (z , E) dI ∝ s dz ∂z E 2κ = − SS ∑ |ψSSn(0)|2 e−2κSSz ϵ En

(3)

Here, the additional term, ϕMO, is the MO contribution to the ϕap measurement. The term in brackets in eq 3 follows naturally from the equation for the density of states at the surface (ρs), shown in eq 4:15

−

3424

2κMO ϵ

E MO(z − δ)

∑ |ψMOn(δ)|2 e−2κ En

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shown in Figure 3c (here lighter shades correspond to higher dI/dz). Using DFT predictions of the orbital shape, the black outlines in Figure 3c, a mask of the orbital lobes was manually applied to the experimental dI/dz image, Figure 5a. The individual lobes are labeled for convenient identification. A major benefit in this is to help emphasize the portions of the dI/dz image resulting predominantly from the molecular orbitals, as opposed to substrate states. Thus, the resulting image shows the dI/dz data only for regions where the MO amplitude is large, i.e. corresponding to ϕMO, eq 3. Figure 5b shows a computational dI/dz image of a single 4-aza-8fluorotryptanthrin molecule that was calculated for only the LUMO. That is, no substrate was involved in the calculation. The excellent agreement between these images is immediately apparent. The γ1* lobe is the brightest of the lobes in both images. The next highest intensity is the β1* lobe. Other lobes show general agreement as well. For instance, the α1*, α2*, and α3* lobes are dim in both images. Indeed, the α2* lobe is essentially nonvisible in Figure 5b; although nonzero values are present in the data, they are generally too dim to see in a grayscale image, and it is the dimmest feature in the experimental result as well (Figure 5a). Even fine details within each lobe are apparent and agree quite well between experiment and theory. For instance, within the β1* lobe, the left side is brighter in both Figures 5a and 5b. For lobe γ2*, the left side is brighter in both. Lobe γ3* over the indolo carbonyl is brighter near the upper edge of the lobe in both images. Apart from these correlations, there are minor subtle differences that are possibly due to the influence of the substrate states on the experimental measurements. This excellent agreement provides substantial corroboration that the spatial disposition of the predominantly contributing electron state dominates the ϕap where the MO lobes contribute predominantly to the measurement, i.e., where the tip is directly over the lobe of an MO. It is also important to keep in mind that in the regions where the MO is present, the dI/dz images do not correlate to either the topography of the molecule or the ESP. This leaves the MO states as the only remaining influence on the dI/dz image. Thus, it is clear that in these regions ϕap is dominated by the ϕMO term in eq 3.

Thus, the two terms may be considered as separate contributions to dI/dz, and thus to ϕap, thereby justifying the separation of barrier terms in eq 3. With this in mind, it follows that the measured ϕap will depend on which set of states has a larger contribution at a particular location, assuming no change in E or ϵ, which is correct for these experiments. In particular, if ψSSn(δ) ≪ ψMOn(δ), then it can be inferred that the MO term will predominantly contribute to the tunneling, and ϕ(qs) ≪ ϕMO assuming similar κSS, κMO. This seems very likely considering the strong distance dependence of tunneling currents in general. Conversely, in regions where the MO wave function amplitude is low, the opposite relationship will exist and the substrate states will dominate ϕap. Thus, these results suggest that ϕap is more heavily influenced by the spatial disposition of the MOs where the tunneling is orbital-mediated, i.e., when the tip is over an orbital. However, ϕap is more heavily influenced by the ESP of the molecule where the MOs are absent, and thus tunneling is predominantly mediated by the substrate states. The above assumptions are strengthened if it can be shown that a good agreement exists between the measured ϕMO and a theoretically determined version of the measurement. In order to provide a theoretical basis from which to understand these results, the authors developed a new computational approach that provides simulated dI/dz images; a detailed description of the theoretical/computational method can be found elsewhere.52 Figure 5 shows a comparison of experimental results and computational results. Figure 5a is a gray scale reproduction of the single molecule dI/dz image

4. CONCLUSIONS From these data, it is clear that variations in ϕap for these types of adlayers depend on more than just topographic or surface charge effects. In fact, it appears that there are two separate modes of ϕap variation, depending on what sort of electron states are predominantly contributing to tunneling. Where the MO lobes are absent, the ϕap variation follows the electrostatic potential across the molecule, as expected from traditional theory. However, when the tunneling is mediated by MOs present in the adlayer, the ϕap response follows neither electrostatic nor topographic phenomena, suggesting that these measurements are dominated by a separate mechanism. Based on these results, the authors propose that the variation in ϕap in the regions of the MOs is simply the result of the spatial disposition of the MO state itself. Furthermore, this is corroborated by the computational results that consider only the spatial attributes of the MOs. It is interesting that the ϕap values vary across the molecule itself. In a strict sense, this should not be the case, since the MO is a single state, with a consistent energy difference from the barrier throughout (U − E, where U is the potential of the barrierthe energy of a free electron for an isolated molecule).

Figure 5. Comparison of the experimental dI/dz data with a computational dI/dz image. (a) The dI/dz data from Figure 3c are presented here in grayscale. The data are masked in order to display only measurements collected directly over the LUMO lobes. (b) A computational dI/dz image that was created using the novel convolution method cited in the text. It is important to note that the computational dI/dz image only takes into account the spatial distribution of the LUMO. The two images show remarkable agreement suggesting that the MO contribution dominates the measured dI/dz in regions where it displays large amplitudes. 3425

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The Journal of Physical Chemistry C

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However, the spatial disposition of the MO can apparently vary significantly across the molecule. Since the sorts of measurements described here are really spatial measurements, with the ϕap inferred from these spatial measurements, the measured ϕap will respond to this variation in disposition. Thus, where the MO is present, the measurement does not necessarily represent a true barrier energy in the traditional sense, as much as it represents the spatial disposition of the MO states that are the primary contributors to electron tunneling in those locations. We are pursuing these measurements in the hope that they might become useful for predicting molecular behaviors. A correlation between reactivity and electron barrier energy seems reasonable since an event such as reduction would be facilitated and guided regiochemically by lower LUMO energy barriers, as this would allow a more energetically favorable path for entry of the electron into the MO. Alternatively, if the variations of ϕap are simply due to the state disposition, a lower ϕap would correspond to a more disperse state, which would make a reduction event more likely in that location since there would be a greater wave function overlap from a reducing agent available for the electron to enter the LUMO. Conversely, a region of higher ϕap corresponds to either a larger energy barrier to a reduction event and/or a contracted MO in that region which would correspond to a reduced wave function overlap for the transfer of an electron by a reducing agent or both; in either event, the probability of a reduction event would be lower. Therefore, it is conceptually reasonable to pursue these measurements in an attempt to gauge or predict chemical reactivity.

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AUTHOR INFORMATION

Corresponding Author

*Tel., (321) 674-7350; e-mail, jolson@fit.edu. Present Address †

Center for Computational Science, Department of Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine, Miami, FL 33176. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the National Science Foundation (grant #1058427) for their generous financial support for this research.

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REFERENCES

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DOI: 10.1021/acs.jpcc.5b11959 J. Phys. Chem. C 2016, 120, 3420−3427