J. Phys. Chem. C 2008, 112, 14545–14548
14545
Scanning Tunneling Microscopy of 8-Fluoroindolo[2,1-b]quinazolin-6,12-dione (8-Fluorotryptanthrin) at the Graphite-Solution Interface: Fully Resolved Molecular Orbitals Rachel E. Gilman, Mark J. Novak, J. Clayton Baum, and Joel A. Olson* Department of Chemistry, Florida Institute of Technology, 150 West UniVersity BouleVard, Melbourne, Florida 32901 ReceiVed: June 18, 2008
Scanning tunneling microscopy was used to observe 8-fluorotryptanthrin, an analogue of indolo[2,1b]quinazolin-6,12-dione (tryptanthrin). Images were collected of molecules adsorbed at the solution-HOPG (highly oriented pyrolytic graphite) interface, displaying submolecular resolution. Measurements at negative bias are provided where individual molecular orbital lobes were observed, correlating directly to the density functional theory model of the highest occupied molecular orbital (HOMO). Molecules measured approximately 1.3 nm in length. Separate surface domains, displaying an offset of approximately 0.36 nm, were observed from parallel rows of molecules. Two-dimensional enantiomers were observed occupying the domain boundary. Raised features observed in the image suggest that molecules of 8-fluorotryptanthrin experience adsorptioninduced stereoisomerization, similar to its parent compound. Introduction Indolo[2,1-b]quinazolin-6,12-dione (tryptanthrin, 1) and its analogues have generated interest as potential therapeutic agents due to their easy synthesis, stability, and activity against a variety of pathogenic organisms.1-5 Although inhibitory concentrations (IC50’s) down to the low nanogram per milliliter range have been reported, their mechanism(s) at the molecular level remains unknown. Ultimately, pharmacological and toxicological events depend upon favorable interactions between a xenobiotic and a receptor as the initial trigger. These interactions typically involve the highest occupied and lowest unoccupied molecular orbitals (HOMO and LUMO, respectively). Therefore, a study of tryptanthrin 1 and its analogues was undertaken utilizing scanning tunneling microscopy (STM), an increasingly useful technique for studying the geometric and electronic properties of compounds from a molecular orbital perspective.6-11 Continuing from our initial work on the parent tryptanthrin 1,12 we report herein STM imaging of 8-fluorotryptanthrin 2, along with density functional theory (DFT) modeling of this compound as a theoretical framework to assist in interpreting the electronic characteristics of the adsorbed compound. Negative bias measurements provided well-resolved images at submolecular resolution which correlated very closely with the DFT generated HOMO of 2. Salient features of the experimentally observed molecular orbital include several lobes separated by clearly defined nodes, along with an observed nonplanar molecular conformation apparently resulting from molecule-molecule interactions on the surface, similar to the adsorption-induced stereoisomerization observed in the previous study on 1.12 Additionally, the images show interesting two-dimensional (2D) crystallographic features such as 2-D enantiomers that occupy lattice mismatches between surface domains. Experimental Methods 8-Fluorotryptanthrin was synthesized in a single step using commercially available isatoic anhydride and 5-fluoroisatin * Corresponding author. E-mail:
[email protected].
according to the protocol of Mitscher et al.13 A saturated solution of 8-fluorotryptanthrin was prepared by mixing 21 mg of 8-fluorotryptanthrin into 1.5 mL of 1-phenyloctane (Aldrich, Milwaukee, WI). Monolayers were observed from the 8-fluorotryptanthrin adsorbing spontaneously onto freshly cleaved highly oriented pyrolytic graphite (HOPG, GE Advanced Ceramics, Strongsville, OH, grade STM-1). The STM tip was mechanically prepared from Pt-Ir wire (85-15%, Alfa Aesar, Ward Hill, MA). A drop of the 8-fluorotryptanthrin solution was placed onto the newly cleaved HOPG surface, and the STM tip was immersed directly into the solution in order to image the molecular monolayer at the solution-graphite interface. The STM consisted of a Molecular Imaging PicoSPM scanning head (Phoenix, AZ) and RHK SPM1000 control electronics unit (Troy, MI). STM images were collected in constant current mode (1 nA, -0.800 V sample bias), under ambient conditions, using hardware slope correction. The images were processed using an x-offset background correction and a smoothing convolution filter. For the computational studies, calculations were performed with Spartan’04 software14 using B3LYP15,16 DFT with the 6-31G* basis set. Complete geometry optimization of gas-phase 8-fluorotryptanthrin resulted in a planar structure. Results and Discussion Figure 1a shows an STM image of a monolayer of 8-fluorotryptanthrin on an HOPG substrate (7 nm × 7 nm). Individual molecules can be clearly discerned in the image, one of which is identified by a box. As expected, the molecules measure approximately 1.3 nm in length. The image shows two separate horizontally aligned surface domains that are labeled in Figure
10.1021/jp8053668 CCC: $40.75 2008 American Chemical Society Published on Web 08/20/2008
14546 J. Phys. Chem. C, Vol. 112, No. 37, 2008
Gilman et al.
Figure 1. (a) 7 nm × 7 nm STM image of a monolayer of 8-fluorotryptanthrin. The imaged region contains two labeled surface domains, the rows of which display a lattice mismatch of ∼0.36 nm indicated by the dashed lines. A row of 2-D enantiomer molecules, labeled mismatch, are observed to occupy the boundary between the domains. An additional row of 2-D enantiomers is observed at the top of the imaged region. The box contains a single molecule. The features labeled A and B show the two types of raised features observed in the molecules. (b) A schematic of the molecular arrangement of the molecules in the left portion of (a).
1a along the right edge of the image. The upper domain is three molecules high. Two rows of molecules of the lower domain are visible in the image, and the lower domain (presumably) extends beyond the bottom edge of the imaged region. The rows of molecules in the two surface domains are parallel but exhibit an offset between the domains that measures ∼0.36 nm, as indicated by the dashed lines in Figure 1a. At the boundary of the mismatch, a horizontal row of molecules (labeled “mismatch” in Figure 1a) that appear differently than those in the domains can be observed. A careful evaluation of the mismatch molecules reveals that they are actually 2-D enantiomers of the molecules in the surface domains. The expression of chirality at surfaces has been well-described previously.17-23 In this case, since the only plane of symmetry of the molecule occurs through the molecular plane, the surface itself acts to break the symmetry. In order to more clearly display the arrangement of the molecules, Figure 1b shows a schematic of the left-hand portion of Figure 1a. In Figure 1b the lattice mismatch is shown by dashed lines. The domains and mismatch molecules are labeled as in Figure 1a. The mismatch parameter of ∼0.36 nm may be the result of the influence of the underlying HOPG surface, whose hexagonal centers occur 0.32 nm apart. The difference in these values may be due to the small amount of thermal drift observed in these images. This suggests that there may be an energetically optimum position for the 8-fluorotryptanthrin on the HOPG surface. Thus, the two domains may have formed separately, but in registry with portions of the HOPG surface that were offset by a single HOPG hexagonal unit. Where the domains meet, the energy of the mismatch is minimized by the inclusion of the 2-D enantiomer which fits between the domains with less steric interaction than would an additional row of either of the domains. However, until a thorough computational study is performed, this hypothesis must be considered speculative. Figure 2a shows an expanded view of the individual molecule identified by the box in Figure 1a. There are several features
observed in the individual molecule indicated by arrows. Figure 2b shows the DFT calculated HOMO for 8-fluorotryptanthrin showing surfaces with an isovalue ) 0.32. In Figure 2b, each lobe of the HOMO is labeled corresponding to the features in Figure 2a. For convenience sake, we have labeled the lobes with numerically subscripted characters R-δ corresponding to lobes associated with the A-D rings, respectively, as labeled in the structures for 1 and 2; this is the naming convention for the rings as established in the original patent for 1.5 It is clear that the submolecular features in Figure 2a can be directly correlated to the individual lobes of the HOMO. The primary exception to this correlation is the lobe in Figure 2b labeled δ3. The δ3 lobe is located directly over the fluorine atom and is apparently significantly diminished in Figure 2a; an arrow labeled δ3 is shown in Figure 2a where the feature would be expected based on the location of the lobe in Figure 2b. The diminished nature of the δ3 lobe is consistent with the DFT results where it occurs ∼0.7 nm lower than the other lobes of the HOMO; see the Supporting Information. Others have reported STM of polycyclic aromatic compounds at the vacuum-HOPG interface, and they were able to image general molecular orbital features such as nodes that run through the molecules.7,11,24,25 Additionally, individual MO lobes have been observed in phthalocyanines at the solution-HOPG interface.26 The data presented herein constitute the first report of STM of a tryptanthrin analogue where the resolution is high enough to correlate features in the STM image of the adsorbed compound to individual lobes of the pertinent computed molecular orbital. The ability to resolve individual lobes of the MOs is a crucial step toward better understanding the biological activities of these compounds. Another interesting feature of Figure 1a is that several of the molecules show regions with larger apparent heights than the rest of the molecule. Since these features occur at different locations in different individual molecules observed on the surface, it is unlikely that they are purely the result of variations
STM Imaging of 8-Fluorotryptanthrin
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Figure 2. (a) Expanded view of the molecule shown by the box in Figure 1a. Individual features are clearly discernible and are individually labeled. (b) Results of DFT calculations showing the computed HOMO of 8-fluorotryptanthrin with lobes labeled corresponding to the features shown in (a). Direct correspondence of individual lobes is clearly evident.
in the HOMO that would affect the tunneling matrix element. For instance, a comparison of the boxed molecule in Figure 1a to the molecule immediately to its left shows that the brighter feature in the D ring of the molecule on the left is not present in the boxed molecule. Since these are identical molecules, the differences in apparent height cannot be attributed to variations in the density of states across each molecule. Were this the case, the raised features would regularly manifest themselves in all of the molecules on the surface, including the 2-D enantiomers of the mismatch rows (since the HOMO is symmetric about the plane of the molecule). Furthermore, it is unlikely that the raised features are the result of a Moire interaction with the HOPG surface since such patterns usually display a periodicity greater than that of a molecular monolayer. The authors attribute these raised features to nonplanar conformations assumed by the molecules within the surface layer. For purposes of height measurements, the origin has been arbitrarily assigned to the highest point of the R2 lobe within each molecule. Two different types of raised features can be observed in Figure 1a. The first, labeled “A” in Figure 1a, is seen in most of the molecules in the surface domains and measures ∼0.3 nm (averaged over all of the domain molecules) in height; height values for each lobe are provided in the Supporting Information. This appears to manifest itself as an increased apparent height of the δ1 lobe. The second type of raised feature observed in Figure 1a is labeled “B” and measures ∼0.25 nm in height. This feature is observed only in the 2-D enantiomers present at the mismatch sites. Here the R1, and in some cases the R2, lobes are raised away from the plane of the surface (recall that the mismatch molecules are the 2-D enantiomers of the molecule shown in Figure 2, and therefore they are flipped horizontally). Since the lobes of the HOMO for the planar molecule are symmetric about the plane of the molecule, for convenience the same lobe notation is used for the 2-D enantiomer, though in its mirrorimage form. These phenomena appear to be similar to the adsorptioninduced stereoisomerization of the parent compound (1) that has already been reported by the authors.12 In the cited study, DFT calculations indicated that conformational deformations
from planarity can require relatively little energy (3.4 kcal/mol to deform the ends of 1 by ∼75 pm from planarity) indicating that these molecules are remarkably supple when adsorbed to HOPG surfaces. Since the apparent height of a feature in an STM image is the result of the spatial and energetic convolution of the electronic states of the surface and the tip, it is very difficult to separate conformational effects from electron state variations. Thus, it is impossible to accurately (and quantitatively) decipher any molecular conformations from these images alone. However, if it is assumed that the apparent height does not significantly vary over the molecule (i.e., the tunneling matrix element is consistent from lobe to lobe), a simplistic conformational model can be constructed to better understand the thermodynamics of the conformers. DFT computations of constrained conformers of 2 were determined based on simple trigonometric considerations of the measured heights of the raised features discussed above. The domain molecules were modeled by constraining the dihedral angle between rings C and D to 173.00° and allowing energy minimization. The constraint angle was chosen from an assessment of the difference in apparent heights between the R1 lobe and the δ1 lobe. The energy of the bent conformation of the domain molecules relative to the planar conformation was computed to be 0.6 kcal/mol. The mismatch molecules were modeled by constraining the dihedral angle between rings A and B to 174.55° and allowing energy minimization. Again, the constraint angle was derived from a trigonometric comparison of the apparent heights of the lobe and the lobe of the mismatch molecules. The energy of the bent conformation of the mismatch molecules relative to the planar conformation was computed to be 0.4 kcal/mol. Computational investigations of planar and conformationally distorted tryptanthrins adsorbed onto HOPG surfaces indicate that the influence of the surface on the energies required to achieve bent conformations can be expected to amount to at most ∼35% of the energy requirement. This approaches the inherent uncertainties associated with DFT energy calculations for such low energy differences. Therefore, the molecular energy differences described in the previous paragraphs can be con-
14548 J. Phys. Chem. C, Vol. 112, No. 37, 2008 sidered reliable for adsorbed 8-fluorotryptanthrin in spite of the fact that they were computed for gas-phase molecules. Since the energetics for these systems is largely adsorptiondriven (typically the energy of adsorption is ∼30 kcal/mol for similar planar polycyclic aromatics as computed by the authors and others),11 the energy required for breaking planarity is thermodynamically available if it allows an increased surface density of adsorbed molecules. In this case, the tendency toward distorted conformations is likely exacerbated by the presence of the mismatch between the surface domains. Specifically, the conformational adjustments could result from the steric interactions that arise from the inclusion of the 2-D enantiomers adsorbed at the mismatch sites. In this case, the energy gained from adsorption at the mismatch sites (12 mismatch molecules over the imaged region at 30 kcal/mol) more than compensates for the energy required for the conformational adjustments (30 domain molecules at 0.6 kcal/mol and 12 2-D enantiomers at 0.4 kcal/mol). The breaking of symmetry due to conformational adjustments from planarity as shown in Figure 1a suggests that these molecules are undergoing adsorption-induced (3-D) stereoisomerization. Furthermore, two types of 3-D stereoisomers are observed, namely, those resulting from the deformation of the domain molecules and those resulting from the deformation of the 2-D enantiomers at the mismatch sites. Both of these stereoisomers are distinct from what has been reported for the parent compound and represent previously unobserved conformations. Interestingly, the stereoisomerization of the parent compound was based on partial sp3 hybridization of the amide nitrogen as might be expected from the structure. For the 8-fluorotryptanthrin, the stereoisomerization appears to not significantly involve the amide nitrogen but, rather, carbon atoms of the two phenyl rings. Also, the stereoisomerization for the parent compound was observed uniformly across the imaged region and included stereoisomers that were either enantiomers, or nearly so. For the current case, only one enantiomer of each type was observed. Although the stereoisomerization effects of the parent compound and the 8-fluorotryptanthrin are energetically adsorption-driven, these results suggest that different steric environments can have significant effects upon the conformations assumed by this class of molecules. Conclusions Currently the biological mechanism(s) of tryptanthrins at the cellular and molecular levels remains unknown. Regardless of whether analogues in this class of alkaloids act as mutagens, inhibitors of key metabolic pathway(s), and/or physically (i.e., membrane disrupters), toxicological and pharmacological events at the molecular level depend on favorable HOMO-LUMO interactions between a biological substrate and a receptor as an initial trigger event. Consequently, we are utilizing STM to better understand the geometric and electronic properties of these compounds in order to more reliably predict the potential activity of new analogues in vitro and in vivo from a molecular orbital perspective. Bioavailability issues aside, effort is currently focused on refining current quantitative structure-activity relationship (QSAR) models that have been used to correlate in vitro activity of the tryptanthrins with their redox properties as determined through cyclic voltammetry experiments. Although we cannot exclude the relevance of the planar conformation of tryptanthrin in biological modeling, we now have to take into consideration that their conformational and electronic
Gilman et al. behaviors are more complicated than initially expected, especially in light of our observations that these compounds can readily assume enantiomeric conformations with a low energy barrier. Utilizing data that can be gathered from STM analyses (i.e., tunneling spectroscopies), in combination with known antiparasitic activities of various tryptanthrin analogues, we hope to theoretically construct the electronic and geometric profile of a potential in vivo receptor. The results herein, demonstrating that individual lobes in a molecular orbital can be directly observed, are an important step toward these goals. Supporting Information Available: Raw lobe-by-lobe topographical height measurements of each of the whole molecules observed in Figure 1a, as well as a profile view of the DFT results showing the lower height of the δ3 lobe. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Pitzer, K. K.; Scovill, J. P.; Kyle, D. E.; Gerena, L. United States Patents 99-US22569, 2000018769, 19990928, 2000. (2) Bhattacharjee, A. K.; Hartell, M. G.; Nichols, D. A.; Hicks, R. P.; Stanton, B.; Van Hamont, J. E.; Milhous, W. K. Eur. J. Med. Chem. 2004, 39 (1), 59–67. (3) Bhattacharjee, A. K.; Skanchy, D. J.; Jennings, B.; Hudson, T. H.; Brendle, J. J.; Werbovetz, K. A. Bioorg. Med. Chem. 2002, 10 (6), 1979– 1989. (4) Scovill, J.; Blank, E.; Konnick, M.; Nenortas, E.; Shapiro, T. Antimicrob. Agents Chemother. 2002, 46 (3), 882–883. (5) Baker, W. R.; Mitscher, L. A. United States Patents 94-US13259, 9513807, 19941117, 1995. (6) Gutman, I.; Tomovic, Z.; Muellen, K.; Rabe, J. P. Chem. Phys. Lett. 2004, 397 (4-6), 412–416. (7) Lackinger, M.; Mueller, T.; Gopakumar, T. G.; Mueller, F.; Hietschold, M.; Flynn, G. W. J. Phys. Chem. B 2004, 108 (7), 2279–2284. (8) Lee, H. S.; Iyengar, S.; Musselman, I. H. Anal. Chem. 2001, 73 (22), 5532–5538. (9) Takeuchi, H.; Kawauchi, S.; Ikai, Jpn. J. Appl. Phys., Part 1 1996, 35 (6B), 3754–3758. (10) Toerker, M.; Fritz, T.; Proehl, H.; Gutierrez, R.; Grossmann, F.; Schmidt, R. Phys. ReV. B: Condens. Matter 2002, 65 (24), 245422/1– 245422/8. (11) Florio, G. M.; Werblowsky, T. L.; Mueller, T.; Berne, B. J.; Flynn, G. W. J. Phys. Chem. B 2005, 109 (10), 4520–4532. (12) Novak, M. J.; Baum, J. C.; Buhrow, J. W.; Olson, J. A. Surf. Sci. 2006, 600 (20), L269–L273. (13) Mitscher, L. A.; Wong, W.-C.; DeMeulenaere, T.; Sulko, J.; Drake, S. Heterocycles 1981, 15 (2), 1017–1021. (14) Spartan’04; Wavefunction, Inc.: Irvine, CA. (15) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. B: Condens. Matter 1988, 37 (2), 785–789. (16) Becke, A. D. J. Chem. Phys. 1993, 98 (7), 5648–5652. (17) Humblot, V.; Lorenzo Maria, O.; Baddeley Christopher, J.; Haq, S.; Raval, R. J. Am. Chem. Soc. 2004, 126 (20), 6460–6469. (18) Barlow, S. M.; Raval, R. Surf. Sci. Rep. 2003, 50 (6-8), 201–341. (19) De Feyter, S.; De Schryver, F. C. J. Phys. Chem. B 2005, 109 (10), 4290–4302. (20) Gesquiere, A.; Jonkheijm, P.; Hoeben, F. J. M.; Schenning, A. P. H. J.; De Feyter, S.; De Schryver, F. C.; Meijer, E. W. Nano Lett. 2004, 4 (7), 1175–1179. (21) Zhang, J.; Gesquiere, A.; Sieffert, M.; Klapper, M.; Muellen, K.; De Schryver, F. C.; De Feyter, S. Nano Lett. 2005, 5 (7), 1395–1398. (22) Fasel, R.; Parschau, M.; Ernst, K.-H. Angew. Chem., Int. Ed. 2003, 42 (42), 5178–5181. (23) Huang, T.; Hu, Z.; Wang, B.; Chen, L.; Zhao, A.; Wang, H.; Hou, J. G. J. Phys. Chem. B 2007, 111 (25), 6973–6977. (24) Hou, J. G.; Yang, J.; Wang, H.; Li, Q.; Zeng, C.; Yuan, L.; Bing, W.; Chen, D. M.; Zhu, Q. Nature 2001, 409 (6818), 304–305. (25) Lu, X.; Grobis, M.; Khoo, K. H.; Louie, S. G.; Crommie, M. F. Phys. ReV. Lett. 2003, 90 (9), 096802/1–096802/4. (26) Qiu, X.; Wang, C.; Zeng, Q.; Xu, B.; Yin, S.; Wang, H.; Xu, S.; Bai, C. J. Am. Chem. Soc. 2000, 122 (23), 5550–5556.
JP8053668
