Immobilization of Zinc Porphyrin Complexes on Pyridine

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Immobilization of Zinc Porphyrin Complexes on Pyridine-Functionalized Glass Surfaces Agnieszka Dreas-Wlodarczak,† Michael M€ullneritsch,‡ Thomas Juffmann,‡ Carla Cioffi,§ Markus Arndt,*,‡ and Marcel Mayor*,†,§ †

Karlsruhe Institute of Technology, Institute of Nanotechnology, P.O. Box 3640, D-76021 Karlsruhe, Germany, ‡ Faculty of Physics, University of Vienna, Boltzmanngasse 5, A-1090 Wien, Austria, and §Department of Chemistry, University of Basel, St. Johannsring 19, CH-4056 Basel, Switzerland Received February 11, 2010. Revised Manuscript Received March 10, 2010

In order to immobilize sublimable and fluorescent dye molecules on transparent surfaces for the detection of far field molecular interference experiments, we investigate the potential of pyridine-functionalized glass substrates as coordination sites for the zinc complex of tetraphenylporphyrin (ZnTPP). Borosilicate glass is functionalized with 4-(6-(ethoxydimethylsilyl)hexyloxy)pyridine in order to cover the glass surface with pyridine subunits. ZnTPP molecules are deposited by sublimation through mechanical masks of various sizes in a high-vacuum chamber. The resulting micropatterns are analyzed using epifluorescence microscopy which also allows us to define a measure for the quality of molecular immobilization. We observe a reduced mobility and an increased efficiency for the trapping of ZnTPP on pyridine-functionalized surfaces.

Introduction The generation of molecular nanostructures has become an important field of research for a multitude of different applications ranging from molecular electronics,1,2 over molecular machines,3,4 to light harvesting5 and others. Our present efforts are geared toward tailor-made surface functionalization to reduce the mobility of specific dye chromophores which are expected to become relevant in molecular quantum interference experiments. Already in the past, de Broglie’s wave-particle duality of large molecules was successfully demonstrated in far-field diffraction6 and near-field interferometry using a variety of molecules from C60 over porphyrins7 up to massive fluorinated organic compounds.8 In these experiments, the molecular beam was generated by thermal sublimation and the detection was based on ionization followed by mass spectroscopy. Thermal sublimation, however, usually provides neither very dense nor very directed beams, in particular for high-mass particles. Moreover, ionization detectors are known to be notoriously inefficient for large molecules, with a detection probability often below η < 10-4, and they do not provide the high spatial resolution of optical imaging. To further explore the limits of molecular interference experiments, it is therefore interesting to develop new strategies to localize and to identify molecules with high spatial resolution and *Corresponding authors. E-mail: [email protected] (M.A.); [email protected] (M.M.). (1) Aswal, D.; Lenfant, S.; Guerin, D.; Yakhmi, J.; Vuillaume, D. Anal. Chim. Acta 2006, 568, 84–108. (2) Gomar-Nadal, E.; Puigmarti-Luis, J.; Amabilino, D. B. Chem. Soc. Rev. 2008, 37, 490–504. (3) Koch, N. ChemPhysChem 2007, 8, 1438–1455. (4) Balzani, V.; Credi, A.; Venturi, M. ChemPhysChem 2008, 9, 202-220. (5) Maier, S.; Fendt, L.-A.; Zimmerli, L.; Glatzel, T.; Pfeiffer, O.; Diederich, F.; Meyer, E. Small 2008, 4, 1115–1118. (6) Arndt, M.; Nairz, O.; Voss-Andreae, J.; Keller, C.; van der Zouw, G.; Zeilinger, A. Nature 1999, 401, 680–682. (7) Hackerm€uller, L.; Uttenthaler, S.; Hornberger, K.; Reiger, E.; Brezger, B.; Zeilinger, A.; Arndt, M. Phys. Rev. Lett. 2003, 91, 90408. (8) Gerlich, S.; Hackerm€uller, L.; Hornberger, K.; Stibor, A.; Ulbricht, H.; Goldfarb, F.; Savas, T.; M€uri, M.; Mayor, M.; Arndt, M. Nat. Phys. 2007, 3, 711–715.

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with high sensitivity. Furthermore, the detection method has to be compatible with a high-vacuum (HV) environment. A recent demonstration of surface adsorption for quantum interferometry and lithography9 solved this problem by preparing a highly reactive Si (111) 7  7 surface to capture and immobilize C60 molecules. Imaging of the particles was done using scanning tunneling microscopy (STM), which is capable of seeing every individual particle on atomically flat surfaces under genuine UHV conditions. The high spatial resolution in STM recording comes however at the price of being restricted to small surface areas. Fluorescence is an alternative detection method that allows the investigation of large substrate areas. We have, however, to make sure that our data evaluation is not misled by any migration of the molecules on the surface. The immobilization of fluorescent dye molecules is therefore needed, and the zinc complex of tetraphenylporphyrin (ZnTPP) is particularly appealing, also as a fluorescent core for very massive molecules. Metalloporphyrins were already immobilized on transparent surfaces exposing suitable coordination sites like pyridines,10 amines,11 or cyano groups12 by axial coordination. However, the dye immobilizations in these studies were based on complexations from solutions while a dye deposition in high vacuum is required for our new interferometry imaging system. In the present paper we would like to present our current approach toward a new detection system. In particular, we report the immobilization of ZnTPP dye molecules on suitably functionalized substrates and their subsequent imaging using epifluorescence microscopy.

Results and Discussion Immobilization Concept. The envisaged molecular far-field interference experiment is sketched in Figure 1A. The thermal (9) Juffmann, T.; Truppe, S.; Geyer, P.; Deachapunya, S.; Ulbricht, H.; Arndt, M. Phys. Rev. Lett. 2009, 103, 263601. (10) Da Cruz, F.; Driaf, K.; Berthier, C.; Lameille, J.-M.; Armand, F. Thin Solid Films 1999, 349, 155–161. (11) Zhang, Z.; Hu, R.; Liu, Z. Langmuir 2000, 16, 1158–1162. (12) Li, D. Q.; Moore, L. W.; Swanson, B. I. Langmuir 1994, 10, 1177–1185.

Published on Web 03/18/2010

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Figure 1. (A) Layout of the proposed far-field molecular interference experiment: A molecular beam of ZnTPP derivatives is created in an effusive beam source. The beam is then collimated to a few μrad by apertures A1 and A2 before it passes a diffraction grating G (d = 100 nm). The molecular quantum interference pattern behind the grating is expected to have a period of 10..20 μm (depending on the mass and velocity of the particles) and will be registered upon arrival on the surface S using epifluorescence microscopy. The sample plate of 170 μm thickness is closing the vacuum chamber, and it is therefore pressed onto two Viton sealing rings by the differential pressure between the ambient air and the vacuum. The microscope objective achieves a magnification of 40 at a high numerical aperture without the use of any immersion oil, as this might contaminate the pyridine surface. The immobilization experiments reported here were performed in setup (B), where the molecules were deposited through a mesh M on the glass surface S inside the vacuum, before being analyzed in air. This arrangement facilitated a quicker comparison of different substrates.

molecular beam is first collimated by the apertures A1 and A2 to prepare the transverse coherence that is required to reveal the de Broglie matter wave nature of the flying molecules. At the position of the diffraction grating G each molecule is then delocalized over more than the grating slit separation d, which is taken to be 100 nm. Far-field interference shall then lead to the formation of alternating stripes of reduced and increased molecular density on the sample surface S, which will appear as dark and bright stripes in optical fluorescence. Up to now, far-field diffraction has not been demonstrated with anything more massive than C70. But it is expected that the limit can still be pushed by more than 1 order of magnitude with existing technology in the near future. Optical far-field microscopy provides a wide field of view, and it is, in principle, capable of locating single molecules13 with a resolution down to the nanometer level.14,15 Unfortunately, good beam forming properties are best observed for molecules with small intermolecular interactions, an intrinsic characteristic of the particle that reduces also its interaction with the surface S. But a strong interaction between the molecule and the surface is required to guarantee the instant and permanent binding of the molecule. Surface immobilization can be readily achieved at cryogenic temperatures. But all our experiments will profit from the ease of room temperature analysis, in particular if one wants to keep the option of using the sample plate as the interface between the high-vacuum chamber and the room temperature microscope in air, as required for in situ and online monitoring of the appearing micropatterns (see Figure 1A). Our strategy is to profit from a selective interaction between the deposited dye molecules and the substrate surface. This is provided by the coordination between the central zinc atom of (13) Moerner, W. E.; Fromm, D. P. Rev. Sci. Instrum. 2003, 74, 3597–3619. (14) Hell, S. W. Science 2007, 316, 1153–1158. (15) Yildiz, A.; Forkey, J. N.; McKinney, S. A.; Ha, T.; Goldman, Y. E.; Selvin, P. R. Science 2003, 300, 2061–2065.

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a porphyrin derivative and pyridine subunits exposed on the target surface. For our first tests, the zinc complex of tetraphenylporphyrin (ZnTPP) was used as a commercially available dye. This complex is of particular interest as a substructure of a new generation of tailor-made massive and yet volatile model compounds. A high volatility is important for the successful generation of neutral slow molecular beams, and already earlier we were able to show that this property can be achieved by lateral functionalization with perfluoralkyl chains.8 Surface binding and immobilization, on the other hand, require stable and strong interactions with the substrate. Earlier studies16 showed already the challenges related to the surface deposition on clean glass substrates: the fluorescent pattern tends to quickly fade away because of both lateral diffusion and bleaching of the fluorescent dye molecules. As already mentioned above, the sublimability of the dye structure is a crucial requirement for the formation of an intense molecular beam, and only compounds with small intermolecular interactions such as weak van der Waals forces can be considered. In view of this intrinsic feature of the fluorophore, its poor fixation on the surface is not surprising. The problem of surface diffusion may, however, be overcome by offering a fixing agent on the substrate. Axial coordination between nitrogen atoms in heterocycles and metalloporphyrins is a common motive in nature (e.g., hemoglobin) as well as in supramolecular chemistry.17-20 Multipyridyl structures have been used to preorganize metalloporphyrin-containing reactants.17-19 As far as surfaces are concerned, metalloporphyrins have been (16) Goldfarb, F.; Deachapunya, S.; Stefanov, A.; Stibor, A.; Reiger, E.; Arndt, M. J. Phys.: Conf. Ser. 2005, 19, 125–133. (17) Anderson, S.; Anderson, H.; Sanders, J. J. Chem. Soc., Perkin Trans. 1995, 2255–2267. (18) Anderson, S.; Anderson, H.; Sanders, J. Acc. Chem. Res. 1993, 26, 469–475. (19) Hoffmann, M.; Wilson, C. J.; Odell, B.; Anderson, H. L. Angew. Chem., Int. Ed. 2007, 46, 3122–3125. (20) Weiss, J. J. Inclusion Phenom. Macrocyclic Chem. 2001, 40, 1–22.

DOI: 10.1021/la100638u

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Article Scheme 1. Synthesis of Glass Substrates Exposing Pyridine Subunits 40 and the Proposed Mechanism for Immobilization of ZnTPP Dyes by Complex Formation 50 a

a Reagents and conditions: (a) 5-hexen-1-ol, NaH, DMSO, RT, 18 h, 67%; (b) HSi(CH3)2OCH2CH3, Karstedt’s catalyst, toluene, 24 h, 120 °C, 44%; (c) toluene, 125 °C, 48 h; (d) ZnTPP deposition by sublimation.

immobilized on silica10 and gold21 surfaces, on TiO2 nanoparticles,22 and on silica gel23 by axial coordination to immobilized N-heterocycles like pyridines or imidazoles. As there are no additional chemicals involved in the complexation between the pyridine and the Zn porphyrin, it seems well suited for the immobilization of ZnTPP even under high-vacuum conditions, although some surface exploration may be required before the molecular partners lock in place. Surface Functionalization. Borosilicate glass is transparent in the wavelength range of relevance for the experimental setup displayed in Figure 1A,B, i.e., above 400 nm. In order to covalently link pyridine subunits to the glass substrate, a pyridine derivative with an anchor group for silicon oxide surfaces like e.g. an ethoxysilane was needed. As displayed in Scheme 1, a suitable precursor for the pyridine functionalization of glass substrates was synthesized in two steps. Starting with 4-chloropyridine hydrochloride (1), treatment with 5-hexen-1-ol in dry dimethyl sulfoxide (DMSO) and sodium hydride (NaH) as base provided the pyridine derivative 2 comprising a long chain with a terminal olefin in reasonable yields of 67% after column chromatography (CC). Hydrosilylation of 2 with dimethylethoxysilane was achieved in toluene with catalytic amounts of Pt2(CH2dCHSi(CH3)2OSi(CH3)2CHdCH2)3 (Karstedt’s catalyst) in a pressure tube under argon at 125 °C. The terminally dimethylethoxysilyl-functionalized pyridine derivative 3 was isolated as colorless highly viscous liquid in a yield of 44% by CC. The dimethylethoxysilyl derivative 3 is expected to cover silicon oxide surfaces with a molecular monolayer by reacting with exposed hydroxy functions at the substrate surface. While (21) Zhang, Z.; Hou, S.; Zhu, Z.; Liu, Z. Langmuir 2000, 16, 537–540. (22) Brumbach, M. T.; Boal, A. K.; Wheeler, D. R. Langmuir 2009, 25, 10685– 10690. (23) Geier, G.; Sasaki, T. Tetrahedron 1999, 55, 1859–1870.

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triethoxy- and trichloroalkylsilanes are prone to surface polymerization, the silane 3 with a single ethoxy leaving group and two sterically demanding passive methyl groups should rather yield in covalently linked molecular monolayers exposing the terminal pyridine unit. The covalent immobilization of the pyridine is desirable to keep the functional unit at the surface even in a UHV environment. To functionalize the substrate surface with the dimethylethoxysilyl derivative 3, the oxygen plasma cleaned glass substrate was given into a solution of 3 in dry toluene in a pressure tube. The closed pressure tube was heated to 125 °C for 2 days. After recooling to RT, the glass samples 40 were taken out of the solution and were washed extensively with dry toluene and subsequently with dry CH2Cl2. Remaining solvent traces were finally removed in an argon gas stream. Attempts to investigate the surface functionalization by UV spectroscopy failed due to the UV absorption of the borosilicate glass up to 300 nm. The chemical modification of the surface and the presence of surface pyridine groups were investigated by drop shape analysis and by inspecting the ability of the surface to click ZnTPP. Contact angles of the substrates were recorded in ambient conditions with a KRUSS “easy drop standard” apparatus. The contact angle of a water droplet on the pyridine functionalized glass surface (Figure 2B) was ∼62° and thus considerably larger than ∼27°, i.e., the angle that we observed for a plasma-cleaned glass substrate (Figure 2A). The functionalization of the glass substrate with 3 resulted in the exposure of hydrophobic organic alkylpyridine subunits at the surface of 40 which is reflected by the reduced hydrophilicity of the surface. Interestingly, if a 0.1 M solution of aqueous HCl was used instead of the water droplet (Figure 2C), the contact angle was reduced to ∼28°, pointing at the increased wettability of 40 due to the protonation of the pyridine subunits. Immobilization and Detection. To evaluate the detection sensitivity and the resolution limit of the proposed method, ZnTPP was sublimated and the molecular beam was deposited through a micromechanical mask (G) onto a substrate at 300 K in the highvacuum setup (see Figure 1B). The mask served to define a structure on the surface which could then be imaged. The masks were holey carbon films in copper meshes (G1 = Quantifoil R1.2/1.3: holes with a diameter of 1.2 μm, separated by 2.5 μm; G2 = R2/4: holes with a diameter of 2 μm, separated by 6 μm). The meshes were suspended in a distance of about 100 μm to the glass surface. Given a furnace aperture of about 40  80 μm2 and an aperture-grating distance of 95 cm, the edges of the collimation-limited mesh image should not broaden by more than 10 nm on the substrate. Molecular diffraction plays no measurable role in this geometry. After the evaporation, the glass samples were taken into air to be subjected to microscopy (Zeiss Axioscope). The samples were illuminated with green filtered (excitation filter: BP 550/25, Zeiss) light from a mercury short-arc lamp (HBO 100, Osram). Their fluorescence was collected with a high-resolution objective (Zeiss Achroplan 63, NA = 0.95) and spectrally red-filtered according to the emission spectra of ZnTPP in toluene.22 All images are recorded with an EMCCD camera (Andor IXON 885). In a first experiment, 106 ZnTPP molecules/μm2 were deposited onto a pure and a functionalized surface. The particle numbers are derived from the measured temperature, the given geometry and the published vapor pressure of ZnTPP.24 A long deposition time was chosen (13 h) so that the molecules would have sufficient time to diffuse and cluster if they are not bound by functional groups. The fluorescence images displayed in Figure 3 were (24) Perlovich, G. L.; Golubchikov, O. A.; Klueva, M. E. J. Porphyrins Phthalocyanines 2000, 4, 699–706.

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Figure 2. Contact angle investigations of the different surfaces. The picture shows a water droplet on the oxygen plasma-cleaned glass substrate (A) and on the pyridine-functionalized glass substrate 40 (B). A droplet of a 0.1 M aqueous HCl solution on the pyridinefunctionalized glass substrate 40 (C) reduced the contact angle again. Contact angles of 26.9° (A), 62.4° (B), and 28.3° (C) were obtained by using the Young-Laplace equation for sessile drop fitting.

Figure 3. After the deposition of about 106 ZnTPP molecules per μm2 through a mask, the shadow image of the mask cannot be seen on the untreated glass (A). On a coverslip cleaned in air plasma, strong clustering is still observed within the predefined pattern area (B). Only the pyridine-functionalized surface (C) shows a clear mask image (hole size 2 μm, period 6 μm). Some spurious diffusion over small distances reduces the image contrast when we study an even finer grating (D), with a hole size of 1.2 μm and a period of 2.5 μm. The fine structure can, however, be retrieved on the same surface by limiting the coverage to 700 ZnTPP molecules per μm2 (E). Interestingly, the pattern can only be seen for ZnTPP in the presence of a central coordinating Zn atom. With metal-free H2TPP molecules we consistently observe surface diffusion beyond the mask structure and important clustering (F). The dashed red lines represent typical cross section lines as used to quantify the fluorescence contrast (see Figure 4).

recorded about 30 min after deposition using the setup shown in Figure 1B. On an ordinary borosilicate microscopy coverslip (Hecht Assistant) the molecules form micrometer-sized clusters which wash out any fine details in surface deposited patterns (Figure 3A). Also on the plasma-cleaned sample (Figure 3B) strong clustering is observed, and the 6 μm pattern is smeared out. In contrast to that, well-defined homogeneously fluorescing dots were observed on the pyridine-functionalized surfaces (Figure 3C). Their measured diameter of 2.9 μm (fwhm) still exceeds the collimation-limited expectation of 2 μm, indicating that at a surface coverage of more than a monolayer the molecules are still able to migrate across the geometrical shadow within several hundred nanometers. This is also confirmed by experiments using the mask G1 with 1.2 μm sized open holes. Even on the pyridine-functionalized surface the fine structure could not be fully resolved at 106 molecules/μm2 (Figure 3D). In order to explore the role of mutual immobilization between the deposited ZnTPP molecules, we reduced the exposure time by a factor 1000 to deposit only about 700 molecules/μm2 in a second experiment. Langmuir 2010, 26(13), 10822–10826

Figure 3E shows a pattern of ZnTPP molecules deposited through the smaller mask G1. Its tiny pattern can clearly be resolved at such a low surface coverage and more than half a day of diffusion time. In contrast to that, control experiments with H2TPP displayed strong diffusion on both pyridine-functionalized (Figure 3F) and air plasma-cleaned samples, when the coordinating Zn atom was absent. The remarkable difference in surface mobility between metal-free and Zn-coordinated porphyrin is consistent throughout all experiments and corroborates the important role of the metal atom for the interaction of the entire complex with the surface. To quantify the quality of the immobilization, fluorescence cross sections were taken along the lines of highest modulation (red dashed lines in Figure 3 B,C,E,F). The cluster formation on the oxygen air plasma-cleaned glass substrate (Figure 3B) provided a periodic but spiked light scattering signal (blue dots in Figure 4A) which is attributed to the clustering of initially mobile particles. In contrast to that, ZnTPP formed a high contrast pattern which is attributed to improved immobilization on the pyridine-functionalized sample (Figure 3C). Its fringe contrast DOI: 10.1021/la100638u

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Figure 4. Fluorescence contrast along the lines of highest modulation (see dashed red lines in Figure 3). An increased contrast with a visibility V of 48% was recorded for the ZnTPP deposited through the larger mesh on the pyridine-functionalized surface (red dots in (A)) while the plasma-cleaned surface showed regular spikes related to molecular clustering (blue dots in (A)). A visibility V of 18% was recorded for the pyridine-functionalized sample with ZnTPP deposited through the smaller mesh (red dots in (B)) while the visibility for H2TPP is even far too low to be reliably determined (blue dots in (B)). The solid lines in (A) and (B) correspond to best fit sine functions.

observed for the Soret band. Also, the longest wavelength absorption band was with 553 nm shifted by 4 nm. A 10-fold reduction of the ZnTPP surface density resulted in a sharpening of the absorption band with a further red shift for both peaks (orange line in Figure 5). When a similar number of ZnTPP molecules were sublimed onto a pyridine-functionalized glass sample (blue line in Figure 5), a further sharpening of the Soret band and an additional bathochromic shift to 439 nm were observed. Interestingly, a shift of the Soret band by about 10 nm was also earlier observed in toluene for a Zn porphyrin upon complexation with pyridine.26 The increasing shift of the absorption line thus points to an increased interaction with the surface and is also consistent with the view that the pyridine-functionalized surface can coordinate to the Zn atom of ZnTPP.

Figure 5. Absorption spectra of various glass surfaces decorated with ZnTPP. The toluene spectrum is taken from ref 25. The other spectra were analyzed in a Perkin-Elmer Lambda 900 spectrometer. All spectra are normalized to the strongest absorption peak in the range of 420 nm < λ < 440 nm. The interaction with the surface and other molecules shifts and broadens the lines. The shift increases with the decreasing layer thickness and additionally on the pyridine-functionalized surface.

V = (Smax - Smin)/(Smax þ Smin) amounts to 48% (Figure 4A; red dots = measurement, red line = simulation). The same strategy was applied to compare the samples in Figure 3E,F which were dye-coated through the smaller mesh. As shown in Figure 4B, a 20-fold increase of the visibility V was observed for the ZnTPP compared to the H2TPP. Furthermore, the interaction of the porphyrin with the underlying substrate was corroborated by optical absorption spectroscopy. For this purposes, samples of various concentrations of ZnTPP molecules on borosilicate glass were prepared. At a concentration of 66  106 ZnTPP molecules/μm2 the transmission of the sample is reduced to about 1%. We therefore take this coverage as a good approximation to bulk ZnTPP (red line in Figure 5). Compared to the absorption spectrum of ZnTPP in toluene,22 a broadening and a bathochromic red shift of 9 nm was 10826 DOI: 10.1021/la100638u

Conclusion In summary, our studies support the hypothesis that immobilization of Zn porphyrins is assisted by the formation of complexes between the central Zn atom and the functional surface. Binding on the submicrometer range has been shown. This is well sufficient for far-field molecular interference with particles in the range of up to and even beyond 10 000 amu. In combination with the synthesis of sublimable massive metalloporphyrins, the here presented detection approach becomes a promising detector component for molecular interference experiments. Acknowledgment. Support from the ESF EuroQuasar program MIME, the FWF program Z149-N16, the Centre for Functional Nanostructures (CFN) of the DFG within the project C3.8, the EU through the project FUNMOL (number 213382) and the NanoSciEþ program MaECENAS is gratefully acknowledged. Supporting Information Available: Synthetic protocols and characterization of the pyridine derivatives 2 and 3 and the pyridine-functionalized borosilicate glass sample 40 . This material is available free of charge via the Internet at http://pubs.acs.org. (25) Du, H.; Fuh, R.; Li, J.; Corkan, L.; Lindsey, J. Photochem. Photobiol. 1998, 68, 141–142. (26) Mamardashvili, G. M.; Kulikova, O. M. Russ. J. Coord. Chem. 2006, 32, 756–760.

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