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Co-Registered Molecular Logic Gate with a COReleasing Molecule Triggered by Light and Peroxide Upendar Reddy Gandra, Jörg Axthelm, Patrick Hoffmann, Nandaraj Taye, Steve Glaeser, Helmar Görls, Samantha L Hopkins, Winfried Plass, Ute Neugebauer, Sylvestre Bonnet, and Alexander Schiller J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b00867 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 27, 2017
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Co-Registered Molecular Logic Gate with a CO-Releasing Molecule Triggered by Light and Peroxide Upendar Reddy G,† Jörg Axthelm,† Patrick Hoffmann,‡ Nandaraj Taye,∥ Steve Gläser,† Helmar Görls,† Samantha L. Hopkins,⊥ Winfried Plass,† Ute Neugebauer,‡ Sylvestre Bonnet,⊥ Alexander Schiller*† †
Institute for Inorganic and Analytical Chemistry (IAAC), Friedrich Schiller University Jena, Humboldtstr. 8, D-07743 Jena, Germany ‡
Leibniz Institute of Photonic Technology, Albert-Einstein-Str. 9, D-07745 Jena, Germany
∥Chromatin ⊥Leiden
and Disease Biology Laboratory, National Center for Cell Science, 411007 Pune, India
Institute of Chemistry, Leiden University, Einsteinweg 55 2333 CC, Leiden, Netherlands
Supporting Information Placeholder ABSTRACT: Co-registered molecular logic gates combine two different in- and outputs, such as light and matter. We introduce a biocompatible CO-releasing molecule (CORM A) as Mn(I) tricarbonyl complex with the ligand 5-(dimethylamino)-N, Nbis(pyridin-2-ylmethyl) naphthalene-1-sulfonamide (L). CO release is chaperoned by turn-on fluorescence and can be triggered by light (405 nm) as well as with hydrogen peroxide in aqueous phosphate buffer. Complex A behaves as a logic OR gate via co-registering the inputs of irradiation (light) and peroxide (matter) into the concomitant outputs fluorescence (light) and CO (matter). Cell viability assays confirm the low toxicity of A toward different human cell lines. The CORM has been used to track the inclusion of A into cancer cells.
Molecular logic gates involve chemical and/or optical in- and outputs. Prasanna de Silva et al. showed two decades ago that molecular fluorescent probes for ions could function as logic gates.1 As a result, molecule-based computation could be established in supramolecular and materials chemistry.2 Even applications of molecule-based computation have been found in the design of smart materials,3 in the delivery/activation of drugs,3 and in clinical diagnostics.3 However, structural integration and concatenation of molecular logic gates in circuits represents one of the biggest challenges.4 We demonstrated the flexibility and implementation power of a fluorescent IMPLICATION gate with the wiring algorithm approach (sugar computer) on a tic-tac-toe automaton.5 Another important challenge is the successful integration of optical and chemical in- and outputs into logic gates. Only very few molecular logic gates can co-register and process effectively two different in- and outputs, such as light and matter.4,6 For example, the fluorescent output shows the calculation process, whereas the output matter can be used as an input for the following chemical logic gate (concatenation). Co-registering inputs of light and matter in molecular logic gates and processing them into the concomitant outputs of light and matter are rare in literature.7a However, combining matter and light in inputs (e.g. light and glutathione) is widely available in chemically activated photodynamic therapy (PDT).7a Apart from molecular logic and
computing5 we develop carbon monoxide and nitric oxide releasing molecules (CORMs and NORMs) and materials (CORMAs and NORMAs).8,9 Optically and chemically activated CORMs with fluorescent dyes display excellent co-registered molecular logic gates because they can process concomitantly electromagnetic and chemical information.
Scheme 1. Route of synthesis and molecular structures of the CORMs A and B
Reaction conditions: (i) Mn(CO)5Br, (ii) AgOSO2CF3, acetone, 60 ° C, 1.5 h. Solvent molecules, triflate anions and hydrogens in crystal structures are omitted for clarity; gray = carbon, blue = nitrogen, red = oxygen, purple = manganese and yellow = sulfur. Up to now, such CORMs have not yet been interpreted as logic gates. CORMs were originally introduced by Motterlini and coworkers to provide highly toxic carbon monoxide (CO) in a controlled manner.10 However, CO is classified as an endogenous vital messenger molecule in mammals, such as hydrogen sulfide (H2S) or nitric oxide (NO). Surprisingly, at lower doses, CO imparts anti-inflammation effects, improves organ transplantation survival rates, inhibits bacterial growth and induces anti-cancer
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activity in mammalian physiology.11 As a consequence, CORMs that deliver CO utilizing controlled release conditions are of great interest,12 such as transition metal carbonyl complexes13, organic photo-CORMs14 and nanocarrier CORMs.15 Ford and coworkers have reported a trackable photo-CORM,16a [Re(bpy)(CO)3(thp)] (thp = tris-(hydroxymethyl) phosphine), which exhibits a change in the emission wavelength upon liberation of one CO molecule under photolysis (405 nm). Mascharak et al. demonstrated luminescent photo-CORM with 2-pyridyl)-benzothiazole having Re(I) tricarbonyl core.16b However, these CORMs suffer from poor solubility in buffered aqueous solutions and lower stability due to bidentate N,N’-chelate ligands. Therefore, novel strategies for the construction of fluorescent CORMs with improved water solubility and stability are necessary. In this study, we have tethered a dansyl fluorophore to dipicolylamine (dpa). This ligand binds to a manganese(I) tricarbonyl core (complex A and complex B as control, Scheme 1). Complex A in d6 electron configuration (low spin) is nonfluorescent due to the Mn(I) center with three electronwithdrawing CO ligands. Once the CO molecules are released from A, fluorescence of the dansyl moiety is restored. In addition, we demonstrate for the first time a chemically-triggered COreleasing molecule (CT-CORM) with complex A. Mn(I) complexes can be easily oxidized to higher oxidation states using hydrogen peroxide.17 Here, we show that matter (H2O2) and light (405 nm) trigger the CO release from the Mn(I) tricarbonyl complex A, thus matter and light are used as inputs for a molecular logic OR gate. The concomitant output is fluorescence at 514 nm and release of CO (Figure 4). The CO could be used as signal to concatenate the OR gate with another logic gate sensitive to CO. To the best of our knowledge this is the first report of a chemically induced CO-release with a turn-on luminescence response. In addition, no CORM has been yet interpreted as logic gate. Analytical and spectroscopic data confirmed the desired success in synthesis and purity of the new CORMs: A and B as well as intermediates and inactive products (see the SI for details). The CORM B was synthesized for unambiguous assignment of the spectral responses of A upon release of CO molecules as well as the influence of the -SO2 unit (Scheme 1). The solid-state X-ray crystal structures of the triflate salts of A and B show all facially arranged carbonyl ligands.
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was attributed to a ligand-centered transition with significant metal-to-ligand charge transfer (MLCT) contribution, whereas in the case of B only one distinct absorption band at 285 nm was observed (see the SI for details). Upon excitation at 365 nm, ligand L showed a strong emission band with a λmax of 588 nm (ΦL = 0.09, Figure 1a). A hypsochromic shift (∆λF) of 74 nm of the emission maxima was observed upon binding of L to the tricarbonyl Mn(I) fragment with anticipated quenching of the dansyl-based fluorescence (Figure 1b). The weak emission band with a λmax of ~ 514 nm was assigned to a new CT-based process due to the formation of complex A including electron withdrawing CO molecules at the Mn(I) core. However, B could not show any emission above 500 nm suggesting that the emission observed for the L and A is a dansyl based charge transfer transition. No detectable change was observed in the electronic spectral pattern of A either in the dark or in presence of ambient light over a course of 12 h and 24 h, respectively (see the SI for details). However, photolysis of solution containing A at intervals of 20 s resulted in reduction of the intensity of absorption band at 366 nm with the corresponding appearance of a new band at 332 nm (see the SI for details). CORM A showed a very weak luminescence at 514 nm (λex = 365 nm, ΦA = 0.0108). Interestingly, upon photolysis at intervals of 20 s, an increase of the intensity at 514 nm (ΦA = 0.0998) was observed. This could be attributed to the liberation of three CO molecules. The light-induced release of CO molecules from A and B was confirmed using the myoglobin assay.9a After irradiation of the solution at 405 nm, the absorption of deoxy-Mb at 557 nm decreased and the typical peaks of MbCO at 540 and 577 nm grew in the Q-band region (Figure 2a). This confirmed the conversion of Deoxy-Mb into Mb-CO. All CO molecules of A and B were released after 1.2 min at similar experimental conditions suggesting that there is no significant effect of the -SO2 and/or dansyl unit on CO release of A.
Figure 2. (a) Conversion of reduced Mb to Mb-CO in a mixture of A (15 µM) and reduced Mb (60 µM) in phosphate buffer (pH 7.4) upon exposure to light (λirr = 405 nm; 8 mWcm-2). (b) The fluorescence intensity monitoring the rise in the emission spectrum at 514 nm following exposure to UV light vs. time intervals of 20 s. Monoexponential fitting is represented by a solid line (apparent rate constant kCO = 22 s-1). Figure 1. (a) Electronic absorption spectrum of A (green line) and absorption luminescence spectrum (λ= 365 nm) of L (red line). (b) Luminescence spectrum of L and changes in luminescence spectra of A before and after (A′) photolysis (405 nm; 8 mWcm2 ). The electronic absorption spectrum of L shows two distinct bands at 252 and 332 nm (Figure 1a). The band at 252 nm was attributed to a charge transfer (CT) transition involving the Namine as a donor and the dansyl moiety as an acceptor. The other band at 332 nm was assigned as dansyl-based CT transition.18 Upon formation of A, the band at 332 nm becomes broad and shifts to 366 nm, which
The kinetic analysis of the CO release upon photolysis of a solution of A at 405 nm gave a first-order CO-releasing rate constant kCO = 22 s–1 (Figure 2b).9b The light-induced CO release was accompanied by the formation of an inactive product, which was also characterized by UV-Vis, ESR, NMR and IR spectroscopy as well as ESI-MS (see the SI for details). To identify the magnetism and oxidation state of the Mn core in the inactive product, we performed X-band ESR spectroscopy. The X-band ESR spectrum (at 293 K) of the photolyzed solution of A exhibits a six-line spectrum indicative of a paramagnetic Mn(II) species. Further, the paramagnetic nature of the product was shown by 1H NMR. The signals for all protons of A became broad upon irradiation. This
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confirms that the Mn(II) species (d5, high spin) is still bound to the dpa ligand. The photolyzed solution was evaporated to dryness and the residue was subjected to IR spectroscopy. The solid displayed no CO stretching vibration anymore. Unfortunately, we were not able to isolate or synthesize the inactive product as Mn(II) complex. Mononuclear Mn(I) complexes are known to be oxidized by hydrogen peroxide (H2O2) owing to the high reduction potentials of the Mn species.17 We used a solution of hydrogen peroxide (40 µM) as a trigger for the release of CO from A. We have recorded the luminescence spectra of A in presence of H2O2 (Figure 3). Upon excitation at 365 nm, a gradual increase in the emission intensity at 514 nm was observed with time, similar to the luminescence response of A under photolytic conditions. The enhancement in dansyl-based emission intensity with the addition of H2O2 resulted presumably from the release of CO molecules followed by the formation of a Mn(II) species. The H2O2-induced CO release also formed an inactive product. Its properties were similar to the inactive product obtained from light induced CO release. The apparent rate of CO release (kCO = 18 min–1) via H2O2 (40 µM) is significantly slower compared to light triggered release (kCO = 22 s–1). In addition, H2O2-induced CO release was checked via a portable CO sensor.9a We observed similar CO release with NaOCl.
the molecular OR gate with the inputs light and H2O2 and the concomitant outputs fluorescence (514 nm) and release of CO. (c) Logic circuit diagram of the co-registered logic OR gate. The turn-on luminescence response of A before and after photoactivated CO release prompted us to track the inclusion of the CORM and CO release through confocal laser scanning microscopy (CLSM) in vitro with HCT116 colon cancer cells (Figure 5).18 The CLSM images showed a “turn-on” fluorescence displayed in the green channel by the cells after illumination which reveals the release of CO from A. Furthermore, cell viability studies in living HepaRG® (progenitor cells) and LX-2 cells (stellate cells) confirmed low cytotoxicity of A (Figure 5a & b). We varied the concentrations of A from 9 to 500 µM under dark and pre-illuminated (365 nm) conditions with 24 h of incubation (see the SI for details). The concentration values at which 50% of the cell population displayed an effect (EC50) are similar to each other; 73 ± 16 µM (73 ± 5.7 µM) and 88 ± 3 µM (114 ± 4.6 µM) for HepaRG® and LX-2, respectively. The EC50 values of A against five human cancer cell lines were similar under dark and photolytic (455 nm, 3 J cm–2) conditions, ranging from ~10 to 30 µM possibly due to the increased levels of reactive oxygen species (ROS) in cancer cells.19 Although, the non-cancerous MRC-5 cells also displayed analogous dark and photolysis EC50 values, a significantly higher EC50 of ~80 µM was observed compared to the cancerous cell lines resulting in a significant therapeutic index.
Figure 3. (a) Time dependent emission enhancement of A (15 µM) (λexc = 365 nm) in the presence of hydrogen peroxide (40 µM) in PBS buffer. (b) Plot of fluorescence intensity at 514 nm vs. time (solid line as monoexponential fitting, apparent rate constant kCO = 18 min–1). According to these results, we have constructed a molecular OR gate with A (Figure 4a). We assigned light and matter (H2O2) as two inputs and the concomitant fluorescence and CO release as outputs. The truth table reveals the necessary combinations of an OR gate (Figure 4b). OR gates are logic functions in full adders for the construction of computers.5 CORM A in combination with light and matter can now be used as co-registered OR gate; fluorescence implies a visible output, whereas the concomitant output CO can be used as an input for a concatenated chemical logic gate.4
Figure 5. Confocal (fluorescence) microscopy images of HCT116 colon cancer cells incubated with 10 µM of A for 30 min at 37 °C (λex = 365 nm; λem = 510 nm; (i) & (iii) bright field images. (ii) image without light exposure, (iv) image recorded after three 10 s pulses of low power LED light (405 nm). Cell viability in percentage in (a) HepaRG® and (b) LX-2 cell line. POS = positive control. Overall, we have developed a novel, stable, and biocompatible CORM A with turn-on fluorescence response upon release of CO. It is both photoactive (405 nm) as well as chemically active (H2O2) toward CO release in aerated aqueous media. The inactive product after CO release was found to be a ligated Mn(II) species. Matter and light were inputs for a molecular co-registered logic OR gate. The concomitant output was fluorescence at 514 nm and release of CO. Additionally, CORM A is the “turn-on” photoCORM that allows one to track and confirm CO delivery in cellular matrices.
ASSOCIATED CONTENT Figure 4. (a) Fluorescence spectra of A in presence of two inputs: light (405 nm) and hydrogen peroxide (40 µM). (b) Truth table of
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Experimental procedures and characterization data for all new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author
[email protected] Notes
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(15) Kunz, P. C.; Meyer, H.; Barthel, J.; Sollazzo, S.; Schmidt, A. M.; Janiak, C. Chem. Commun. 2013, 49, 4896. (16) (a) Pierri, A. E.; Pallaoro, A.; Wu, G.; Ford, P. C. J. Am. Chem. Soc. 2012, 134, 18197. (b) Carrington, S. J.; Chakraborty, I.; Bernard, J. M.; Mascharak, P. K. Inorg. Chem. 2016, 55, 7852. (17) Yamaduchi, K. S.; Sawyer, D. T. Isr. J. Chem. 1985, 25, 164. (18) Reddy, G. U.; Agarwalla, H.; Taye, N.; Ghorai, S.; Chattopadhyay, S.; Das, A. Chem. Commun. 2014, 50, 9899. (19) Burns, N. A.; Ahmad, I. A.; Zhu. Y.; Oberley. L W.; Spitz, D. R, Biochem J. 2009, 418, 29.
The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by a grant from German Research Foundation (DFG) for supporting FOR 1738 (grant number SCHI 1175/2-2). A. S. thanks the DFG for a Heisenberg fellowship (grant numbers SCHI 1175/4-1 and SCHI 1175/5-1). We thank Florian Reinhardt for ESR measurements and A.P. de Silva for helpful discussions.
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