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Fluorescence probing of the ferric Fenton reaction via novel chelation† Dhiraj P. Murale,a Sudesh T. Manjare,ab Yoon-Sup Leea and David G. Churchill*a

Received 14th September 2013, Accepted 25th October 2013 DOI: 10.1039/c3cc47038e www.rsc.org/chemcomm

A new probe-chelator PET dyad was synthesised, which can be used to detect Fe3+ via fluorescence enhancement to discriminate between Fe2+ and Fe3+ via Fenton chemistry involving hydrogen peroxide. This is the first BODIPY which works as a 2 : 1 multiplexer for Fe3+, Fe2+ and H2O2.

Neurodegenerative disease research is an area with numerous open questions with often important considerations related to the involvement of iron and reactive oxygen species (ROS), which engages researchers from inorganic, organic, biological and analytical backgrounds.1 For instance, the imbalance of ferric ion concentration may indicate the loss of biological metal ion homeostasis which may possibly give rise, as a chemical trigger, to neurological disorders such as Parkinson’s and Alzheimer’s diseases;2 cancerous states may also be correlated with such ion concentrations. Fenton chemistry is a very well-known concept with direct connections to in vivo ferrous and ferric ion concentrations.3 Ferrous and ferric ions are involved in the generation of hydroxyl and peroxide radicals in the presence of hydrogen peroxide; this redox pair is thus very important and ubiquitous in biological systems.4 Iron can stimulate the reactive oxygen species, which induces mitochondrial dysfunction and DNA fragmentation to degrade the cellular contents and lipid peroxidation.5 Iron is one of the most abundant and vital trace elements in human life; in a healthy adult human the average total iron content is B4 g (70% in hemoglobin, 25% stored). As iron is involved in many biological processes, the selective and accurate detection of Fe3+ is a notable and important challenge along with its differentiation from Fe2+. Ferrous and ferric ions are involved in the generation of ROS species, formally identified as ‘‘hydroxyl’’ and peroxide radicals, in the well-known process known as Fenton chemistry.3

a

Molecular Logic Gate Laboratory, Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon, 305-701, Republic of Korea. E-mail: [email protected]; Fax: +82-42-350-2810; Tel: +82-42-350-2845 b Center for Catalytic Hydrocarbon Functionalizations, Institute for Basic Science (IBS), 373-1 Guseong-dong, Yuseong-gu, Daejeon, 305-701, Republic of Korea † Electronic supplementary information (ESI) available: Experimental details, in-line spectral data, and MS spectra. See DOI: 10.1039/c3cc47038e

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A mechanism proposed by Bray and Gorin involves a highly reactive iron–oxo complex such as the ferryl ion [FeIVO]2+ as the oxidative intermediate.6 Kremer has suggested that a ferryl complex acts as the key intermediate in the Fenton reaction based on a highvalent iron(IV)–oxo complex [FeIVO]2+.7 Baerends also produced compelling computational evidence that a high-valent ferryl complex [FeIVO]2+ is easily formed from aqueous Fe2+ and H2O2 in water and suggested that it may be the key active intermediate in the ubiquitous reaction.8 However, it has yet to be usefully employed in molecular fluorescent probing so as to monitor soluble iron levels.

Molecular logic gating has emerged as a guiding construct to demonstrate how interactions of probes and analytes, whether single or multiple, can be connected conceptually to a digital Boolean logic.9 Molecular logic gates (AND, OR, NAND, XOR, XNOR, INHIBIT, IMPLICATION, etc.) have been well-studied as off-shoots of chemosensing studies, and frequently involve a single, small molecular probe bearing selectivity for one or more analytes (chemical input) and producing different signal outputs (optical outputs). ‘‘Multiplexing’’ is a main and important molecular logic gate poorly studied to date; it has been purported to be the only way to reduce the cost of molecular logic gatebased devices. Multiplexers include the 2 : 1 and 1 : 2 versions. In 2007 Andreasson et al. reported the first 2 : 1 molecular multiplexer.10 There exist few probes to discriminate between Fe2+ and Fe3+.11 There are a number of probes for the ‘‘turn-on’’ detection of ferric ions. Among these, BODIPY-based probes have been reported. One metal binding approach involves a chelation pocket with one weakly coordinating thienyl group;12a separately, reaction-based quinone formation was also reported;12b the oxidation of hydroxylamines is another example of a novel modality;12c Rurack et al. have reported Fe3+ detection using a crown ether receptor.12d Herein, we present the first BODIPY 2 : 1 multiplexer for Fe3+, Fe2+ and H2O2. In an effort to discriminate Fe3+ and Fe2+, we synthesized a novel BODIPY-based

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Fig. 1 The detailed mechanism of probe 3 with biologically important analytes serving as multiple inputs.

probe bearing a novel [Ophenol, Nt-amine, Se, Se] chelation pocket. This chelator exhibited a large improvement in desired chemosensing properties over a previously reported [Ophenol, Ntert-amine, X] (X = Npy, Sth, Ofuryl)-based analog.13 The former binding site can selectively detect Fe3+ via fluorescence enhancement, which allows entry into Fenton chemistry interplay wherein discrimination between Fe3+ and Fe2 is cleanly and conveniently made. Hydrogen peroxide can function both as a reductant or as an oxidant (Fig. 1). Overall, this probe has an advantage of detecting multiple biologically important inputs over other reported probes. Through the chelation design provided in 2, we synthesized the non-fluorescent dye conjugates that undergo an active photoinduced electron transfer (PET) between the phenyl-4-aminobased donor group and the BODIPY fluorophore acceptor group in the unchelated state. In the synthesis, secondary amine (1)14 and 4hydroxybenzaldehyde were used to obtain aldehyde 2 under Mannich conditions; the BODIPY15 reaction sequence involved (i) 2,4dimethylpyrrole, catalytic TFA, (ii) DDQ and finally (iii) triethylamine and BF3–OEt2 to afford the corresponding BODIPY species 3 (Scheme 1). The probe was characterized by 1H- and 13C-NMR spectroscopy, and mass spectrometry. The chelation site in 3 was tested with different metal ions, many of which are biologically relevant (1.0  10 6 M, in 50 : 50, water : DMSO). For this study, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Pb2+, Zn2+, Ni2+, Hg2+, Na+, K+, Ag+, and Fe3+ were used as their perchlorates salts, revealing selective detection of Fe3+ with a 15-fold ‘‘turn-on’’ response compared to that for the probe. A detection limit of 9.63  10 5 M was determined. However, there was also some response to Ag+ (Fig. 2). As a result of this screening, we observed the selectivity of the probe for Fe3+ but not for Fe2+. These results encouraged us to screen the 3 Fe2+ ensemble with hydrogen peroxide wherein a dramatic increase in fluorescence intensity was determined similar to that found for 3Fe3+. This indicates a facile and apparent Fe2+/Fe3+ conversion in the presence of hydrogen peroxide. When testing 3Fe3+ with hydrogen peroxide, there was a decrease in fluorescence intensity indicating rapid

Scheme 1

Synthesis of the probe.

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Fig. 2 (A) Emission spectrum of probe 3 (1.0  10 6, in 50 : 50, water : DMSO) with Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Pb2+, Zn2+, Ni2+, Hg2+, Na+, K+, Ag+, and Fe3+ metal ions (333 mM in water). (B) Emission spectrum of probe 3 (1.0  10 6, in 50 : 50, water : DMSO) with increasing concentration of Fe3+ ions (0 mM to 166 mM).

Fig. 3 (A) Changes in emission intensity of 3Fe2+ in the presence of hydrogen peroxide. (B) Changes in emission intensity of 3Fe3+ in the presence of hydrogen peroxide, demonstrating Fenton chemistry probing; using probes (1.0  10 6, in 50 : 50, water : DMSO) and analytes (66.7 mM in water).

conversion of Fe3+ to Fe2+, again related to species formed via Fenton chemistry, this time the reverse cycle (vide supra). These results are strong in support of the compound as a basic structure with which to discern the existence and extent of Fenton chemistry (Fig. 3). Also Job analysis showed 1 : 1 binding stoichiometry (Fig. S16, ESI†). From the emissive studies mentioned above, molecular logic gate 2 : 1 multiplexing was interpreted. Herein, the probe has been established as a molecular system that exhibits combinational logic gate properties based on three biologically-important inputs of [Fe3+], [Fe2+] and H2O2 (Fig. 4). As per our knowledge, this is the first probe to most clearly reveal 2 : 1 multiplexing characteristics for biological analytes such as [Fe3+], [Fe2+], and H2O2 that constitute the starting materials in Fenton chemical reactions. Also, it is the first such BODIPY 2 : 1 multiplexer. The truth table was provided for the response of these inputs in which output ‘1’ is defined as fluorescence enhancement and ‘0’ denotes no (or little) change in emission intensity.

Fig. 4 (top and bottom right) Proposed three-input 2 : 1 multiplexer logic gate and truth table. (bottom left) A = 3, B = 3 + Fe3+, C = 3 + Fe3+ + H2O2, D = 3 + Fe2+, E = 3 + Fe2+ + H2O2.

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Experimental data pointed to the ability of 3 to bind, discriminate, and transform Fe2+ and Fe3+ when in the presence of hydrogen peroxide. Importantly, it has been reported in the computational chemical literature that aqueous Fe2+ and H2O2 generate the FeIV-oxo complex as the most active intermediate. Therefore, we studied the Fenton chemistry route computationally, using density functional theory (DFT) calculations beginning with Fe2+ coordinated to compound 3 (full molecule), hydrogen peroxide and a discretely bound water molecule (Gaussian 09; B3LYP method with 6-311g* basis set for Se and Fe only and 6-31g* basis set for all other atoms). Additionally, calculations were performed using water as solvent. These calculations helped vividly depict the unique Fen+ chelation site which allows for the observed chemical/photosensing mechanism (Fig. 5 and ESI†). Because of the importance of the fluorescence responses that arise from analyte changes, HOMO–LUMO levels were computed. The complexed probe 3 was also calculated. Importantly, the active intermediate FeIV-oxo complex was obtained by formally breaking the O–O bond in [3FeII(OH2)(O2H2)]+. A linear transition of the oxygen–oxygen distance of the bound peroxide shows formation of a complex with an Fe–OH bond (Fig. 5). Then, the hydroxyl radical abstracts a proximal hydrogen from the bound water to afford an outersphere water molecule and a high-valent species [3FeIV(OH)2]+. Next, a hydrogen atom from one hydroxyl ligand completes the formation of a bound water and the stable [3FeIVQO(OH2)]+ ferryl complex (Fig. 5). O–O bond breaking was determined to be exothermic by B6 kcal mol 1; the activation barrier bore the same pattern as that reported for [FeII(H2O)5(H2O2)]2+.8 The energy difference between [3FeII(OH2)(O2H2)]+ and [3FeIVQO(OH2)]+ is 25 kcal mol 1 (Fig. 5), approximately the same as that reported in the literature.8 These results suggested that the high-valent [3FeIVQO(OH2)2]+ complex may be favored under aqueous conditions and responsible for the observed contributions to PET elimination. As per our knowledge, this is the first study of Fenton chemistry of a fluorescent probe in which the ferryl group is implicated. When inspecting the HOMO–LUMO diagrams from one step in the potential energy surface to the next, there are several places in which electron cloud shifting occurs implying dramatic changes

Fig. 5 The computed energies for the transformation of [3FeII(OH2)(O2H2)]+ to [3FeIVQO(OH2)]+ as a function of O  O bond length/intermolecular distance. Above, only coordination spheres and reaction sites are shown. For full diagrams of the complexes, see the ESI.† H = 0 kcal mol 1 is assigned to the energy of geometry optimized form of [3FeIVQO(OH2)]+.

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in photophysical characteristics of the system shown experimentally. When the O–O bond distance extends from 1.46 to 1.86 Å, the general patterns of the LUMO + 1 and LUMO are found to reverse (ESI†). When the O  O distance moves from 1.86 to 2.00 Å, the general patterns of the HOMO and HOMO 1 are found to reverse. Finally, when the O  O distance extends from 2.45 to 2.64 Å, the electron density found in the LUMO + 1 pushes into the BODIPY body, whereas the HOMO 1 rearranges onto the meso group (ESI†). In conclusion we have synthesized a new molecular probe which can bind Fe3+ and afford a 15-fold ‘‘turn-on’’ response and a detection limit of 9.63  10 5 M. From a logic gating perspective, this probe can be used as a 2 : 1 molecular multiplexer for studying the ferric Fenton reaction. Finally, formation of the ferryl group ([FeIVO]2+) as the oxidative intermediate via a DFT study was confirmed and was in excellent agreement geometrically and energetically to that found by Baerends.8 As per our knowledge this is the first fluorescence-based probe involving a 2 : 1 molecular multiplexer that holds potential for further development in studying the Fenton reaction in real neuronal systems with required modifications to address the solubility issue.

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Chem. Commun., 2014, 50, 359--361 | 361

Fluorescence probing of the ferric Fenton reaction via novel chelation.

A new probe-chelator PET dyad was synthesised, which can be used to detect Fe(3+)via fluorescence enhancement to discriminate between Fe(2+) and Fe(3+...
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