This tutorial review describes recent progress in modeling the active sites of carboxylate-rich non-heme diiron enzymes that activate dioxygen to carry out several key reactions in nature. The chemistry of soluble methane monooxygenase, which catalyzes the selective oxidation of methane to methanol, is of particular interest for (bio)technological applications. Novel synthetic diiron complexes that mimic structural, and, to a lesser extent, functional features of these diiron enzymes are discussed. The chemistry of the enzymes is also briefly summarized. A particular focus of this review is on models that mimic characteristics of the diiron systems that were previously not emphasized, including systems that contain (i) aqua ligands, (ii) different substrates tethered to the ligand framework, (iii) dendrimers attached to carboxylates to mimic the protein environment, (iv) two N-donors in a syn-orientation with respect to the iron-iron vector, and (v) a N-rich ligand environment capable of accessing oxygenated high-valent diiron intermediates.

The mechanism by which dipicolylamine (DPA) chelate-appended fluorophores respond to zinc was investigated by the synthesis and study of five new analogs of the 2′,7′-dichlorofluorescein-based Zn 2+ sensor Zinpyr-1 (ZP1). With the use of absorption and emission spectroscopy in combination with potentiometric titrations, a detailed molecular picture has emerged of the Zn 2+ and H + binding properties of the ZP1 family of sensors. The two separate N 3 O donor atom sets on ZP1 each converge to form binding pockets in which all four heteroatoms participate in coordination to either Zn 2+ or protons. The position of the pyridyl group nitrogen atom, 2-pyridyl or 4-pyridyl, has a large impact on the fluorescence response of the dyes to protons despite relatively small changes in pK a values. The fluorescence quenching effects of such multifunctional electron-donating units are often taken as a whole. Despite the structural complexity of ZP1, however, we provide evidence that the pyridyl arms of the DPA appendages participate in the quenching process, in addition to the contribution from the tertiary nitrogen amine atom. Potentiometric titrations reveal ZP1 dissociation constants (K d ) for Zn 2+ of 0.04 pM and 1.2 nM for binding to the first and second binding pockets of the ligand, respectively, the second of which correlates with the value observed by fluorescence titration. This result demonstrates that both binding pockets of this symmetric, ditopic sensor need to be occupied in order for full fluorescence turn-on to be achieved. These results have significant implications for the design and implementation of fluorescent sensors for studies of mobile zinc ions in biology.

Substitution of one pyridine by pyrazine in each DPA appendage of ZP1, leads to a new zinc sensor, ZPP1, with a modified background fluorescence and zinc affinity. ZPP1 exhibits a two-step zinc response during fluorescence titrations, which leads to a new method for zinc quantification. The ability of ZPP1 to image and quantitate zinc was demonstrated in pancreatic Min6 cells.

An iron(II) complex with a pyridine-containing 14-membered macrocyclic (PyMAC) ligand L1 (L1 = 2,7,12-trimethyl-3,7,11,17-tetra-azabicyclo[11.3.1]heptadeca-1(17),13,15-triene), 1, was prepared and characterized. Complex 1 contains low-spin iron(II) in a pseudo-octahedral geometry as determined by X-ray crystallography. Magnetic susceptibility measurements (298 K, Evans method) and Mössbauer spectroscopy (90 K, δ = 0.50(2) mm/s, ΔE(Q) = 0.78(2) mm/s) confirmed that the low-spin configuration of Fe(II) is retained in liquid and frozen acetonitrile solutions. Cyclic voltammetry revealed a reversible one-electron oxidation/reduction of the iron center in 1, with E(1/2)(Fe(III)/Fe(II)) = 0.49 V vs Fc(+)/Fc, a value very similar to the half-wave potentials of related macrocyclic complexes. Complex 1 catalyzed the epoxidation of cyclooctene and other olefins with H(2)O(2). Low-temperature stopped-flow kinetic studies demonstrated the formation of an iron(IV)-oxo intermediate in the reaction of 1 with H(2)O(2) and concomitant partial ligand oxidation. A soluble iodine(V) oxidant, isopropyl 2-iodoxybenzoate, was found to be an excellent oxygen atom donor for generating Fe(IV)-oxo intermediates for additional spectroscopic (UV-vis in CH(3)CN: λ(max) = 705 nm, ε ≈ 240 M(-1) cm(-1); Mössbauer: δ = 0.03(2) mm/s, ΔE(Q) = 2.00(2) mm/s) and kinetic studies. The electrophilic character of the (L1)Fe(IV)═O intermediate was established in rapid (k(2) = 26.5 M(-1) s(-1) for oxidation of PPh(3) at 0 °C), associative (ΔH(‡) = 53 kJ/mol, ΔS(‡) = -25 J/K mol) oxidation of substituted triarylphosphines (electron-donating substituents increased the reaction rate, with a negative value of Hammet's parameter ρ = -1.05). Similar double-mixing kinetic experiments demonstrated somewhat slower (k(2) = 0.17 M(-1) s(-1) at 0 °C), clean, second-order oxidation of cyclooctene into epoxide with preformed (L1)Fe(IV)═O that could be generated from (L1)Fe(II) and H(2)O(2) or isopropyl 2-iodoxybenzoate. Independently determined rates of ferryl(IV) formation and its subsequent reaction with cyclooctene confirmed that the Fe(IV)-oxo species, (L1)Fe(IV)═O, is a kinetically competent intermediate for cyclooctene epoxidation with H(2)O(2) at room temperature. Partial ligand oxidation of (L1)Fe(IV)═O occurs over time in oxidative media, reducing the oxidizing ability of the ferryl species; the macrocyclic nature of the ligand is retained, resulting in ferryl(IV) complexes with Schiff base PyMACs. NH-groups of the PyMAC ligand assist the oxygen atom transfer from ferryl(IV) intermediates to olefin substrates.

The active sites of metalloenzymes are often deeply buried inside a hydrophobic protein sheath, which protects them from undesirable hydrolysis and polymerization reactions, allowing them to achieve their normal functions. In order to mimic the hydrophobic environment of the active sites in bacterial monooxygenases, diiron (

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The spectroscopic and proton- and Zn(II)-binding properties of two new members of the Zinpyr family of fluorescent sensors are reported. In ZP1B and ZP3B, a (2-picolyl)(4-picolyl)amine (2,4-DPA) moiety is installed in place of the di(2-picolyl)amine (2,2-DPA) ligand used in the parent sensors ZP1 and ZP3. This modification has the benefit of both lowering the proton-induced turn-on at physiological pH levels and altering the Zn(II) affinity so as to detect only the most concentrated stores of this ion in biological samples. Comparison of the proton affinities of all four sensors, as determined by potentiometric titrations, contributes to our understanding of the solution properties of this family of sensors.

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