Pradip K. Mascharak

Structural and functional models of nitrile hydratase

Abstract Nitrile hydratase, the enzyme involved in microbial nitrile assimilation, comprises either one non-heme low-spin iron(III) or a non-corrinoid cobalt(III) center at the active site. Two carboxamido nitrogens, one cysteine-sulfur, and two sulfurs from a cysteine-sulfenic (Cys-SO) and a cysteine-sulfinic (Cys-SO 2 ) acid moiety respectively constitute the donor set around the metal center of this hydrolytic enzyme. Binding of one molecule of NO at the sixth site of the iron(III) center modulates the activity of the enzyme. It is suggested that a metal-bound hydroxide could be involved in the hydration of nitriles. Attack of water (or hydroxide) on a metal-bound nitrile is another possibility. During the past few years, modeling work by several groups have provided a great deal of insight into the structure and function of nitrile hydratase. Structural and spectroscopic studies on iron(III) and cobalt(III) complexes of various designed S,N-containing ligands have shown that the unusual coordination structure of the M(III) site (a) raises its potential and shuts off any redox activity, (b) allows binding of NO at the sixth site and oxidative modification of the bound S donors, and (c) brings the p K a of metal-bound water close to 7. Attempts to synthesize catalytically active model complexes have met with limited success. This review includes the results and implications of the modeling studies reported so far.

Design strategies to improve the sensitivity of photoactive metal carbonyl complexes (photoCORMs) to visible light and their potential as CO-donors to biological targets.

The recent surprising discovery of the beneficial effects of carbon monoxide (CO) in mammalian physiology has drawn attention toward site-specific delivery of CO to biological targets. To avoid difficulties in handling of this noxious gas in hospital settings, researchers have focused their attention on metal carbonyl complexes as CO-releasing molecules (CORMs). Because further control of such CO delivery through light-triggering can be achieved with photoactive metal carbonyl complexes (photoCORMs), we and other groups have attempted to isolate such complexes in the past few years. Typical metal carbonyl complexes release CO when exposed to UV light, a fact that often deters their use in biological systems. From the very beginning, our effort therefore was directed toward identifying design principles that could lead to photoCORMs that release CO upon illumination with low-power (5-15 mW/cm(2)) visible and near-IR light. In our work, we have utilized Mn(I), Re(I), and Ru(II) centers (all d(6) ground state configuration) to ensure overall stability of the carbonyl complexes. We also hypothesized that transfer of electron density from the electron-rich metal centers to π* MOs of the ligand frame via strong metal-to-ligand charge transfer (MLCT) transitions in the visible/near-IR region would weaken metal-CO back-bonding and promote rapid CO photorelease. This expectation has been realized in a series of carbonyl complexes derived from a variety of designed ligands and smart choice of ligand/coligand combinations. Several principles have emerged from our systematic approach to the design of principal ligands and the choice of auxiliary ligands (in addition to the number of CO) in synthesizing these photoCORMs. In each case, density functional theory (DFT) and time-dependent DFT (TDDFT) study afforded insight into the dependence of the CO photorelease from a particular photoCORM on the wavelength of light. Results of these theoretical studies indicate that extended conjugation in the principal ligand frames as well as the nature of the donor groups lower the energy of the lowest unoccupied MOs (LUMOs) while auxiliary ligands like PPh3 and Br(-) modulate the energy of the occupied orbitals depending on their strong σ- or π-donating abilities. As a consequence, the ligand/coligand combination dictates the energy of the MLCT bands of the resulting carbonyl complexes. The rate of CO photorelease can be altered further by proper disposition of the coligands in the coordination sphere to initiate trans-effect or alter the extent of π back-bonding in the metal-CO bonds. Addition of more CO ligands blue shift the MLCT bands, while intersystem crossing impedes labilization of metal-CO bonds in several Re(I) and Ru(II) carbonyl complexes. We anticipate that our design principles will provide help in the future design of photoCORMs that could eventually find use in clinical studies.

Coordination of carboxamido nitrogen to tervalent iron:insight into a new chapter of iron chemistry

Although coordination of carboxamido nitrogen to Fe(III) center has been assumed to be improbable, research work during the past few years has demonstrated that Fe(III) complexes with ligated carboxamido nitrogens can be readily synthesized. The Fe(III)–Namido bond distances lie in the range of 1.8–2.2 A in the various low spin and high spin Fe(III) complexes. These complexes are stable in aqueous media and their redox parameters indicate that the carboxamido nitrogens provide significant stability to the Fe(III) center.

Manganese carbonyls bearing tripodal polypyridine ligands as photoactive carbon monoxide-releasing molecules.

The recently discovered cytoprotective action of CO has raised interest in exogenous CO-releasing materials (CORMs) such as metal carbonyls (CO complexes of transition metals). To achieve control on CO delivery with metal carbonyls, we synthesized and characterized three Mn(I) carbonyls, namely, [Mn(tpa)(CO)(3)]ClO(4) [1, where tpa = tris(2-pyridyl)amine], [Mn(dpa)(CO)(3)]Br [2, where dpa = N,N-bis(2-pyridylmethyl)amine], and [Mn(pqa)(CO)(3)]ClO(4) [3, where pqa = (2-pyridylmethyl)(2-quinolylmethyl)amine], by crystallography and various spectroscopic techniques. All three carbonyls are sensitive to light and release CO when illuminated with low-power UV (5-10 mW) and visible (λ > 350 nm, ~100 mW) light. The sensitivity of 1-3 to light has been assessed with respect to the number of pyridine groups in their ligand frames. When a pyridine ring is replaced with quinoline, extended conjugation in the ligand frame increases the absorptivity and makes the resulting carbonyl 3 more sensitive to visible light. These photosensitive CORMs (photoCORMs) have been employed to deliver CO to myoglobin under the control of light. The superior stability of 3 in aqueous media makes it a photoCORM suitable for inducing vasorelaxation in mouse aortic muscle rings.

Light-triggered eradication of Acinetobacter baumannii by means of NO delivery from a porous material with an entrapped metal nitrosyl.

A photoactive manganese nitrosyl, namely [Mn(PaPy(3))(NO)](ClO(4)) ({Mn-NO}), has been loaded into the columnar pores of an MCM-41 host. Strong interaction between the polar nitrosyl and the -OH groups on the host wall leads to excellent entrapment of the NO donor within the porous host. With the aluminosilicate-based host (Al-MCM-41), the loading is further enhanced due to electrostatic interaction of the cationic species with the aluminum sites. The extent of loading has been determined via analytical techniques including N(2) adsorption/desorption isometry. Powder X-ray diffraction studies on the loaded materials afford patterns typical of an ordered mesoporous silicate consisting of a hexagonal array of unidimensional channels (with slight loss of crystallinity). Elemental mapping of the loaded particles confirms the incorporation of {Mn-NO} into the porous MCM-41 structure and attests to the homogeneity of the guest molecule distribution throughout individual particles. When suspensions of the loaded materials in saline solution are exposed to low-power (10-100 mW) visible light, rapid release of NO is observed. With continuous exposure, a steady release of 50-80 μM of NO is attained with 5 mg of material/mL buffer within 5 min, and the NO flux is maintained for a period of ~60 min. Rapid bursts of 5-10 μM NO are noted with short light pulses. Loss of either the nitrosyl or its photoproduct(s) from these materials in biological media is minimal over long periods of time. The NO release profiles suggest potential use of these powdery biocompatible materials as NO donors where the delivery of NO (a strong antibiotic) could be controlled via the exposure of light. Such prediction has been confirmed with the successful eradication of both drug-susceptible and drug-resistant Acinetobacter baumannii in a soft-tissue infection model through light-triggered NO delivery.

Chemistry of iron(III) complexes of N,N′-bis(2-hydroxyphenyl)-pyridine-2,6-dicarboxamide: seven-coordinate iron(III) complexes ligated to deprotonated carboxamido nitrogens

Abstract As part of our continuing effort to develop the chemistry of iron(III) complexes of ligands that employ carboxamido nitrogens to bind the metal center, we have synthesized the designed ligand N,N′-bis(2-hydroxyphenyl)pyridine-2,6-dicarboxamide (POPYH4, Hs are dissociable phenolic and carboxamido hydrogens). Reactions of the completely deprotonated POPY4− with certain iron(III) starting materials in DMF afford pentagonal bipyramidal complexes of the type [Fe(POPY)X2]n− (X=1-MeIm, SCN−), in which the pentadentate POPY4− ligand occupies the equatorial plane. When smaller amounts of base are used, the partially deprotonated POPYH2 2− ligand gives rise to the tetrahedral bis complex [Fe(POPYH2)2]− in which only the phenolic oxygens of the ligand are bound to iron(III). With [Fe2OCl6]2−, one obtains [Fe(POPYH2)(Cl)]2O, a new species that contains a (μ-oxo)diiron(III) core. Here also, POPYH2 2− employs the phenolic oxygens to bind iron and acts as a bidentate ligand. The structures of these complexes have been determined by X-ray crystallography. The conditions for interconversions among the four complexes have also been elucidated. Both [Fe(POPY)X2]n− complexes with coordinated carboxamido nitrogens are stable in air and in aqueous solution. The redox properties of these species indicate that ligated carboxamido nitrogens provide enhanced stability to iron(III) centers.

A New Approach for Studying Fast Biological Reactions Involving Nitric Oxide: Generation of NO Using Photolabile Ruthenium and Manganese NO Donors

Abstract Nitric oxide (NO) is recognized as one of the major players in various biochemical processes, including blood pressure, neurotransmission and immune responses. However, experimental studies involving NO are often limited by difficulties associated with the use of NO gas, including its toxicity and precise control over NO concentration. Moreover, the reactions of NO with biological molecules, which frequently occur on time scales of microseconds or faster, are limited by the millisecond time scale of conventional stopped-flow techniques. Here we present a new approach for studying rapid biological reactions involving NO. The method is based on designed ruthenium and manganese nitrosyls, [Ru(PaPy3)(NO)](BF4)2 and [Mn(PaPy3)(NO)](ClO4) (PaPy3H = N,N–bis(2-pyridylmethyl)amine-N-ethyl-2-pyridine-2-carboxamide), which upon photolysis produce NO on a fast time scale. The kinetics of the binding of the photogenerated NO to reduced cytochrome c oxidase (CcO) and myoglobin (Mb) was investigated using time-resolved optical absorption spectroscopy. The NO was found to bind to reduced CcO with an apparent lifetime of 77 μs using the [Mn(PaPy3)(NO)]+ complex; the corresponding rate is 10–20 times faster than can be detected by conventional stopped-flow methods. Second-order rate constants of ∼1 × 108 M−1 s−1 and ∼3 × 107 M−1 s−1 were determined for NO binding to reduced CcO and Mb, respectively. The generation of NO by photolysis of these complexes circumvents the rate limitation of stopped-flow techniques and offers a novel alternative to study other fast biological reactions involving NO.

Photodelivery of CO by designed PhotoCORMs: correlation between absorption in the visible region and metal-CO bond labilization in carbonyl complexes.

The therapeutic potential of photoactive CO‐releasing molecules (photoCORMs) have called for close examination of the roles of the ligand(s) and the central metal atoms on the overall photochemical labilization of the metal–CO bonds. Along this line, we have synthesized four metal complexes, namely, [MnBr(azpy)(CO)3] (1), [Mn(azpy)(CO)3(PPh3)]ClO4 (2), [ReBr(azpy)(CO)3] (3), and [Re(azpy)(CO)3(PPh3)]ClO4 (4), derived from 2‐phenylazopyridine. These complexes were characterized by spectroscopic and crystallographic studies. Although both 1 and 3 exhibit strong metal‐to‐ligand charge‐transfer bands in the 500–600 nm region, only 1 photoreleases CO upon illumination with visible light. Results of theoretical studies were used to gain insight into this surprising difference. Strong spin‐orbit coupling (prominent in heavy metals) appears to promote intersystem crossing to a triplet state in 3, a step that discourages CO release upon illumination with visible light. Slow release of CO from 2 and 4 also indicates that strong σ‐donating ligands, such as Br−, accelerate the rate of CO photorelease relative to π‐acid ligands, such as PPh3.

Synthesis and Characterization of a "Turn-On" photoCORM for Trackable CO Delivery to Biological Targets

A designed photoactive CO releasing molecule (photoCORM), namely, fac-[MnBr(CO)3(pbt)] (1, pbt = 2-(2-pyridyl)benzothiazole), promotes CO-induced death of MDA-MB-231 human breast cancer cells upon illumination with broadband visible light. The CO release from this photoCORM can be tracked by rise in fluorescence within the cellular matrix due to deligation of the pbt ligand. The results of this study suggest the potential of 1 in eradication of cancer cells through CO delivery.

Triggered dye release via photodissociation of nitric oxide from designed ruthenium nitrosyls: turn-ON fluorescence signaling of nitric oxide delivery.

Two new fluorescein-tethered nitrosyls derived from designed tetradentate ligands with carboxamido-N donors have been synthesized and characterized by spectroscopic techniques. These two diamagnetic {Ru-NO}(6) nitrosyls, namely, [(Me(2)bpb)Ru(NO)(FlEt)] (1-FlEt, Me(2)bpb = 1,2-bis(pyridine-2-carboxamido)5-dimethylbenzene, FlEt = fluorescein ethyl ester) and [((OMe)(2)IQ1)Ru(NO)(FlEt)] (2-FlEt, (OMe)(2)IQ1 = 1,2-bis(isoquinoline-1-carboxamido)-4,5-dimethoxybenzene), display NO stretching frequencies (ν(NO)) at 1846 and 1832 cm(-1) in addition to their FlEt carbonyl stretching frequencies (ν(CO)) at 1715 and 1712 cm(-1), respectively. Coordination of the dye ligand enhances the absorptivity and NO photolability of these two nitrosyls in the visible region (450-600 nm) of light. Exposure to visible light promotes rapid loss of NO from both {Ru-NO}(6) nitrosyls to generate Ru(III) photoproducts in dry aprotic solvents, such as MeCN and DMF. The FlEt(-) moiety remains bound to the paramagnetic Ru(III) center in such cases, and hence, the photoproducts exhibit very weak fluorescence from the dye unit. In the presence of water, the Ru(III) photoproducts undergo further aquation and loss of the FlEt(-) moiety via protonation. These steps lead to turn-ON fluorescence (from the free FlEt unit) and provide a visual signal of the NO photorelease from 1-FlEt and 2-FlEt in aqueous media.