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Stephen J Lippard

Publications and source records attributed to Stephen J Lippard.

At least 73 records · Page 4Linked to original sources
PubMed2004

Determination by X-ray absorption spectroscopy of the Fe-Fe separation in the oxidized form of the hydroxylase of methane monooxygenase alone and in the presence of MMOD.

The diiron active site in the hydroxylase of Methylococcus capsulatus (Bath) methane monooxygenase (MMOH) has been studied in the oxidized form by X-ray absorption spectroscopy (XAS). Previous investigations by XAS and X-ray crystallography have identified two different distances (3.0 and 3.4 angstroms) between the two Fe atoms in the dinuclear site. The present study has employed a systematic extended X-ray absorption fine structure (EXAFS) fitting methodology, utilizing known and simulated active site and relevant model structures, to determine unambiguously the Fe-Fe separation in the oxidized form of MMOH. Consistent and unique fits were only possible for an Fe-Fe distance of 3.0 angstroms. This methodology was then applied to study potential changes in the active site local structure in the presence of MMOD, a protein of unknown function in multicomponent MMO. Fe K-edge and EXAFS analyses revealed negligible changes in the diiron site electronic and geometric structure upon addition of MMOD to oxidized MMOH.

Binding Sites↗
PubMed2004

Cobalt chemistry with mixed aminotroponiminate salicylaldiminate ligands: synthesis, characterization, and nitric oxide reactivity.

A new class of mixed aminotroponimine salicylaldimine ligands and their corresponding cobalt(II) complexes are reported. This work expands the family of cobalt(II) aminotroponiminato complexes to include salicylaldiminate and derivatized fluorescein moieties. The H2iPrSATI-n (n = 3, 4) ligands 3 and 4, respectively, contain an aminotroponimine moiety and a salicylaldimine fragment connected with an alkyl linker. In the H2iPrFATI-n (n = 3, 4) ligands 5 and 6, a derivatized fluorescein replaces the salicylaldimine fragment. The cobalt(II) complexes [Co(iPrSATI-3)] (7) and [Co2(iPrSATI-4)(2)] (9) were prepared and structurally characterized. The reaction of NO with both complexes ultimately results in the formation of a dinitrogen-containing species. The mononitrosyl, [Co(iPrSATI-3)(NO)] (8), was isolated and characterized. The reactivity of [Co(iPrFATI-3)] (10) and [Co(iPrFATI-4)] (11) with NO mimics that observed for the salicylaldimine derivatives, as monitored by solution IR spectroscopy. When followed by fluorescence spectroscopy, reaction of 11 with NO evoked a 3-fold increase in emission intensity after 22 h.

Journal Article↗
PubMed2004

Catalytic oxidative ring opening of THF promoted by a carboxylate-bridged diiron complex, triarylphosphines, and dioxygen.

The catalytic oxidation of triphenylphosphine in the presence of dioxygen by the diiron(II) complex [Fe(2)(micro-O(2)CAr(Tol))(2)(Me(3)TACN)(2)(MeCN)(2)](OTf)(2) (1), where (-)O(2)CAr(Tol) = 2,6-di(p-tolyl)benzoate and Me(3)TACN = 1,4,7-trimethyl-1,4,7-triazacyclononane, has been investigated. The corresponding diiron(III) complex, [Fe(2)(micro-O)(micro-O(2)CAr(Tol))(2)(Me(3)TACN)(2)](OTf)(2) (2), the only detectable iron-containing species during the course of the reaction, can itself promote the reaction. Phosphine oxidation is coupled to the catalytic oxidation of THF solvent to afford, selectively, the C-C bond-cleavage product 3-hydroxypropylformate, an unprecedented transformation. After consumption of the phosphine, solvent oxidation continues but results in the products 2-hydroperoxytetrahydrofuran, butyrolactone, and butyrolactol. The similarities of the reaction pathways observed in the presence and absence of catalyst, as well as (18)O labeling, solvent dependence, and radical probe experiments, provide evidence that the oxidation is initiated by a metal-centered H-atom abstraction from THF. A mechanism for catalysis is proposed that accounts for the coupled oxidation of the phosphine and the THF ring-opening reaction.

Carboxylic Acids↗
PubMed2004

Identification of nuclear proteins that interact with platinum-modified DNA by photoaffinity labeling.

Interactions between cellular proteins and cisplatin-modified DNA are important in determining the anticancer activity of the drug. To develop a general approach for identifying proteins that mediate cellular responses to cisplatin, photoreactive cisplatin analogues having a tethered benzophenone moiety were prepared and used to form the major 1,2-intrastrand platinum-DNA cross-links. Upon irradiation of the platinated DNA dissolved in a HeLa nuclear extract, the appended photolabile benzophenone group generates a highly reactive species that binds irreversibly to cellular proteins that interact with the probe. Several DNA-protein cross-linked adducts were identified that may function in the cellular processing of cisplatin-DNA adducts. Of these, PARP-1 had not previously been demonstrated directly to contact Pt-DNA cross-links in human cells.

Base Sequence↗
PubMed2004

Dirhodium tetracarboxylate scaffolds as reversible fluorescence-based nitric oxide sensors.

We report the synthesis and characterization of dirhodium tetracarboxylate complexes [Rh(2)(mu-O(2)CR)(4)(L)(2)], with R = Me and L = dansyl-imidazole (Ds-im) or dansyl-piperazine (Ds-pip). The fluorophores coordinate to the axial sites of the dirhodium core through the imidazole or piperazine N-atom and emit only weakly when excited at 365 or 345 nm for the Ds-im and Ds-pip complexes, respectively. These fluorophore-containing complexes were investigated for their ability to elicit a fluorescence response in the presence of NO. An immediate increase in fluorescence emission of greater than 15-fold occurs when NO is admitted to solutions containing [Rh(2)(mu-O(2)CMe)(4)] and Ds-pip or Ds-im. In both systems, the fluorescence response, which arises by NO-induced displacement of the axially coordinated fluorophore, is reversible with a sensitivity of approximately 4 microM. The related dinitrosyl complexes [Rh(2)(mu-O(2)CR)(4)(NO)(2)], where R = Me, Et, or n-Pr, were prepared, structurally characterized, and found to be air-stable, losing NO upon standing in solution. Sequestration of a methylene chloride solution of the Ds-pip complex from aqueous media by a NO-permeable membrane allows for fluorescence detection of NO for potential applications in biological fluids.

Biosensing Techniques↗
PubMed2004

Synthesis and characterization of zinc sensors based on a monosubstituted fluorescein platform.

The synthesis of a new fluorescein carboxaldehyde asymmetrically substituted on the xanthene (top) ring is reported. This molecule is a key precursor for two of three monofunctionally derivatized fluorescein-based Zn(II) sensors presented in this work. Detailed preparative routes to, and photophysical characterization of, these sensors are described. The sensors are based on the previously reported ZP4 motif (Burdette, S. C.; Frederickson, C. J.; Bu, W.; Lippard, S. J. J. Am. Chem. Soc. 2003, 125, 1778-1787) and incorporate a di(2-picolyl)amine-containing aniline-derivatized ligand framework. By varying the nature of the substituent (X) para to the aniline nitrogen atom, which is responsible for PET quenching of the unbound ZP dye, we investigated the extent to which such electronic tuning might improve the fluorescent properties of asymmetrical ZP sensors. Although a comparison of probes with X = H, F, Cl, OMe reveals that the photophysical behavior of these dyes is not readily predictable, our methodology illustrates the ease with which aniline-based ligands may be linked to fluorescein dyes.

Biosensing Techniques↗
PubMed2004

Crystal structure of the toluene/o-xylene monooxygenase hydroxylase from Pseudomonas stutzeri OX1. Insight into the substrate specificity, substrate channeling, and active site tuning of multicomponent monooxygenases.

The four-component toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 is capable of oxidizing arenes, alkenes, and haloalkanes at a carboxylate-bridged diiron center similar to that of soluble methane monooxygenase (sMMO). The remarkable variety of substrates accommodated by ToMO invites applications ranging from bioremediation to the regio- and enantiospecific oxidation of hydrocarbons on an industrial scale. We report here the crystal structures of the ToMO hydroxylase (ToMOH), azido ToMOH, and ToMOH containing the product analogue 4-bromophenol to 2.3 A or greater resolution. The catalytic diiron(III) core resembles that of the sMMO hydroxylase, but aspects of the alpha2beta2gamma2 tertiary structure are notably different. Of particular interest is a 6-10 A-wide channel of approximately 35 A in length extending from the active site to the protein surface. The presence of three bromophenol molecules in this space confirms this route as a pathway for substrate entrance and product egress. An analysis of the ToMOH active site cavity offers insights into the different substrate specificities of multicomponent monooxygenases and explains the behavior of mutant forms of homologous enzymes described in the literature.

Azides↗
PubMed2004

Oxidation of sulfide, phosphine, and benzyl substrates tethered to N-donor pyridine ligands in carboxylate-bridged diiron(II) complexes.

Substituted pyridines were employed to prepare a series of terphenylcarboxylate-bridged diiron(II) compounds to mimic aspects of the chemistry at the active sites of bacterial multicomponent monooxygenases, including soluble methane monooxygenase (sMMO) and toluene monooxygenase (ToMO). Complexes of general formula [Fe2(O2CArTol)4L], L = 2, 3, or 4-pyridyldiphenylphosphine, 2-pyridylphenylsulfide, or 2-benzylpyridine and ArTol = 2,6-di(p-tolyl)benzoate, were synthesized and characterized by X-ray crystallography. Upon exposure of these compounds to dioxygen, ligand oxidation ensued and, in one case, proceeded catalytically.

Benzyl Compounds↗
PubMed2004

Synthesis and characterization of carboxylate-rich complexes having the [Fe2(mu-OH)2(mu-O2CR)]3+ and [Fe2(mu-O)(mu-O2CR)]3+ cores of O2-dependent diiron enzymes.

The {Fe2(mu-OH)2(mu-O2CR)}3+ and {Fe2(mu-O)(mu-O2CR)}3+ cores of the carboxylate-bridged diiron(III) centers in the enzyme active sites were reproduced by small molecule model complexes that were prepared through direct oxygenation of the mononuclear iron(II) complexes. Upon oxygenation of [Fe(O2CArTol)2(Hdmpz)2], where -O2CArTol is 2,6-di(p-tolyl)benzoate and Hdmpz is 3,5-dimethylpyrazole, [Fe2(mu-OH)2(mu-O2CArTol)(O2CArTol)3(OH2)(Hdmpz)2] was generated and characterized to share close physical properties with sMMOHox, including delta = 0.45 (2) mm/s, DeltaEQ = 1.21 (2) mm/s, and J = -7.2 (2) cm-1. The compound [Fe2(mu-O)(mu-O2CAr4-FPh)(O2CAr4-FPh)3(Hdmpz)3], where -O2CAr4-FPh is 2,6-di(4-fluorophenyl)benzoate, with delta = 0.51 (2) mm/s, DeltaEQ = 1.26 (2) mm/s, and J = -117.4 (1) cm-1, was isolated as the oxygenation product of [Fe(O2CAr4-FPh)2(Hdmpz)2].

Binding Sites↗
PubMed2004

Dioxygen activation in methane monooxygenase: a theoretical study.

Using broken-symmetry unrestricted Density Functional Theory, the mechanism of enzymatic dioxygen activation by the hydroxylase component of soluble methane monooxygenase (MMOH) is determined to atomic detail. After a thorough examination of mechanistic alternatives, an optimal pathway was identified. The diiron(II) state H(red) reacts with dioxygen to give a ferromagnetically coupled diiron(II,III) H(superoxo) structure, which undergoes intersystem crossing to the antiferromagnetic surface and affords H(peroxo), a symmetric diiron(III) unit with a nonplanar mu-eta(2):eta(2)-O(2)(2)(-) binding mode. Homolytic cleavage of the O-O bond yields the catalytically competent intermediate Q, which has a di (mu-oxo)diiron(IV) core. A carboxylate shift involving Glu243 is essential to the formation of the symmetric H(peroxo) and Q structures. Both thermodynamic and kinetic features agree well with experimental data, and computed spin-exchange coupling constants are in accord with spectroscopic values. Evidence is presented for pH-independent decay of H(red) and H(peroxo). Key electron-transfer steps that occur in the course of generating Q from H(red) are also detailed and interpreted. In contrast to prior theoretical studies, a requisite large model has been employed, electron spins and couplings have been treated in a quantitative manner, potential energy surfaces have been extensively explored, and quantitative total energies have been determined along the reaction pathway.

Bacterial Proteins↗
PubMed2004

Cisplatin-induced post-translational modification of histones H3 and H4.

The anti-cancer drug cisplatin kills cells by damaging DNA and inducing apoptosis. Understanding the detailed mechanisms by which cancer cells respond to cisplatin has the potential to improve substantially platinum-based therapy. Post-translational modification of histones alters chromatin structure, facilitating the binding of nuclear factors that mediate DNA repair, transcription, and other processes. In the present study, we have investigated the effects of cisplatin treatment on histone post-translational modification in cancer cells. We discovered that specific phosphorylation of histone H3 at Ser-10, mediated by the p38 MAPK pathway, is induced in response to cisplatin treatment. In addition, hyperacetylation of histone H4 was caused by drug treatment. These findings revealed a link between cisplatin administration and chromosomal structural alterations, providing mechanistic information about how cells respond to platinum-induced stress.

Breast Neoplasms↗
PubMed2004

Synthesis and characterization of Cu(2)(I,I), Cu(2)(I,II), and Cu(2)(II,II) compounds supported by two phthalazine-based ligands: influence of a hydrophobic pocket.

The copper coordination chemistry of two phthalazine-based ligands of differing steric bulk was investigated. A family of dinuclear complexes were prepared from reactions of [Cu(2)(bdptz)(MeCN)(2)](OTf)(2), 1(OTf)(2), where bdptz = 1,4-bis(2,2'-dipyridylmethyl)phthalazine. Treatment of 1(OTf)(2) with NaO(2)CCH(3) afforded the class I mixed-valent compound [Cu(2)(bdptz)(2)](OTf)(3), 2(OTf)(3), by disproportionation of Cu(I). Compound 2(OTf)(3) displays an electron paramagnetic resonance spectrum, with g( parallel ) = 2.25 (A( parallel ) = 169 G) and g( perpendicular ) = 2.06, and exhibits a reversible redox wave at -452 mV versus Cp(2)Fe(+)/Cp(2)Fe. The complex [Cu(2)(bdptz)(micro-OH)(MeCN)(2)](OTf)(3), 3(OTf)(3), was prepared by chemical oxidation of 1 with AgOTf, and exposure of 1 to dioxygen afforded [Cu(2)(bdptz)(micro-OH)(2)](2)(OTs)(4), 4(OTs)(4), which can also be obtained directly from [Cu(H(2)O)(6)](OTs)(2). In compound [Cu(2)(bdptz)(micro-vpy)](OTf)(2), 5(OTf)(2), where vpy = 2-vinylpyridine, the vpy ligand bridges the two Cu(I) centers by using both its pyridine nitrogen and the olefin as donor functionalities. The sterically hindered compounds [Cu(2)(Ph(4)bdptz)(MeCN)(2)](OTf)(2), 6(OTf)(2), and [Cu(2)(Ph(4)bdptz)(micro-O(2)CCH(3))](OTf), 7(OTf), were also synthesized, where Ph(4)bdptz = 1,4-bis[bis(6-phenyl-2-pyridyl)methyl]phthalazine. Complexes 1-7 were characterized structurally by X-ray crystallography. In 6 and 7, the four phenyl rings form a hydrophobic pocket that houses the acetonitrile and acetate ligands. Complex 6 displays two reversible redox waves with E(1/2) values of +41 and +516 mV versus Cp(2)Fe(+)/Cp(2)Fe. Analysis of oxygenated solutions of 6 by electrospray ionization mass spectrometry reveals probable aromatic hydroxylation of the Ph(4)bdptz ligand. The different chemical and electrochemical behavior of 1 versus 6 highlights the influence of a hydrophobic binding pocket on the stability and reactivity of the dicopper(I) centers.

Copper↗
PubMed2004

A tautomeric zinc sensor for ratiometric fluorescence imaging: application to nitric oxide-induced release of intracellular zinc.

Zinc is an essential metal ion for human growth and development, the disruption of cellular Zn(2+) homeostasis being implicated in several major disorders including Alzheimer's disease, diabetes, and cancer. The molecular mechanisms of Zn(2+) physiology and pathology are insufficiently understood, however, owing in part to the lack of tools for measuring changes in intracellular Zn(2+) concentrations with high spatial and temporal fidelity. To address this critical need, we have synthesized, characterized, and applied an intracellular fluorescent probe for the ratiometric imaging of Zn(2+) based on a tautomeric seminaphthofluorescein platform. Zin-naphthopyr 1 (ZNP1) affords single-excitation, dual-emission ratiometric detection of intracellular Zn(2+) through Zn(2+)-controlled switching between fluorescein and naphthofluorescein tautomeric forms. The probe features visible excitation and emission profiles, excellent selectivity responses for Zn(2+) over competing Ca(2+) and Mg(2+) ions at intracellular concentrations, a dissociation constant (K(d)) for Zn(2+) of <1 nM, and an 18-fold increase in fluorescence emission intensity ratio (lambda(624)/lambda(528)) upon zinc binding. We demonstrate the value of the ZNP1 platform for biological applications by imaging changes in intracellular [Zn(2+)] in living mammalian cells. Included is the ratiometric detection of endogenous pools of intracellular Zn(2+) after NO-induced release of Zn(2+) from cellular metalloproteins. We anticipate that ZNP1 and related probes should find utility for interrogating the biology of Zn(2+).

Animals↗
PubMed2004

Bright fluorescent chemosensor platforms for imaging endogenous pools of neuronal zinc.

A series of new fluorescent Zinpyr (ZP) chemosensors based on the fluorescein platform have been prepared and evaluated for imaging neuronal Zn(2+). A systematic synthetic survey of electronegative substitution patterns on a homologous ZP scaffold provides a basis for tuning the fluorescence responses of "off-on" photoinduced electron transfer (PET) probes by controlling fluorophore pK(a) values and attendant proton-induced interfering fluorescence of the metal-free (apo) probes at physiological pH. We further establish the value of these improved optical tools for interrogating the metalloneurochemistry of Zn(2+); the novel ZP3 fluorophore images endogenous stores of Zn(2+) in live hippocampal neurons and slices, including the first fluorescence detection of Zn(2+) in isolated dentate gyrus cultures. Our findings reveal that careful control of fluorophore pK(a) can minimize proton-induced fluorescence of the apo probes and that electronegative substitution offers a general strategy for tuning PET chemosensors for cellular studies. In addition to providing improved optical tools for Zn(2+) in the neurosciences, these results afford a rational starting point for creating superior fluorescent probes for biological applications.

Biosensing Techniques↗
PubMed2004

Synthesis, characterization, and cytotoxicity of a series of estrogen-tethered platinum(IV) complexes.

Several estrogen-tethered platinum(IV) complexes were prepared and characterized by ESI-MS and (1)H NMR spectroscopy. Their design was inspired by the observation that estrogen receptor-positive cells exposed to the hormone are sensitized to cisplatin. Intracellular reduction of bis-estrogen-cis-diamminedichloroplatinum(IV), BEP(n) (where n = 1-5 methylene groups between Pt and estrogen), occurs to afford cisplatin and two equivalents of the linker-modified estrogen. The ability of BEP(n) to induce overexpression of HMGB1 was established by immunofluorescence microscopy. The cytotoxicity of the compounds was evaluated in ER(+) MCF-7 and ER(-) HCC-1937 human breast cancer cell lines. BEP3 selectively induces overexpression of HMGB1 in MCF-7 cells, compared to HCC-1937 cells, and enhances their sensitivity (IC(50) = 2.1 +/- 0.4 microM versus 3.7 +/- 0.9 microM, respectively) to the compound. The difference in compound activities and the potential of compounds of this class for treating breast and ovarian cancer are discussed.

Breast Neoplasms↗
PubMed2004

Membrane-permeable and -impermeable sensors of the Zinpyr family and their application to imaging of hippocampal zinc in vivo.

Esterification of fluorescent biosensors is a common strategy used to trap probes within the cell. Zinpyr-1 (ZP1) is a fluorescein-based bright fluorescent sensor for divalent zinc that is cell permeable without prior modification. We describe here the synthesis and characterization of ZP1 sensors containing a carboxylic acid or ethyl ester functionality at the 5 or 6 position of the fluorescein. The presence of an electronegative carboxylate decreases the proton-induced background fluorescence of the probe by lowering the pKa of the benzylic amines responsible for fluorescence quenching. The charged species ZP1(6-CO2-) is membrane-impermeant, whereas the permeability of the neutral ZP1(5/6-CO2Et) is similar to that of the parent sensor. Intracranial microinfusion of ZP1(6-CO2Et) into rat hippocampus produces reduced staining of vesicular zinc in neuropil and very clear delineation of zinc-positive injured neuronal somata and dendrites as compared with ZP1.

Animals↗