Biomedical subjects

Stephen J Lippard

Publications and source records attributed to Stephen J Lippard.

At least 19 recordsLinked to original sources

PubMed2007

Metal-based turn-on fluorescent probes for sensing nitric oxide.

Nitric oxide, a reactive free radical, regulates a variety of biological processes. The absence of tools to detect NO directly, rapidly, specifically, and selectively motivated us to synthesize metal-based fluorescent probes to visualize the presence of NO. We prepared and investigated Co(II), Fe(II), Ru(II), Rh(II), and Cu(II) complexes as turn-on fluorescent NO sensors. Our exploration has provided insight into how the interaction of transition-metal centers with nitric oxide can be utilized for NO sensing.

Fluorescent Dyes↗

PubMed2006

Zinspy sensors with enhanced dynamic range for imaging neuronal cell zinc uptake and mobilization.

Thiophene moieties were incorporated into previously described Zinspy (ZS) fluorescent Zn(II) sensor motifs (Nolan, E. M.; Lippard, S. J. Inorg. Chem. 2004, 43, 8310-8317) to provide enhanced fluorescence properties, low-micromolar dissociation constants for Zn(II), and improved Zn(II) selectivity. Halogenation of the xanthenone and benzoate moieties of the fluorescein platform systematically modulates the excitation and emission profiles, pH-dependent fluorescence, Zn(II) affinity, and Zn(II) complexation rates, offering a general strategy for tuning multiple properties of xanthenone-based metal ion sensors. Extensive biological studies in cultured cells and primary neuronal cultures demonstrate 2-{6-hydroxy-3-oxo-4,5-bis[(pyridin-2-ylmethylthiophen-2-ylmethylamino)methyl]-3H-xanthen-9-yl}benzoic acid (ZS5) to be a versatile imaging tool for detecting Zn(II) in vivo. ZS5 localizes to the mitochondria of HeLa cells and allows visualization of glutamate-mediated Zn(II) uptake in dendrites and Zn(II) release resulting from nitrosative stress in neurons.

Animals↗

PubMed2006

X-ray structure of a hydroxylase-regulatory protein complex from a hydrocarbon-oxidizing multicomponent monooxygenase, Pseudomonas sp. OX1 phenol hydroxylase.

Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 A resolution. PHM binds in a canyon on one side of the (alphabetagamma)2 PHH dimer, contacting alpha-subunit helices A, E, and F approximately 12 A above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more pi-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an approximately 6 A pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O2 activation, suggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH alpha-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket. Comparisons between the ToMOH and PHH structures provide insights into their substrate regiospecificities.

Binding Sites↗

PubMed2006

X-ray crystal structures of manganese(II)-reconstituted and native toluene/o-xylene monooxygenase hydroxylase reveal rotamer shifts in conserved residues and an enhanced view of the protein interior.

We report the X-ray crystal structures of native and manganese(II)-reconstituted toluene/o-xylene monooxygenase hydroxylase (ToMOH) from Pseudomonas stutzeri OX1 to 1.85 and 2.20 A resolution, respectively. The structures reveal that reduction of the dimetallic active site is accompanied by a carboxylate shift and alteration of the coordination environment for dioxygen binding and activation. A rotamer shift in a strategically placed asparagine 202 accompanies dimetallic center reduction and is proposed to influence protein component interactions. This rotamer shift is conserved between ToMOH and the corresponding residue in methane monooxygenase hydroxylase (MMOH). Previously unidentified hydrophobic pockets similar to those present in MMOH are assigned.

Crystallography, X-Ray↗

PubMed2006

Midrange affinity fluorescent Zn(II) sensors of the Zinpyr family: syntheses, characterization, and biological imaging applications.

The syntheses and photophysical characterization of ZP9, 2-{2-chloro-6-hydroxy-3-oxo-5-[(2-{[pyridin-2-ylmethyl-(1H-pyrrol-2-ylmethyl)amino]methyl}phenylamino)methyl]-3H-xanthen-9-yl}benzoic acid, and ZP10, 2-{2-chloro-6-hydroxy-5-[(2-{[(1-methyl-1H-pyrrol-2-ylmethyl)pyridin-2-ylmethylamino]methyl}phenylamino)methyl]-3-oxo-3H-xanthen-9-yl}benzoic acid, two asymmetrically derivatized fluorescein-based dyes, are described. These sensors each contain an aniline-based ligand moiety functionalized with a pyridyl-amine-pyrrole group and have dissociation constants for Zn(II) in the sub-micromolar (ZP9) and low-micromolar (ZP10) range, which we define as "midrange". They give approximately 12- (ZP9) and approximately 7-fold (ZP10) fluorescence turn-on immediately following Zn(II) addition at neutral pH and exhibit improved selectivity for Zn(II) compared to the di-(2-picolyl)amine-based Zinpyr (ZP) sensors. Confocal microscopy studies indicate that such asymmetrical fluorescein-based probes are cell permeable and Zn(II) responsive in vivo.

Aniline Compounds↗

PubMed2006

Conjugated metallopolymers for fluorescent turn-on detection of nitric oxide.

A series of eight pi-conjugated polymers (CPs) composed of phenylenevinylene, phenyleneethynylene, fluorene, and thiophene derivatives have been prepared with bipyridyl or terpyridyl substituents within the pi-conjugated backbone or at side-chain positions. These ligand-modified CPs serve as macromolecular scaffolds for conducting metallopolymers. The optical and photoluminescent properties of the polymers and corresponding copper(II) metallopolymers were investigated. Copper(II) is a highly efficient quencher of CP emission (75-100% quenching). CPs featuring bipyridyl units within the CP backbone are quenched more efficiently than those with terpyridyl units. The copper(II) metallopolymer undergoes reduction to the corresponding copper(I) species upon reaction with nitric oxide, with concomitant changes in integrated emission ranging from a 50% decrease to a 320% increase. The positive emission response is largest when Cu(II) was bound to the CP through bipyridyl units within the backbone, making these materials the best candidates for NO sensing by a turn-on emission mechanism.

Copper↗

PubMed2006

Direct nitric oxide detection in aqueous solution by copper(II) fluorescein complexes.

A series of FL(n) (n = 1-5) ligands, where FL(n) is a fluorescein modified with a functionalized 8-aminoquinoline group as a copper-binding moiety, were synthesized, and the chemical and photophysical properties of the free ligands and their copper complexes were investigated. UV-visible spectroscopy revealed a 1:1 binding stoichiometry for the Cu(II) complexes of FL(1), FL(3), and FL(5) in pH 7.0 buffered aqueous solutions. The reactions of FL(2) or FL(4) with CuCl(2), however, appear to produce a mixture of 1:1 and 1:2 complexes, as suggested by Job's plots. These binding modes were modeled by the synthesis and X-ray crystal structure determination of Cu(II) complexes of 2-[(quinolin-8-ylamino)methyl]phenol (modL), employed as a surrogate of the FL(n) ligand family. Two kinds of crystals, [Cu(modL)(2)](BF(4))(2) and [Cu(2)(modL')(2)(CH(3)OH)](BF(4))(2) (modL' = 2-[(quinolin-8-ylamino)methyl]phenolate), were obtained. The structures suggest that one oxygen and two nitrogen atoms of the FL(n) ligands most likely bind to Cu(II). Introduction of nitric oxide (NO) to pH 7.0 buffered aqueous solutions of Cu(FL(n)) (1 microM CuCl(2) and 1 microM FL(n)) at 37 degrees C induces an increase in fluorescence. The fluorescence response of Cu(FL(n)) to NO is direct and specific, which is a significant improvement over commercially available small molecule-based probes that are capable of detecting NO only indirectly. The NO-triggered fluorescence increase of Cu(FL(5)) occurs by reduction of Cu(II) to Cu(I) with concomitant dissociation of the N-nitrosated fluorophore ligand from copper. Spectroscopic and product analyses of the reaction of the FL(5) copper complex with NO indicated that the N-nitrosated fluorescein ligand (FL(5)-NO) is the species responsible for fluorescence turn-on. Density functional theory (DFT) calculations of FL(5) versus FL(5)-NO reveal how N-nitrosation of the fluorophore ligand brings about the fluorescence increase. The copper-based probes described in the present work form the basis for real-time detection of nitric oxide production in living cells.

Aminoquinolines↗

PubMed2006

Fluorescent nitric oxide detection by copper complexes bearing anthracenyl and dansyl fluorophore ligands.

Anthrancenyl and dansyl fluorophore ligands [AnCH2pipCS(2)K (1), Ds-Hen (2), Ds-HAMP (3), Ds-HAQ (4), and Ds-HAPP (5)] were prepared for coordination to Cu(II). Five Cu complexes, [Cu(AnCH2pipCS2)2] (6), [Cu(Ds-en)2] (7), [Cu(Ds-AMP)2] (8), [Cu(Ds-AQ)2] (9), and [Cu(Ds-APP)(OTf)] (10), were synthesized as candidates for detecting nitric oxide (NO) by fluorescence and characterized by X-ray crystallography. A decrease in fluorescence compared to that of the free ligands (1-5) was measured following the formation of the corresponding five Cu(II) complexes 6-10. Fluorescence was restored in the presence of NO in CH3OH/CH2Cl2 solutions of the compounds. Complexes 7, 8, and 10 exhibited a fluorescence response to NO in pH 7.0 or 9.0 buffered aqueous solutions. Spectroscopic studies revealed that NO-induced fluorescence enhancement in these Cu(II) complexes occurs by reduction to Cu(I). The present studies demonstrate that Cu(II) complexes are effective as fluorescent probes for detecting NO in both organic and aqueous environments.

Anthracenes↗

PubMed2006

Luminescent properties of water-soluble conjugated metallopolymers and their application to fluorescent nitric oxide detection.

Water-soluble pi-conjugated polymers (CPs) incorporating 5,5'-(2,2'-dipyridyl) (CP1) or 6,6'-(2,2'-dipyridyl) (CP2) units within the pi-conjugated backbone were prepared as scaffolds for macromolecular metal complexation. The response of CP emission to a range of metal ions was investigated in water, 10 mM aqueous sodium dodecyl sulfate, and acetonitrile/water (95:5). Cupric ions are the most efficient quenchers of CP emission, with K(SV) = 1.1 x 10(5) and 5.2 x 10(4) M(-1) in water for CP1a (40% bipyridyl monomer units) and CP1b (20% bipyridyl monomer units), respectively. Quenching is approximately twice as effective in acetonitrile/water (95:5) (K(SV) = 3.1 x 10(5) M(-1) for CP1a and 1.1 x 10(5) M(-1) for CP1b). Partial restoration of emission was observed upon exposure of Cu(II)-CP solutions to excess NO(g) in acetonitrile/water (95:5) or 10 mM SDS(aq).

2,2'-Dipyridyl↗

PubMed2006

Characterization of the particulate methane monooxygenase metal centers in multiple redox states by X-ray absorption spectroscopy.

The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-A resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO(iso)), chemically reduced (pMMO(red)), and chemically oxidized (pMMO(ox)) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO(iso) contains both Cu(I) and Cu(II) and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu-Cu interaction in all redox forms of the enzyme. The Cu-Cu distance increases from 2.51 to 2.65 A upon reduction, concomitant with an increase in the average Cu-O/N bond lengths. Appropriate Cu2 model complexes were used to refine and validate the EXAFS fitting protocols for pMMO(iso). Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe(III), is consistent with heme impurities. These findings are complementary to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions.

Copper↗

PubMed2006

Organic cation transporters are determinants of oxaliplatin cytotoxicity.

Although the platinum-based anticancer drugs cisplatin, carboplatin, and oxaliplatin have similar DNA-binding properties, only oxaliplatin is active against colorectal tumors. The mechanisms for this tumor specificity of platinum-based compounds are poorly understood but could be related to differences in uptake. This study shows that the human organic cation transporters (OCT) 1 and 2 (SLC22A1 and SLC22A2) markedly increase oxaliplatin, but not cisplatin or carboplatin, accumulation and cytotoxicity in transfected cells, indicating that oxaliplatin is an excellent substrate of these transporters. The cytotoxicity of oxaliplatin was greater than that of cisplatin in six colon cancer cell lines [mean +/- SE of IC(50) in the six cell lines, 3.9 +/- 1.4 micromol/L (oxaliplatin) versus 11 +/- 2.0 micromol/L (cisplatin)] but was reduced by an OCT inhibitor, cimetidine, to a level similar to, or even lower than that of, cisplatin (29 +/- 11 micromol/L for oxaliplatin versus 19 +/- 4.3 micromol/L for cisplatin). Structure-activity studies indicated that organic functionalities on nonleaving groups coordinated to platinum are critical for selective uptake by OCTs. These results indicate that OCT1 and OCT2 are major determinants of the anticancer activity of oxaliplatin and may contribute to its antitumor specificity. They also strongly suggest that expression of OCTs in tumors should be investigated as markers for selecting specific platinum-based therapies in individual patients. The development of new anticancer drugs, specifically targeted to OCTs, represents a novel strategy for targeted drug therapy. The results of the present structure-activity studies indicate specific tactics for realizing this goal.

Animals↗

PubMed2006

Polymer-bound dirhodium tetracarboxylate films for fluorescent detection of nitric oxide.

A polymer-bound dirhodium complex, [{Rh2(O2CCH3)3(Ds-pip)}n(O2C-P)], was prepared via ligand exchange of [Rh2(O2CCH3)4] with the side chains of a methyl methacrylate/methacrylic acid copolymer (O2C-P) followed by axial coordination of the fluorophore, N-dansylpiperazine (Ds-pip). Emission from Ds-pip is quenched when coordinated to the dirhodium complex but can be restored upon displacement by analytes. Exposure of [{Rh2(O2CCH3)3(Ds-pip)}n(O2C-P)] films to aqueous nitric oxide (NO) evokes a 2.2-fold increase in integrated emission. The polymer matrix excludes potentially interfering analytes including reactive oxygen or nitrogen species, which cannot readily permeate the film.

Carboxylic Acids↗

PubMed2006

Water induces a structural conversion and accelerates the oxygenation of carboxylate-bridged non-heme diiron enzyme synthetic analogues.

Recently, we reported the synthesis of a carboxylate-rich non-heme diiron enzyme model compound [Fe2(mu-O2CAr(Tol))4(4-CNPy)2] (1), where (-)O(2)CAr(Tol) is 2,6-di-p-tolylbenzoate and 4-CNPy is 4-cyanopyridine (Yoon, S.; Lippard, S. J. J. Am. Chem. Soc. 2005, 127, 8386-8397). A metal-to-ligand charge-transfer band in the visible region of the optical absorption spectrum involving the nitrogen-donor ligand endowed this complex with a distinctive red color that facilitated analysis of its chemistry. Following this strategy, we prepared and characterized two related isomeric complexes, windmill (3) and paddlewheel (4) species having the formula [Fe2(O2CAr(Tol))4(4-AcPy)2], where 4-AcPy is 4-acetylpyridine. In anhydrous solvents, 1 and 4 adopt paddlewheel structures, but upon the addition of water, they convert to aquated forms, windmill structures having the composition [Fe2(mu-O2CAr(Tol))2(O2CAr(Tol))2(4-RPy)2(H2O)2]. This conversion is favored at low temperature and was studied by NMR spectroscopy. A kinetic analysis of the aquation reaction was undertaken by stopped-flow measurements between 198 and 223 K for both 1 and 4, which revealed a first-order dependence on both the diiron compound and water. The oxygenation rates for the water-containing complexes are much faster than those for the corresponding anhydrous complexes, being 20-fold faster for 4 and 10-fold more rapid for 1. The presence or absence of water had little effect on the activation enthalpies, suggesting that the loss of water may not be necessary prior to dioxygen binding in the transition state.

Carboxylic Acids↗

PubMed2006

Analogues of Zinpyr-1 provide insight into the mechanism of zinc sensing.

Three compounds structurally related to the fluorescent zinc sensor Zinpyr-1 (ZP1) have been synthesized and characterized. In each of these ZinAlkylPyr (ZAP) analogues, an alkyl group (methyl, benzyl) replaces one of the metal-binding picolyl moieties in ZP1. The methyl-for-picolyl substitutions in ZAP1 and ZAP2 have a negligible effect on the optical spectrum of the fluorophore but elevate the quantum yields (Phi = 0.82 (ZAP1), 0.74 (ZAP2)) to values near that of Zn2+-saturated ZP1 (Phi = 0.92). The benzyl-for-picolyl substitution in ZAP3 similarly enhances the quantum yield (Phi = 0.52) relative to that of metal-free ZP1 (Phi = 0.38). As previously observed for methylated ZP1 sensors, methylation of the 6-position of the pyridyl ring diminishes the emission by lowering both the molar extinction coefficient and the quantum yield. Although these new ZAP compounds cannot detect Zn2+ fluorimetrically at neutral pH, complexation of Zn2+ does occur, as evidenced by sizable changes in the optical spectra. The ZAP1-3 probes can detect Zn2+ fluorimetrically at pH 9, indicating that proton-induced background emission obscures any Zn2+-induced fluorescence at pH 7. The tertiary amine groups in ZAP1-3 are less basic than those in ZP1, which implies that the additional pyridine rings are responsible for the emissive response to Zn2+ at pH 7.0.

Fluoresceins↗

PubMed2006

Mechanistic studies of the oxidative N-dealkylation of a substrate tethered to carboxylate-bridged diiron(II) complexes, [Fe2(mu-O2CAr(Tol))2(O2CAr(Tol))2(N,N-Bn2en)2].

Carboxylate-bridged diiron(II) centers activate dioxygen for the selective oxidation of hydrocarbon substrates in bacterial multicomponent monooxygenases. Synthetic analogues of these systems exist in which substrate fragments tethered to the diiron(II) core through attachment to an N-donor ligand are oxidized by transient species that arise following the introduction of O2 into the system. The present study describes the results of experiments designed to probe mechanistic details of these oxidative N-dealkylation reactions. A series of diiron(II) complexes with ligands N,N-(4-R-Bn)Bnen, where en is ethylenediamine, Bn is benzyl, and R-Bn is benzyl with a para-directing group R = Cl, F, CH3, t-Bu, or OCH3, were prepared. A Hammett plot of the oxygenation product distributions of these complexes, determined by gas chromatographic analysis, reveals a small positive slope of rho = +0.48. Kinetic isotope effect (KIE(intra)) values for oxygenation of [Fe2(mu-O2CAr(Tol))2(O2CAr(Tol))2(N,N-(C6H5CDH)2en)2] and [Fe2(mu-O2CAr(Tol))2(O2CAr(Tol))2(N,N-(C6H5CD2)(C6H5CH2)en)2] are 1.3(1) and 2.2(2) at 23 degrees C, respectively. The positive slope rho and low KIE(intra) values are consistent with a mechanism involving one-electron transfer from the dangling nitrogen atom in N,N-Bn2en to a transient electrophilic diiron intermediate, followed by proton transfer and rearrangement to eliminate benzaldehyde.

Binding Sites↗

PubMed2006

Dioxygen activation at non-heme diiron centers: characterization of intermediates in a mutant form of toluene/o-xylene monooxygenase hydroxylase.

We report the generation and characterization of an intermediate in a mutant form of the toluene/o-xylene monooxygenase hydroxylase component from Pseudomonas stutzeri OX1. The reaction of chemically reduced I100W variant in the presence of the coupling protein, ToMOD, with dioxygen was monitored by stopped-flow UV/visible spectroscopy. Rapid-freeze quench (RFQ) samples were also generated for EPR and Mössbauer spectroscopy. A transient species is observed in the UV/visible spectrum with an absorption maximum at 500 nm. EPR and Mössbauer spectra of RFQ samples identified this species as a diiron(III,IV) cluster spin-coupled to a neutral W radical. A diamagnetic precursor to the mixed-valent diiron(III,IV) was also observed at an earlier time point, with Mössbauer parameters typical of high-spin FeIII. We have tentatively assigned this antiferromagnetically coupled diiron(III) intermediate as a peroxo-bridged cluster, and this complex has also been observed in preliminary studies of the wild-type hydroxylase.

Catalysis↗

PubMed2006

Visualization of nitric oxide in living cells by a copper-based fluorescent probe.

Nitric oxide (NO) serves as a messenger for cellular signaling. To visualize NO in living cells, we synthesized a turn-on fluorescent probe for use in combination with microscopy. Unlike existing fluorescent sensors, the construct--a Cu(II) complex of a fluorescein modified with an appended metal-chelating ligand (FL)--directly and immediately images NO rather than a derivative reactive nitrogen species. Using spectroscopic and mass spectrometric methods, we established that the mechanism of the reaction responsible for the NO-induced fluorescence involves reduction of the complex to Cu(I) with release of the nitrosated ligand, which occurs irreversibly. We detected NO produced by both constitutive and inducible NO synthases (cNOS and iNOS, respectively) in live neurons and macrophages in a concentration- and time-dependent manner by using the Cu(II)-based imaging agent. Both the sensitivity to nanomolar concentrations of NO and the spatiotemporal information provided by this complex demonstrate its value for numerous biological applications.

Animals↗

PubMed2006

Interaction of nitric oxide with tetrathiolato iron(II) complexes: relevance to the reaction pathways of iron nitrosyls in sulfur-rich biological coordination environments.

The mechanism of formation of dinitrosyl iron complexes (DNICs) coordinated by cysteine residues at iron-sulfur protein sites has received little attention in the chemical literature. As a logical first step toward elucidating this mechanism and characterizing new iron-nitrosyl intermediates, we investigated the interaction of NO (g) and NO+ with iron-sulfur complexes chosen to mimic sulfur-rich iron sites in biology. The reaction of NO (g) with [Fe(StBu)4]2- cleanly affords the mononitrosyl complex, [Fe(StBu)3(NO)]- (1), a previously unknown species evoked in this chemistry. Reaction of [Fe(StBu)4]2- with NO derivatives, such as NO+, yields the corresponding dinitrosyl S-bridged Roussin red ester [Fe2(mu-StBu)2(NO)4] (2). The nitrosyl complexes 1 and 2 can chemically convert to the DNIC, [Fe(StBu)2(NO)2]- (3). The results should aid in the spectroscopic identification and elucidation of reaction pathways for the nitrosylation of iron in biologically related sulfur-rich coordination environments.

Crystallography, X-Ray↗