Daniela Buccella

Visualization of peroxynitrite-induced changes of labile Zn2+ in the endoplasmic reticulum with benzoresorufin-based fluorescent probes.

Zn(2+) plays essential roles in biology, and the homeostasis of Zn(2+) is tightly regulated in all cells. Subcellular distribution and trafficking of labile Zn(2+), and its inter-relation with reactive nitrogen species, are poorly understood due to the scarcity of appropriate imaging tools. We report a new family of red-emitting fluorescent sensors for labile Zn(2+), ZBR1-3, based on a benzoresorufin platform functionalized with dipicolylamine or picolylamine-derived metal binding groups. In combination, the pendant amines and fluorophore afford an [N3O] binding motif that resembles that of previously reported fluorescein-based sensors of the Zinpyr family, reproducing well their binding capabilities and yielding comparable Kd values in the sub-nanomolar and picomolar ranges. The ZBR sensors display up to 8.4-fold emission fluorescence enhancement upon Zn(2+) binding in the cuvette, with similar responses obtained in live cells using standard wide-field fluorescence microscopy imaging. The new sensors localize spontaneously in the endoplasmic reticulum (ER) of various tested cell lines, allowing for organelle-specific monitoring of zinc levels in live cells. Study of ER zinc levels in neural stem cells treated with a peroxynitrite generator, Sin-1, revealed an immediate decrease in labile Zn(2+) thus providing evidence for a direct connection between ER stress and ER Zn(2+) homeostasis.

Detection of Nitric Oxide and Nitroxyl with Benzoresorufin-Based Fluorescent Sensors

A new family of benzoresorufin-based copper complexes for fluorescence detection of NO and HNO is reported. The copper complexes, CuBRNO1-3, elicit 1.5-4.8-fold emission enhancement in response to NO and HNO. The three sensors differ in the nature of the metal-binding site. The photophysical properties of these sensors are investigated with assistance from density functional theory calculations. The fluorescence turn-on observed upon reaction with HNO is an unexpected result that is discussed in detail. The utility of the new sensors for detecting HNO and NO in HeLa cells and RAW 264.7 macrophages is demonstrated.

A Stable Tetraalkyl Complex of Nickel(IV)

membered ring can be exploited in ring-opening metathesis polymerization (ROMP). To characterize the strain-based reactivity more thoroughly, we have examined the behavior of 1 in the presence of homogeneous nickel(0). The study of reactions of alkenes with complexes of zerovalent nickel has a long and colorful history; we show herein that the strained alkene 1 allows us to create and crystallographically study the first tetraalkyl complex of nickel(IV). We prepared 1 by the method described previously. It is a mixture of two enantiomers defined by the helicity arising from the trans double bond in the eight-membered ring. To solubilize the Ni precursor, [Ni(cod)2] (cod= 1,5-cyclooctadiene) is treated with one equivalent of tri(tert-butyl)phosphine (PtBu3) in cyclohexane to give a pale yellow solution, presumably of [Ni(cod)(PtBu3)]. [4] When a cyclohexane solution of 1 is added to this Ni complex, the resulting solution darkens to maroon, and over time pale yellow crystals deposit. The crystallographically determined structure of this nickel alkene complex tris((5Z,11E)-dibenzo[a,e]cyclooctatetraene)nickel(0) (2) is shown in Figure 1a.

Mononuclear and dinuclear molybdenum and tungsten complexes of p-tert-butyltetrathiacalix[4]arene and p-tert-butyltetrasulfonylcalix[4]arene: facile cleavage of the calixarene ligand framework by nickel.

The reactivity of p-tert-butyltetrathiacalix[4]arene, [S4CalixBut(OH)4], and p-tert-butyltetrasulfonylcalix[4]arene, [(SO2)4CalixBut(OH)4], toward Mo(PMe3)5H2, Mo(PMe3)6, and W(PMe3)4(eta2-CH2PMe2)H has been used to synthesize a series of mononuclear molybdenum and tungsten calixarene compounds that feature both coordinatively saturated and unsaturated metal centers, such as [S4CalixBut(OH)2(O)2]M(PMe3)3H2 (M = Mo, W), [(SO2)4CalixBut(OH)2(O)2]M(PMe3)3H2, [S4CalixBut(OH)2(O)2]Mo(PMe3)3, [(SO2)4CalixBut(OH)2(O)2]Mo(PMe3)3, and [(SO2)4CalixBut(OH)(O)3]M(PMe3)3H. Comparison with the related {[CalixBut(OH)2(O)2]M} complexes indicates that the chemistry of the system is strongly influenced by the nature of the calixarene linker, that is, CH2, S, and SO2. For example, in contrast to the methylene-bridged calixarene system, the thiacalixarene and sulfonylcalixarene systems readily coordinate a second metal center to form homo- and heterodinuclear complexes, namely {[S4CalixBut(O)4]}[M(PMe3)3H2]2, {[(SO2)4CalixBut(O)4]}[Mo(PMe3)3H2]2 and {[S4CalixBut(O)4]}[Mo(PMe3)3H2][W(PMe3)3H2]. Of most interest, incorporation of nickel into [S4CalixBut(OH)2(O)2]M(PMe3)3H2 using Ni(PMe3)4 results in cleavage of a C- bond to give [(SArButOH)(SArButO)3][M(PMe3)3H2][Ni(PMe3)2], an observation that is of relevance to the role that nickel plays in hydrodesulfurization catalysis.

Monovalent indium in a sulfur-rich coordination environment: synthesis, structure and reactivity of tris(2-mercapto-1-tert-butylimidazolyl)hydroborato indium, [TmBut]In

[Tm(Bu(t))]In, the first structurally-characterized monovalent indium compound that features a sulfur-rich coordination environment, has been synthesized via treatment of InCl with [Tm(Bu(t))]K; in contrast to the thallium counterpart, the lone pair of [Tm(Bu(t))]In is a site of reactivity, thereby allowing formation of [Tm(Bu(t))]In-->B(C(6)F(5))(3) and [Tm(Bu(t))]In(kappa(2)-S(4)) upon treatment with B(C(6)F(5))(3) and S(8), respectively.

Reactions of Strained Hydrocarbons with Alkene and Alkyne Metathesis Catalysts

Here we describe the metathesis reactions of a strained eight-membered ring that contains both alkene and alkyne functionality. We find that the alkyne metathesis catalyst produces polymer through a ring-opening alkyne metathesis reaction that is driven by the strain release from the monomer. The strained monomer provides unusual reactivity with ruthenium-based alkene metathesis catalysts. We isolate a discrete trimeric species a Dewar benzene derivative that is locked in this form through an unsaturated cyclophane strap.

Molecular Structures of Thimerosal (Merthiolate) and Other Arylthiolate Mercury Alkyl Compounds

The molecular structure of sodium ethylmercury thiosalicylate (also known as thimerosal and Merthiolate) and related arylthiolate mercury alkyl compounds, namely PhSHgMe and PhSHgEt, have been determined by single crystal X-ray diffraction. (1)H NMR spectroscopic studies indicate that the appearance of the (199)Hg mercury satellites of the ethyl group of thimerosal is highly dependent on the magnetic field and the viscosity of the solvent as a consequence of relaxation due to chemical shift anisotropy.

Reactivity of Mo(PMe3)6 towards benzothiophene and selenophenes: new pathways relevant to hydrodesulfurization.

Mo(PMe(3))(6) cleaves a C-S bond of benzothiophene to give (kappa(2)-CHCHC(6)H(4)S)Mo(PMe(3))(4), which rapidly isomerizes to the olefin-thiophenolate and 1-metallacyclopropene-thiophenolate complexes, (kappa(1),eta(2)-CH(2)CHC(6)H(4)S)Mo(PMe(3))(3)(eta(2)-CH(2)PMe(2)) and (kappa(1),eta(2)-CH(2)CC(6)H(4)S)Mo(PMe(3))(4). The latter two molecules result from a series of hydrogen transfers and are differentiated according to whether the termini of the organic fragments coordinate as olefin or eta(2)-vinyl ligands, respectively. The reactions between Mo(PMe(3))(6) and selenophenes proceed differently from those of the corresponding thiophenes. For example, whereas Mo(PMe(3))(6) reacts with thiophene to give eta(5)-thiophene and butadiene-thiolate complexes, (eta(5)-C(4)H(4)S)Mo(PMe(3))(3) and (eta(5)-C(4)H(5)S)Mo(PMe(3))(2)(eta(2)-CH(2)PMe(2)), selenophene affords the metallacyclopentadiene complex [(kappa(2)-C(4)H(4))Mo(PMe(3))(3)(Se)](2)[Mo(PMe(3))(4)] in which the selenium has been completely abstracted from the selenophene moiety. Likewise, in addition to (kappa(1),eta(2)-CH(2)CC(6)H(4)Se)Mo(PMe(3))(4) and (kappa(1),eta(2)-CH(2)CHC(6)H(4)Se)Mo(PMe(3))(3)(eta(2)-CH(2)PMe(2)), which are counterparts of the species observed in the benzothiophene reaction, the reaction of Mo(PMe(3))(6) with benzoselenophene yields products resulting from C-C coupling, namely [kappa(2),eta(4)-Se(C(6)H(4))(CH)(4)(C(6)H(4))Se]Mo(PMe(3))(2) and [mu-Se(C(6)H(4))(CH)C(CH)(2)(C(6)H(4))](mu-Se)[Mo(PMe(3))(2)][Mo(PMe(3))(2)H].

Helical (5Z, 11E)-Dibenzo[a,e]cyclooctatetrene: A Spring-Loaded Monomer†

The introduction of a kink into an annulene cycle through having one of its double bonds in the trans configuration has severe consequences on the structure and stability of the resulting annulene. Modeling indicates that the p systems of either the [6]-annulene 1 or [8]-annulene 2 should wind into a helical conformation. The computed strain energy for 1 is circa 107 kcalmol , and for 2 is approximately 21 kcal mol . Molecule 1 is probably too unstable at room temperature to allow it to be isolated and studied. Herein we present a method to create the [8]-annulene 3, a benzannulated version of 2. Single crystal X-ray diffraction analysis of 3 reveals that it is a helix. The strain of this ring structure makes it functional as a monomer for ring opening metathesis polymerization (ROMP), yielding a new type of living, regioregular oligophenylene vinylene. Scheme 1 shows the method used to prepare 3, in which ophthaldehyde reacts with the 1,2-bis(triphenylphosphonium ylide) at room temperature. The isolated yield of 3 on a 20 mmol scale is about 20%. Annulene 3 is stable for weeks. The strain energy we calculate using density functional theory (DFT) for 3 relative to the cis,cis-dibenzo[a,e]cyclooctatetraene is about 18 kcalmol . The stabilization owing to benzannulation, along with the steric protection provided by the aromatic rings, allows us to isolate 3. Previous studies on the synthesis of dibenzo[a,e]cyclooctatetraene have not reported the presence of 3, and focus exclusively on the cis,cis isomer. Using H NMR spectroscopy, we observe only a trace amount of cis,cisdibenzo[a,e]cyclooctatetraene. The only compounds we could isolate from the reaction mixture were annulene 3, a small amount of isomerized cis,cis-dibenzo[a,e]cyclooctatetraene, and a substance which has been difficult to characterize and that we tentatively assign as the dimer 4 (Scheme 1). We suspect that earlier published work also likely produced 3, but in those experiments 3 had isomerized during workup and/or purification. Emphasizing both the kinetic stability and the thermodynamic instability of 3, we found that either UV irradiation, iodine, or traces of acid readily (and quantitatively) convert 3 into the more stable cis,cis-dibenzo[a,e]cyclooctatetraene. Wittig and co-workers synthesized dibenzo[a,e]cyclooctatetraene by a synthetic route that involved multiple Hofmann eliminations, and found that the material contained anomalous IR stretches. These resonances would be reasonable for a strained, trans double bond, such as the one found in trans-cyclooctene, but the structure was assigned to the more stable cis,cis-dibenzo[a,e]cyclooctatetraene. Subsequent studies, which prepared dibenzo[a,e]cyclooctatetraene by different methods, did not observe these anomalous IR stretching vibrations. Further comScheme 1. Double Wittig reaction to produce 3 and 4. Reaction conditions: a) LiOEt in EtOH, DMF, slow addition over 6 h.

Formation of Ternary Complexes with MgATP: Effects on the Detection of Mg2+ in Biological Samples by Bidentate Fluorescent Sensors

Fluorescent indicators based on β-keto-acid bidentate coordination motifs display superior metal selectivity profiles compared to current o-aminophenol-N,N,O-triacetic acid (APTRA) based chelators for the study of biological magnesium. These low denticity chelators, however, may allow for the formation of ternary complexes with Mg2+ and common ligands present in the cellular milieu. In this work, absorption, fluorescence, and NMR spectroscopy were employed to study the interaction of turn-on and ratiometric fluorescent indicators based on 4-oxo-4H-quinolizine-3-carboxylic acid with Mg2+ and ATP, the most abundant chelator of biological magnesium, thus revealing the formation of ternary complexes under conditions relevant to fluorescence imaging. The formation of ternary species elicits comparable or greater optical changes than those attributed to the formation of binary complexes alone. Dissociation of the fluorescent indicators from both ternary and binary species have apparent equilibrium constants in the low millimolar range at pH 7 and 25 °C. These results suggest that these bidentate sensors are incapable of distinguishing between free Mg2+ and MgATP based on ratio or intensity-based steady-state fluorescence measurements, thus posing challenges in the interpretation of results from fluorescence imaging of magnesium in nucleotide-rich biological samples.