Synthesis and solution studies of the complexes of trivalent lanthanides with the tetraazamacrocycle TETA-(PO)(2)

A new potentially multidentate hexaprotic ligand H(6)[TETA-(PO)(2)] has been prepared by reaction of ethylenediamine-N,N'-diacetic acid (EDDA), paraformaldehyde, and phosphinic acid; its coordination properties with three lanthanide ions (La(3+), Gd(3+), and Lu(3+)) have been explored. The structures of the complexes were studied in aqueous solution by potentiometric pH titrations and by (31)P NMR spectroscopy. Four acidity constants were determined potentiometrically in the range 2.5 < pH < 14. The four measured pK(a) values can be divided into two groups, and within each group the initial deprotonation was found to have little effect on the second. Variable temperature (31)P and (31)P[(1)H] EXSY NMR spectra showed that, for [Lu(TETA-(PO)(2))](3-), the two phosphorus atoms exist in different chemical environments and undergo an exchange process which is very fast on the NMR time scale at room temperature. This result is consistent with one of the phosphinate residues coordinating the metal ion and exchanging with a free analogue. In the case of [La(TETA-(PO)(2))](3-), only one temperature invariant signal is observed in (31)P NMR spectra; it corresponds to both phosphinate residues remaining uncoordinated to La(3+). The stability of [Ln(TETA-(PO)(2))](3-) has an order of La(3+) > Gd(3+) > Lu(3+). The coordination of one phosphinate residue to Lu(3+) brings the metal ion closer to the plane of four nitrogens and farther from the four carboxylate arms, resulting in [Lu(TETA-(PO)(2))](3-) having a lower stability than the corresponding La(3+) and Gd(3+) complexes. A pM-pH distribution diagram showed that introducing two phosphinate groups into TETA renders [Gd(TETA-(PO)(2))](3-) more stable than [Gd(TETA)](-). The selectivity factor of the ligand for Gd(3+) vs Ca(2+), Zn(2+), and Cu(2+) has been calculated, and the hydration number for [Dy(TETA-(PO)(2))](3-) has been measured by (17)O NMR spectroscopy to be zero.     

Fast reduction of a copper center in laccase by nitric oxide and formation of a peroxide intermediate

The rapid reduction of one of the copper atoms (type 2) of tree laccase by nitric oxide (NO) has been detected. Addition of NO to native laccase in the presence of oxygen leads to EPR changes consistent with fast reduction and slow reoxidation of this metal center. These events are paralleled by optical changes that are reminiscent of formation and decay of the peroxide intermediate in a fraction of the enzyme population. Formation of this species is only possible if the trinuclear copper cluster (type 2 plus type 3) is fully reduced. This condition can only be met if, as suggested previously, a fraction of the enzyme contains both type 3 coppers already reduced before addition of NO. Our data are consistent with this assumption. We have suggested recently that fast reduction of copper is the mechanism by which NO interacts with the oxidized dinuclear center in cytochrome c oxidase. The present experiments using laccase strongly support this view and suggest this reaction as a general mechanism by which copper proteins interact with NO. In addition, this provides an unexploited way to produce a stable peroxide intermediate in copper oxidases in which the full complement of copper atoms is present. This enables the O-O scission step in the catalytic cycle to be studied by electron addition to the peroxide derivative through the native electron entry site, type 1 copper.     

Study of the carbonyl products of terpene/OH radical reactions: detection of the 2,4-DNPH derivatives by HPLC-MS

The oxidation of the terpenes - and -pinene, limonene and ³-carene by hydroxyl radicals has been investigated in a fast-flow reactor coupled to a liquid nitrogen trap for collecting the carbonyl compounds. Identification of the products was performed via 2,4-dinitrophenylhydrazone (DNPH) derivatization of the carbonyls to form the mono- and di-DNPH derivatives, which were analysed by high-performance liquid chromatographic (HPLC)-DAD (diode array detector) and HPLC-mass spectrometry (HPLC-MS). Both electrospray ionization [ESI(–)] and atmospheric pressure chemical ionization [APCI(–)] were suitable for the detection of the DNPH derivatives of formaldehyde, acetaldehyde, myrtanal, campholene aldehyde, perillaldehyde, acetone, nopinone, trans-4-hydroxynopinone and 4-acetyl-1-methylcyclohexene. Also the mono-DNPH derivatives of the dicarbonyl compounds pinonaldehyde, endolim and caronaldehyde could be identified. The MS² spectra generated in the ion trap of the mass spectrometer allowed us to distinguish between aldehydes and ketones on the basis of the characteristic fragment ion m/z 163 for the aldehydes. For the quantitative analysis of the mono-DNPH derivatives, ESI(–) in combination with single ion monitoring (SIM) detection showed the lowest detection limits. For the quantification of the dicarbonyl compounds, the acid-sensitive di-DNPH derivatives had to be formed by keeping the acidity in the acid-catalysed derivatization reaction at about 1.7 mM H₂SO₄. Detection of these dicarbonyl compounds can only be performed by APCI(–) with somewhat lesser sensitivity than by HPLC-DAD.     

Assay development and high-throughput screening of caspases in microfluidic format

Caspase proteases are familiar targets in drug discovery. A common format for screening to identify caspase inhibitors employs fluorogenic or colorimetric tetra-peptide substrates in 96, 384, or 1536 -well microtiter plates. The primary motivation for increasing the number of wells per plate is to reduce the reagent cost per test and increase the throughput of HTS operations. There are significant challenges, however, to moving into or beyond the 1536-well format, such as submicroliter liquid handling, liquid evaporation, increased surface area-to-volume ratios, and the potential for artifacts and interference from small air-borne particles such as lint. Therefore, HTS scientists remain keenly interested in technologies that offer alternatives to the ever-shrinking microtiter plate well. Microfluidic assay technology represents an attractive option that, in theory, consumes only subnanoliter volumes of reagents per test. We have successfully employed a microfluidic assay technology in fluorogenic screening assays for several caspase isoforms utilizing the Caliper Technologies Labchip platform. Caspase-3 is used as a representative case to describe microfluidic assay development and initial high-throughput screening results. In addition, microfluidic screening and plate-based screening are compared in terms of reagent consumption, data quality, and ease of operation.     

Incorporation of a flexible, pyridine-functionalized Diaza-crown ether into discrete supramolecules via coordination-driven self-assembly

A flexible, pyridine-functionalized diaza-crown ether was self-assembled into discrete supramolecules of differing stoichiometries upon combination with various organoplatinum molecules. They are characterized by electrospray ionization mass spectrometry and (31)P[(1)H] and (1)H NMR. In one case, (1)H-(1)H NOE enhancements of a [1 + 1] assembled structure demonstrate the puckered shape of the macrocyclic ring. Despite its inherent flexibility, the dipyridyl-substituted 18-membered diaza-crown ligand prefers to self-assemble into closed systems when reacted with platinum-containing acceptors.     

SPECTRAL PERTURBATIONS OF THE AEQUOREA GREEN-FLUORESCENT PROTEIN

Abstract— In the jellyfish Aequorea, the green-fluorescent protein (GFP) functions as the in vivo bio-luminescence emitter via energy transfer from the photoprotein aequorin. Accumulated evidence has indicated that the Aequorea GFP is a relatively inflexible protein. Present evidence, however, indicates that the chromophore environment is readily accessible to a variety of external perturbants. Native Aequorea GFP has an absorbance maximum at 395 nm and a shoulder at 470 nm. In low ionic strength buffer at neutral pH and room temperature the 395/470 nm absorbance ratio is about 2.0. We show that this ratio is highly variable depending upon temperature, ionic strength, protein concentration, and pH. A maximum ratio of 6.5 (at a protein concentration of 18.6 mg/m/) and minimum of 0.42 (at a pH of 12.2) have been measured. In the latter case, the resulting absorption and excitation spectra resemble those of Renilla GFP in spectral shape (but not wavelength maximum). In all cases as the perturbant is varied the resulting spectra pass through a sharp isosbestic point, suggesting a relatively simple two-state mechanism. These spectral perturbations are fully reversible. On the basis of these results, we suggest that the chromophore binding site is conformationally flexible. pH-Dependent changes in the near-UV and visible circular dichroism spectra plus spectrophotometric titration of tyrosine residues lend additional support to this hypothesis.     

Intra- and intermolecular Fe-catalyzed dicarbofunctionalization of vinyl cyclopropanes

Design and implementation of the first (asymmetric) Fe-catalyzed intra- and intermolecular difunctionalization of vinyl cyclopropanes (VCPs) with alkyl halides and aryl Grignard reagents has been realized via a mechanistically driven approach. Mechanistic studies support the diffusion of alkyl radical intermediates out of the solvent cage to participate in an intra- or intermolecular radical cascade with a range of VCPs followed by re-entering the Fe radical cross-coupling cycle to undergo (stereo)selective C(sp²)–C(sp³) bond formation. This work provides a proof-of-concept of the use of vinyl cyclopropanes as synthetically useful 1,5-synthons in Fe-catalyzed conjunctive cross-couplings with alkyl halides and aryl/vinyl Grignard reagents. Overall, we provide new design principles for Fe-mediated radical processes and underscore the potential of using combined computations and experiments to accelerate the development of challenging transformations.

Design and implementation of the first (asymmetric) Fe-catalyzed intra- and intermolecular difunctionalization of vinyl cyclopropanes (VCPs) with alkyl halides and aryl Grignard reagents has been realized via a mechanistically driven approach.     

Facile Phenylphosphinidene Transfer Reactions from Carbene–Phosphinidene Zinc Complexes

Phosphinidenes [R-P] are convenient P₁ building blocks for the synthesis of a plethora of organophosphorus compounds. Thus far, transition-metal-complexed phosphinidenes have been used for their singlet ground-state reactivity to promote selective addition and insertion reactions. One disadvantage of this approach is that after transfer of the P₁ moiety to the substrate, a challenging demetallation step is required to provide the free phosphine. We report a simple method that enables the Lewis acid promoted transfer of phenylphosphinidene, [PhP], from NHC=PPh adducts (NHC=N-heterocyclic carbene) to various substrates to produce directly uncoordinated phosphorus heterocycles that are difficult to obtain otherwise.     

Unsupervised cell identification on multidimensional X‐ray fluorescence datasets

A novel approach to locate, identify and refine positions and whole areas of cell structures based on elemental contents measured by X‐ray fluorescence microscopy is introduced. It is shown that, by initializing with only a handful of prototypical cell regions, this approach can obtain consistent identification of whole cells, even when cells are overlapping, without training by explicit annotation. It is robust both to different measurements on the same sample and to different initializations. This effort provides a versatile framework to identify targeted cellular structures from datasets too complex for manual analysis, like most X‐ray fluorescence microscopy data. Possible future extensions are also discussed.