Catalytic transesterification of dialkyl phosphates by a bioinspired dicopper(II) macrocyclic complex

For a number of phosphoryltransfer enzymes, including the exonuclease subunit of DNA polymerase I, a mechanism involving two-metal ions and double Lewis-acid activation of the substrate, combined with leaving group stabilization, has been proposed. Inspired by the active site structure of this enzyme, we have designed as a synthetic phosphoryl transfer catalyst the dicopper(II) macrocyclic complex LCu(2). Crystal structures of complexes [(L)Cu(2)(mu-NO(3))(NO(3))](NO(3))(2) (1), [(L)Cu(2)(mu-CO(3))(CH(3)OH)](BF(4))(2) (2), and [(L)Cu(2)(mu-O(2)P(OCH(3))(2))(NO(3))](NO(3))(2) (3) illustrate various possibilities for the interaction of oxoanions with the dicopper(II) site. 1 efficiently promotes the transesterification of dimethyl phosphate (DMP) in CD(3)OD, k(cat) = 2 x 10(-)(4) s(-)(1) at 55 degrees C. 1 is the only available catalyst for the smooth transesterification of highly inert simple dialkyl phosphates. From photometric titrations and the pH dependence of reactivity, we conclude that a complex [(L)Cu(2)(DMP)(OCH(3))](2+) is the reactive species. Steric bulk at the -OR substituents of phosphodiester substrates O(2)P(OR)(2)(-) drastically reduces the reactivity of 1. This is explained with -OR leaving group stabilization by Cu coordination, an interaction which is sensitive to steric crowding at the alpha-C-atom of substituent R. A proposed reaction mechanism related to that of the exonuclease unit of DNA polymerase I is supported by DFT calculations on reaction intermediates. The complex [(L)Cu(3)(mu(3)-OH)(mu-CH(3)O)(2)(CH(3)CN)(2)](ClO(4))(3) (4) incorporates a [Cu(OH)(OCH(3))(2)(CH(3)CN)(2)](-) complex anion, which might be considered as an analogue of the [PO(2)(OCH(3))(2)(OCD(3))](2)(-) transition state (or intermediate) of DMP transesterification catalyzed by LCu(2).     

New approach to calibrating conductivity meters in the low conductivity range

There is currently a major issue with the calibration of conductivity meters used for high purity water: the lack of availability of a reference material or reference methods for low conductivity ranges (conductivity below 1 S cm^–1 at 25.0 °C, resistivity >1 M cm at 25.0 °C). This paper describes the current status of conductivity measurements in high purity water. A new and improved approach, currently being investigated, should allow us to make the calibration of conductivity meters used for low conductivity ranges traceable to the SI.Milipore, Milli-Q and Elix are registered trademarks of Millipore Corporation.     

Functionalized Self-Assembled Monolayers on Gold as Binding Matrices for the Screening of Antibody-Antigen Interactions

Two different types of -substituted alkanethiol/disulfide compounds have been used to prepare monolayer architectures on gold serving as platforms for the immobilization of receptor probe molecules – antibodies. These are: (i) carboxylic acid alkanethiols post-reacted with amino biotin to generate streptavidin surfaces, and (ii) N-hydroxysuccinimide-terminated disulfide surfaces. The properties of the monolayers, with and without attached receptor probe molecules, were analysed using infrared spectroscopy, ellipsometry, fluorescence scanning and atomic force microscopy. Several experimental parameters, such as condensation reagents, additives, probe and target concentrations and immobilization time, were systematically varied to determine the dynamic range and to optimize the sensitivity and signal-to-noise ratio of the biochip platforms. Fluorescence screening using Cy5-labelled antigens finally demonstrated that both surfaces could be successfully employed to immobilize the antibodies. The pros and cons of the two approaches are also discussed.Received November 15, 2002; accepted March 25, 2003 Published online July 28, 2003     

Azobridges from azines, XVII. Intra- and intermolecular [3+2] cycloaddition between nonstabilized azomethineimines and alkenes

Azo compounds 1 and 3 containing a CC-double bond in a parallel but distant position are quatemized by Me₃OBF₄ to 1,MeBF₄ and 3,MeBF₄, whereas MeI produces the cage compounds 2,HX and 4,HX. These [3+2] cycloadducts also are quantitatively formed from 1,MeBF₄ and 3,MeBF₄ with catalytic amounts of azo compounds. Intermolecular [3+2] cycloadditions occur with a mixture of DBH (5) or DBO (8), MeI and a variety of alkenes (→ HI) salts of (6, 7, 9 — 12). The intermediate azomethineimines, if stabilized by a fluorenylidene group, can be isolated (20, 22,24), but not, however, in the presence of a close parallel CC-bond (25 → 26).     

Solubility Enhancement of Low Soluble Biologically Active Compounds by β-Cyclodextrin and Dimethyl-β-cyclodextrin

The solubility enhancement of triflumizole by complexation with -cyclodextrin and with dimethyl--cyclodextrin is compared with respect to the different physico-chemical properties of the host molecules. Although the inclusion reaction constants are rather similar for both complexation reactions, a completely different temperature dependence of the host-guest interaction is observed, which indicates a change of the reaction mechanisms. Moreover, the influence of ethanol as cosolvent is studied.     

Determination of hydrazine, monomethylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine by nonaqueous capillary electrophoresis with amperometric detection

The present study is concerned with the application of nonaqueous capillary electrophoresis (NACE) with electrochemical detection (ED) to the separation and quantitative determination of hydrazine (Hy) and its methyl derivatives. The best performance of NACE-ED was found when using 4 mM sodium acetate/10 mM acetic acid/methanol: acetonitrile = 1:2 as the running buffer, with a bare platinum working electrode set at +1.0 V in an end-column amperometric detection cell. The choice and ratio of suitable solvents for the separation and injection media played an essential role for the performance characteristics of the method. The limits of detection for Hy, methylhydrazine, symmetrical dimethylhydrazine, and unsymmetrical dimethylhydrazine were 5, 2, 12, and 1 ng/mL, respectively. This is between one and two orders of magnitude lower than that achieved by previously reported CE-ED methods in aqueous buffer systems in conjunction with various types of chemically modified electrodes. The practical utility of the new NACE-ED methodology is demonstrated in terms of the determination of traces of Hys in spiked environmental samples containing a wide range of explosives and related compounds.     

Nonaqueous capillary electrophoresis with indirect electrochemical detection

Nonaqueous capillary electrophoresis (NACE) which makes use of organic solvents in place of conventional aqueous electrophoresis buffers is gaining increasing importance among modern separation techniques. Recently, it has been shown that amperometric detection in conjunction with acetonitrile-based NACE offers an extended accessible potential range and an enhanced long-term stability of the amperometric responses generated at solid electrodes. The present contribution takes advantage of the latter aspect to develop reliable systems for NACE with indirect electrochemical detection (IED). In this context, several compounds such as (ferrocenylmethyl)trimethylammonium perchlorate, tris(1,10-phenanthroline)cobalt(III) perchlorate and bis(1,4,7-triazacyclononane)nickel(II) perchlorate were studied regarding their suitability to act as electroactive buffer additives for IED in NACE. The performance characteristics for the respective buffer systems were evaluated. Tetraalkylammonium perchlorates served as model compounds for the optimization of the NACE-IED system. Target analytes choline and acetylcholine could easily be separated and determined by means of NACE-IED. In the case of a buffer system containing 10(-4) M tris(1,10-phenanthroline)cobalt(III) perchlorate the limits of detection were 2.5 x 10(-7) M and 4.6 x 10(-7) M for choline and acetylcholine, respectively. With the elaborated analytical procedure choline could be determined in pharmaceutical preparations.     

Validation studies and proficiency testing

Genetically modified organisms (GMOs) entered the European food market in 1996. Current legislation demands the labeling of food products if they contain <1% GMO, as assessed for each ingredient of the product. To create confidence in the testing methods and to complement enforcement requirements, there is an urgent need for internationally validated methods, which could serve as reference methods. To date, several methods have been submitted to validation trials at an international level; approaches now exist that can be used in different circumstances and for different food matrixes. Moreover, the requirement for the formal validation of methods is clearly accepted; several national and international bodies are active in organizing studies. Further validation studies, especially on the quantitative polymerase chain reaction methods, need to be performed to cover the rising demand for new extraction methods and other background matrixes, as well as for novel GMO constructs.     

Time-resolved fluorescence analysis of the mobile flavin cofactor in p -hydroxybenzoate hydroxylase

Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the ‘in’ conformation with the isoalloxazine ring located in the active site, and the ‘out’ conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the ‘in’ and ‘out’ conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes.