Synthesis, conformational stability and molecular structure of 4-aryl- and 4,5-diaryl-1,8-bis(dimethylamino)naphthalenes.

4-Bromo- and 4,5-dibromo-1,8-bis(dimethylamino)naphthalenes were arylated with arylboronic acids under Suzuki reaction conditions to provide 4-aryl- and 4,5-diaryl-1,8-bis(dimethylamino)naphthalenes, respectively. The interaction of 4,5-dibromo-1,8-bis(dimethylamino)naphthalene with pyridin-3-ylboronic acid was accompanied by heterocyclization leading unexpectedly to the formation of N3,N3,N4,N4-tetramethylacenaphtho[1,2-b]pyridine-3,4-diamine. Dynamic 1H NMR experiments showed fast interconversion between syn and anti conformers of 4,5-diaryl-1,8-bis(dimethylamino)naphthalenes in CDCl3 solution at room temperature. The free energy of the rotational isomerization was determined to be ∼14.0 kcal mol-1 for 4,5-di(m-tolyl) and 4,5-di(naphthalen-2-yl) derivatives. X-ray analysis of 4,5-diaryl-1,8-bis(dimethylamino)naphthalenes revealed a high degree of structural deformation due to internal steric repulsions between both peri-dimethylamino and peri-aryl groups. In crystals, 4,5-di(naphthalen-1-yl)-1,8-bis(dimethylamino)naphthalene molecules exist exclusively in the most stable anti-out form, while for 4,5-di(naphthalen-2-yl) and 4,5-di(m-tolyl) counterparts, only the syn-form is realized. The introduction of two peri-aryl substituents in the 1,8-bis(dimethylamino)naphthalene scaffold affected the basic properties, making the 4,5-diphenyl derivative 0.7 pKa units less basic. The protonation of 4,5-diaryl-1,8-bis(dimethylamino)naphthalenes leads to dramatic changes in their structures. Compared to the corresponding bases, the inter-nitrogen distance in these salts noticeably decreases whereas peri-aromatic rings move away from each other demonstrating the so-called "clothespin effect". This lowers the barriers of syn/anti-isomerization; as a result, protonated molecules with peri-m-tolyl and even peri-(naphthalen-2-yl) substituents exist in crystals as mixtures of rotamers.

Magnetically bistable cobalt-dioxolene complexes with a tetradentate N-donor base.

Synthesis and magnetic characterization of a family of cobalt-dioxolene complexes [(Me2TPA)Co(36-DBCat)] (1), [(Me2TPA)Co(36-DBCat)](PF6) (2) and [(Me2TPA)Co(diox-(OMe)3)](BPh4) (3) (Me2TPA = bis(6-methyl-2-pyridyl)methyl-(2-pyridylmethyl)amine; 36-DBCat = dianion of 3,6-di-tert-butylcatechol; diox-(OMe)3 - 2,5-di-tert-butyl-3,3,4-trimethoxy-6-oxocyclohexa-1,4-dienolate) is reported. The neutral complex 1 is found to form hexa- (CoO2N4, 1a) and pentacoordinated (CoO2N3, 1b) isomers. Variable temperature single crystal X-ray diffraction analysis of 1a and 1b clearly indicates the presence of the high-spin divalent metal ion and the dianionic catecholate form of the dioxolene ligand. Oxidation of 1 by ferrocenium hexafluorophosphate results in the formation of the ionic octahedral complex 2, demonstrating thermally induced valence-tautomeric transition (ls-CoIII-36-DBCat ⇄ hs-CoII-36-DBSQ) in the solid state with T1/2 = 175 K (36-DBSQ = radical-anionic semiquinonate form of the redox-ligand). In contrast, aerial oxidation of 1 is accompanied by changes in the structure of dioxolene resulting in oxocyclohexadienolate ligand and the formation of an ionic complex of high-spin divalent cobalt (3). Compounds 1a, 1b, and 3 are found to demonstrate a field-induced single-ion magnet behavior. The analysis of the electronic structures of 1, 2 and 3 with the aid of DFT and SA-CASSCF/NEVPT2 calculations is also given.

Investigation of Temporal Apparent C4 Sugar Change in Manuka Honey.

New Zealand manuka honeys are known for their propensity to increase apparent C4 sugar content during storage. Depending on the particular storage regime and the initial content of dihydroxyacetone (DHA) in honey, the ready-to-market product often fails the C4 sugar test because of the above phenomenon. We have used DHA labeled with a radioactive 14C isotope in a set of honeys subject to an incubation experiment. These honeys were analyzed for DHA, methylglyoxal (MG), hydroxymethylfurfural (HMF), apparent C4 sugars, and 14C scintillation counts over a period of 18 months. The major conclusion of this experiment is that neither DHA nor MG is responsible for the δ13C shift in the honey protein extract. There must be some other yet unknown substance of manuka honey, which binds to the protein and causes negative δ13C shift. One identified candidate for such a binding is carbon dioxide.

Proton-induced fluorescence in modified quino[7,8-h]quinolines: dual sensing for protons and π-donors.

The synthesis, as well as spectral, structural and photoluminescence properties of dipyrido[3,2-e:2',3'-h]acenaphthene 5 and quinazolino[7,8-h]quinazolines 6 as representatives of the bidentate -N[double bond, length as m-dash]/-N[double bond, length as m-dash] superbases, are reported. These nitrogen bases being more rigid (5) or π-extended (6) analogs of optically-mute quino[7,8-h]quinoline are both active in terms of fluorescence with quantum yields up to φ = 0.71-0.77. At the same time, their luminescence behavior is opposite to that of peri-NMe2/NMe2 naphthalene proton sponges and their hybrid NMe2/-N[double bond, length as m-dash] analogs. Although 5 and 6 exhibit visible region emission upon protonation, for the hybrid systems the fluorescence is manifested only for bases. The most remarkable observation is that the fluorescence of compound 5 can be switched on not only by means of organic or inorganic acids, but also through the formation of chelate complexes with such weak H-donors as water and primary alcohols. It was disclosed that water is present in the complex as a cluster comprising 8 interconnected H2O molecules. Overall, the studied compounds demonstrate a previously unobserved type of dual mode optical response, H-sensing (emission enhancement in 5 and 6 on protonation) and π-sensing (emission quenching in 5H+ and 6H+ on coordination with π-donors). This work seems to be an important contribution to areas such as chemosensorics, the creation of new ligands, hydrogen transfer and some other phenomena representing different types of supramolecular interactions.

Economically Motivated Adulteration of Lemon Juice: Cavity Ring Down Spectroscopy in Comparison with Isotope Ratio Mass Spectrometry: Round-Robin Study.

Background: Economically motivated adulteration (EMA) of foods has become an increasing concern in recent years, with lemon juice as a popular target. Objective and Method: In this work, an optimized preparation procedure for the isolation of citric acid from lemon juice was validated using elemental analyzer-isotope ratio MS (EA-IRMS) to detect adulteration with exogenous citric acid. Additionally, 69 imported lemon juice samples were evaluated using combustion module-cavity ring down spectrometry (CM-CRDS) and compared with the well-established EA-IRMS. Equivalency of CM-CRDS to EA-IRMS was further demonstrated by conducting a round-robin study involving eight laboratories throughout the United States, Canada, and New Zealand. Results: Overall, the results obtained for CM-CRDS were statistically indistinguishable from the results obtained using EA-IRMS for EMA lemon juice analysis. Conclusions: Therefore, CM-CRDS is a viable option for this application. Highlights: The CM-CRDS instrumentation is easy to operate, robust, and provides δ13C values comparable to EA-IRMS for citrate analysis. Through a multi-laboratory exercise, CM-CRDS was shown to be an alternative to EA-IRMS in the detection of economic adulteration of lemon juice.

Lessons learned from inter-laboratory studies of carbon isotope analysis of honey.

Forensic application of carbon isotope ratio measurements of honey and honey protein to investigate the degree of adulteration with high fructose corn syrup or other C4 plant sugars is well established. These measurements must use methods that exhibit suitable performance criteria, particularly with regard to measurement uncertainty and traceability - low levels of adulteration can only be detected by methods that result in suitably small measurement uncertainties such that differences of 1‰ or less can be reliably detected. Inter-laboratory exercises are invaluable to assess the state-of-the art of measurement capabilities of laboratories necessary to achieve such performance criteria. National and designated metrology institutes from a number of countries recently participated in an inter-laboratory assessment (CCQM-K140) of stable carbon isotope ratio determination of bulk honey. The same sample material was distributed to a number of forensic isotope analysis laboratories that could not participate directly in the metrological comparison. The results from these studies have demonstrated that the majority of participants provided isotope delta values with acceptable performance metrics; that all participants ensured traceability of their results; and that where measurement uncertainties were reported; these were fit-for-purpose. A number of the forensic laboratories only reported precision rather than full estimates of measurement uncertainty and this was the major cause of the few instances of questionable performance metrics. Reporting of standard deviations in place of measurement uncertainties is common practice outside metrology institutes and the implications for interpretations of small differences in isotopic compositions are discussed. The results have also highlighted a number of considerations that are useful for organisers of similar inter-laboratory studies in the future.

Direct Comparison of Cavity Ring Down Spectrometry and Isotope Ratio Mass Spectrometry for Detection of Sugar Adulteration in Honey Samples.

In the last several years, economically motivated adulteration (EMA) of foods including honey has received increased attention. The addition of inexpensive sweeteners such as high fructose corn syrup or cane sugar to honey is still encountered despite scientific methods that can routinely detect this type of adulteration. The standard method for detection of these adulterants utilizes isotope ratio mass spectrometry (IRMS); however, this technique requires an elevated degree of technical knowledge for operation as well as a high cost for purchase and maintenance. Cavity ring down spectroscopy (CRDS) has demonstrated potential for this type of analysis and is less expensive with simpler operation. This study evaluates CRDS for the detection of low-cost sweeteners added to honey and compares the performance of CRDS to IRMS. Several honey samples were analyzed, and the advantages and limitations specific to CRDS were evaluated. Overall, the results indicate that CRDS provides a performance comparable to the benchmark technique IRMS for EMA honey analysis.

10-Dimethylamino Derivatives of Benzo[h]quinoline and Benzo[h]quinazolines: Fluorescent Proton Sponge Analogues with Opposed peri-NMe2/-N═ Groups. How to Distinguish between Proton Sponges and Pseudo-Proton Sponges.

For the first time, 10-dimethylamino derivatives of benzo[h]quinoline 6 and benzo[h]quinazoline 7a-e as mixed analogues of archetypal 1,8-bis(dimethylamino)naphthalene ("proton sponge") 1 and quino[7,8-h]quinoline 2a have been examined. Similar to 1 and 2, compounds 6 and 7 display rather high basicity, forming chelated monocations. At the same time, unexpected specifics of the protonated NMe2/-N═ systems consist of a strong shift of the NH proton to the 10-NMe2 group, contrary to the "aniline-pyridine" basicity rule. In case of 4H(+), a rapid migration (in the NMR time scale) of the NH proton between two nitrogen atoms along the N-H···N hydrogen bond was registered at room temperature and frozen below -30 °C with the proton fixed on the NMe2 group. Two different approaches for classification of strong neutral nitrogen organic bases as proton sponges (kinetically inert compounds) or pseudo-proton sponges (kinetically active) are discussed. On this basis, benzoquinoline 6 was identified as staying closer to pseudo-proton sponges while 7a-e to proton sponges due to the presence in their molecules of bulky substituents in the pyrimidine ring. Other remarkable peculiarities of 6 and 7 are their yellow color and luminescence in the visible region distinguishing them from colorless 1 and 2a.

Characterization of a mineral coating of the plant Dyerophytum indicum.

The mineral coating of Dyerophytum indicum Kuntze (Plumbaginaceae) was analyzed by isotope ratio mass spectrometry, X-ray diffraction, thermogravimetric and differential thermal analysis, and scanning electron microscopy/energy-dispersive X-ray spectroscopy methods. Its composition was found to be similar to those of the carbonate mixtures isolated from rotting cacti and speleothems. The coating consisted of three major phases (monohydrocalcite, nesquehonite, and calcite), a minor phase of hydromagnesite, and traces of silica and sylvite. This is the first time that the occurrence of monohydrocalcite, nesquehonite, and hydromagnesite in a living higher plant has been reported. A possible mechanism of the formation of the coating is also discussed.

Interaction of antithyroid drugs with bovine serum albumin: electrophoretic and fluorimetric study.

The pre-equilibrium capillary zone electrophoretic (pre-eq CZE) method to determine association constants of active anionic forms of antithyroid drugs: 6-n-propyl-2-thiouracil (PTU), 6-methyl-2-thiouracil (MTU), 2-thiouracil (TU) with bovine serum albumin (BSA) under physiological pH 7.4 has been developed for the first time. Using the decrease of the selective electrochromatographic peak area of a drug anionic form due to binding with BSA the association constants K of the binary BSA complexes were calculated. It has been found that the binding constants (log K) of BSA with TU, MTU, and PTU are equal to 2.99, 1.85, and 2.11, respectively. The interaction of PTU, MTU, TU, 2-mercapto-1-methylimidazole (MMI), and ethyl-3-methyl-2-thionoimidazoline-1-carboxylate (Carb), which is considered to be a prodrug for MMI, with BSA has been investigated under physiological conditions by means of fluorescence spectroscopy. Fluorescence emission spectra of BSA in the presence of thioamides recorded at 295 nm excitation wavelength clearly show that the studied drugs act as quenchers, except MMI, which acts as quencher when being excited at 280 nm. The 295 nm light excites tryptophan residues, while the 280 nm light excites both tryptophan and tyrosine residues. The binding constants (log K) of BSA with PTU, MTU, TU, MMI, and Carb have been found to be 4.51, 4.30, 4.30, 2.64, and 4.32, respectively.

Oxidase activity of a flavin-dependent thymidylate synthase.

Flavin-dependent thymidylate synthases (FDTS) catalyze the production of dTMP from dUMP and N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (CH(2)H(4)folate). In contrast to human and other classical thymidylate synthases, the activity of FDTS depends on a FAD coenzyme, and its catalytic mechanism is very different. Several human pathogens rely on this recently discovered enzyme, making it an attractive target for novel antibiotics. Like many other flavoenzymes, FDTS can function as an oxidase, which catalyzes the reduction of O(2) to H(2)O(2), using reduced NADPH or other reducing agents. In this study, we exploit the oxidase activity of FDTS from Thermatoga maritima to probe the binding and release features of the substrates and products during its synthase activity. Results from steady-state and single-turnover experiments suggest a sequential kinetic mechanism of substrate binding during FDTS oxidase activity. CH(2)H(4)folate competitively inhibits the oxidase activity, which indicates that CH(2)H(4)folate and O(2) compete for the same reduced and dUMP-activated enzymatic complex (FDTS-FADH(2)-NADP(+)-dUMP). These studies imply that the binding of CH(2)H(4)folate precedes NADP(+) release during FDTS activity. The inhibition constant of CH(2)H(4)folate towards the oxidase activity was determined to be rather small (2 microm), which indicates a tight binding of CH(2)H(4)folate to the FDTS-FADH(2)-NADP(+)-dUMP complex.

The relationships between oxidase and synthase activities of flavin dependent thymidylate synthase (FDTS).

New findings lead to a revised understanding of the substrates' binding order, the role of the substrate as an activator, and the observed lag phase in the FDTS catalyzed reaction.

Thymidyl biosynthesis enzymes as antibiotic targets.

The two long-known "classical" enzymes of uridyl-5-methylation, thymidylate synthase and ribothymidyl synthase, have been joined by two alternative methylation enzymes, flavin-dependent thymidylate synthase and folate-dependent ribothymidyl synthase. These two newly discovered enzymes have much in common: both contain flavin cofactors, utilize methylenetetrahydrofolate as a source of methyl group, and perform thymidylate synthesis via chemical pathways distinct from those of their classic counterparts. Several severe human pathogens (e.g., typhus, anthrax, tuberculosis, and more) depend on these "alternative" enzymes for reproduction. These and other distinctive properties make the alternative enzymes and their corresponding genes appealing targets for new antibiotics.

Proton transfer in gramicidin water wires in phospholipid bilayers: attenuation by phosphoethanolamine.

The transfer of protons in water wires was studied in native gramicidin A (gA), and in the SS- and RR-diastereoisomers of dioxolane-linked gA channels (SS and RR channels). These peptides were incorporated into membranes comprised of distinct combinations of phospholipid headgroups and acyl chains. Quantitative relationships between single channel conductances to H+ (g(H)) and [H+] were determined in distinct phospholipid membranes, and are in remarkable contrast with results previously obtained in monoglyceride membranes. In particular: 1), g(H)-[H+] relationships for the various gA channels in distinct phospholipid membranes are well fitted by single adsorption isotherms. A simple kinetic model assuming mono-occupancy of channels by protons fits said relationships. This does not occur with monoglyceride membranes. 2), Under nonsaturating [H+], g(H) is approximately 1 order of magnitude larger in phospholipid than in monoglyceride membranes. 3), Differences between rates of H+ transfer in various gA channels are still present but considerably attenuated in phospholipid relative to monoglyceride membranes. 4), Charged phospholipid headgroups affect g(H) via changes in [H+] at the membrane/solution interfaces. 5), Phosphoethanolamine groups caused a marked attenuation of g(H) relative to membranes with other phospholipid headgroups. This attenuation is voltage-dependent and tends to saturate H+ currents at voltages larger than 250 mV. This effect is likely to occur by limiting the access and exit of H+ in and out of the channel due to relatively strong oriented H-bonds between waters and phosphoethanolamine groups at channel interfaces. The differential effects of phospholipids on proton transfer could be reasoned by considering solvation effects of side chain residues of gramicidin channels by double acyl chains and by the presence of polar headgroups facilitating the entrance/exit of protons through the channel mouths.

Kinetic isotope effects of proton transfer in aqueous and methanol containing solutions, and in gramicidin A channels.

The electrochemical conductivities of HCL and DCI were measured in: H(2)O and D(2)O; in methanol and fully deuterated methanol; and in water-methanol solutions. The single channel conductances to H(+) (g(H)) and D(+) (g(D)) in various gramicidin A (gA) ion channels incorporated in glycerylmonooleate planar bilayers were also measured. Kinetic isotope effects (KIE) were estimated from the ratio of conductivity measurements. In 1 and 5 M HCl aqueous solutions and in 1 M HCl+3.7 M methanol, the KIE ( approximately 1.35) is not different from values previously determined in dilute acid solutions. This suggests that the mobility of protons in those solutions is largely determined by proton transfer. In 10 M HCl, however, where the mobility of protons is likely to be determined by hydrodynamic diffusion, the measured KIE is considerably larger (1.47). Possible causes for this effect are discussed. The KIE of proton conductivities in 5 and 50 mM HCl in methanol and d-methanol is approximately 1.15. This is considerably smaller than the ratio between conductivities of 5 mM KCl in methanol and d-methanol (1.24). The KIE values (1.22-1.37) for g(H) in gA channels in 1 M HCl are significantly larger than for other monovalent cations and consistent with H(+) transfer. Methanol reduces g(H) in gA channels. The KIE of this effect is not different from the one measured in the absence of methanol. Possible mechanisms for the methanol-induced block of H(+) conductivities in solution and gA channels are discussed.

Proton transfer in gramicidin channels is modulated by the thickness of monoglyceride bilayers.

The thickness of monoglyceride planar bilayers has significant effects on the transfer of protons in both native gramicidin A (gA) and in covalently linked SS- and RR-dioxolane-linked gA proteins. Planar bilayers with various thicknesses were formed from an appropriate combination of monoglyceride with various fatty acid lengths and solvent. Bilayer thicknesses ranged from 25 A (monoolein in squalene) to 54 A (monoeicosenoin in decane). Single-channel conductances to protons (g(H)) were measured in the concentration range of 10-5000 mM HCl. In native gA as well as in RR channels, the shape of the log(g(H))-log([H(+)]) relationships was nonlinear and remained basically unaltered in monoglyceride bilayers with various thicknesses. For both native gA and RR channels, g(H) values were systematically and significantly larger in thin than in thick bilayers. By contrast, the shape of the log(g(H))-log([H(+)]) relationships in the SS channel was linear (with a slope considerably smaller than 1) in thick (>37 A) bilayers. However, in thin (<37 A) bilayers these plots became nonlinear and g(H) values approached those obtained in native gA channels. The linearization of the log-log plots in the SS channel in thick bilayers is a consequence of a dramatic increase (instead of a decrease as in native gA and RR channels) of g(H) in these bilayers in [H(+)] <1 M. The gating characteristics of the various gA channels as a function of bilayer thickness followed the same pattern as described previously. It was noticed, however, that in the thickest monoglyceride bilayer used in this study, both the SS- and RR-dioxolane-linked channels opened in a mode of bursting activity instead of remaining in the open state as in thin bilayers. It is proposed that the thickness of monoglyceride bilayers modulates proton transfer in native gA channels by a combination of factors including the access resistances of channels to H(+), and fluctuations in both the structure of the lipid bilayer and in the distance between gA monomers. The differential effects of relatively thick monoglyceride bilayers on proton transfer in both dioxolane-linked gA channels must relate to distinct interactions between the bilayers and the SS and RR dioxolanes.

Thermodynamic view of activation energies of proton transfer in various gramicidin A channels.

The temperature dependencies (range: 5-45 degrees C) of single-channel proton conductances (g(H)) in native gramicidin A (gA) and in two diastereoisomers (SS and RR) of the dioxolane-linked gA channels were measured in glycerylmonooleate/decane (GMO) and diphytanoylphosphatidylcholine/decane (DiPhPC) bilayers. Linear Arrhenius plots (ln (g(H)) versus K(-1)) were obtained for the native gA and RR channels in both types of bilayers, and for the SS channel in GMO bilayers only. The Arrhenius plot for proton transfer in the SS channel in DiPhPC bilayers had a break in linearity around 20 degrees C. This break seems to occur only when protons are the permeating cations in the SS channel. The activation energies (E(a)) for proton transfer in various gA channels (approximately 15 kJ/mol) are consistent with the rate-limiting step being in the channel and/or at the membrane-channel/solution interface, and not in bulk solution. E(a) values for proton transfer in gA channels are considerably smaller than for the permeation of nonproton currents in gA as well as in various other ion channels. The E(a) values for proton transfer in native gA channels are nearly the same in both GMO and DiPhPC bilayers. In contrast, for the dioxolane linked gA dimers, E(a) values were strongly modulated by the lipid environment. The Gibbs activation free energies (Delta G(#)(o)) for protons in various gA channels are within the range of 27-29 kJ/mol in GMO bilayers and of 20-22 kJ/mol in DiPhPC bilayers. The largest difference between Delta G(#)(o) for proton currents occurs between native gA (or SS channels) and the RR channel. In general, the activation entropy (Delta S) is mostly responsible for the differences between g(H) values in various gA channels, and also in distinct bilayers. However, significant differences between the activation enthalpies (Delta H(#)(o)) for proton transfer in the SS and RR channels occur in distinct membranes.