Molecular Mechanisms in Metal Oxide Nanoparticle-Tryptophan Interactions.

One of the crucial metabolic processes for both plant and animal kingdoms is the oxidation of the amino acid tryptophan (TRP) that regulates plant growth and controls hunger and sleeping patterns in animals. Here, we report revolutionary insights into how this process can be crucially affected by interactions with metal oxide nanoparticles (NPs), creating a toolbox for a plethora of important biomedical and agricultural applications. Molecular mechanisms in TRP-NP interactions were revealed by NMR and optical spectroscopy for ceria and titania and by X-ray single-crystal study and a computational study of model TRP-polyoxometalate complexes, which permitted the visualization of the oxidation mechanism at an atomic level. Nanozyme activity, involving concerted proton and electron transfer to the NP surface for oxides with a high oxidative potential, like CeO2 or WO3, converted TRP in the first step into a tricyclic organic acid belonging to the family of natural plant hormones, auxins. TiO2, a much poorer oxidant, was strongly binding TRP without concurrent oxidation in the dark but oxidized it nonspecifically via the release of reactive oxygen species (ROS) in daylight.

Metabolic labeling of hyaluronan: Biosynthesis and quantitative analysis of 13C,15N-enriched hyaluronan by NMR and MS-based methods.

Hyaluronan (HA), a member of the GAG family of glycans, has many diverse biological functions that vary a lot depending on the length of the HA chain and its concentration. A better understanding of the structure of different-sized HA at the atomic level is therefore crucial to decipher these biological functions. NMR is a method of choice for conformational studies of biomolecules, but there are limitations due to the low natural abundance of the NMR active nuclei 13C and 15N. We describe here the metabolic labeling of HA using the bacterium Streptococcus equi subsp. Zooepidemicus and the subsequent analysis by NMR and mass spectrometry. The level of 13C and 15N isotope enrichment at each position was determined quantitatively by NMR spectroscopy and was further confirmed by high-resolution mass spectrometry analysis. This study provides a valid methodological approach that can be applied to the quantitative assessment of isotopically labeled glycans and will help improve detection capabilities and facilitate future structure-function relationship analysis of complex glycans.

Insights on the reactivity of chondroitin and hyaluronan toward 1,4-butanediol diglycidyl ether.

Hyaluronic acid (HA) cross-linked with 1,4-butanediol diglycidyl ether (BDDE) are hydrogels with many biomedical applications. Degree of substitution, cross-linking and substitution position of the cross-linker might influence the properties of the hydrogels. We showed earlier that the most common substitution position of the cross-linker on the hyaluronan chain was the 4-hydroxyl of N-acetylglucosamine. This result has led us to investigate unsulfated chondroitin (CN) which only differ from HA in the primary structure by the configuration at C4 of the aminoglycan. In the present study, we have investigated (i) the substitution positions of the cross-linker in CN using NMR and LC-MS and compared the results to the data obtained for HA (ii) the effect of alkali on the 13C and 1H chemical shifts in CN and HA (iii) the temperature coefficients and chemical shifts of hydroxyl protons in CN and HA. In CN, the 2-hydroxyl of glucuronic acid and 6-hydroxyl of N-acetylgalactosamine were found to be the major sites of substitution by BDDE. Moreover, while chondroitinase was not able to cleave HA tetrasaccharide substituted at the 4-hydroxyl GlcNAc reducing end by BDDE, it is able to degrade CN-BDDE down to disaccharide units.

A Dual-Promoter Gene Orchestrates the Sucrose-Coordinated Synthesis of Starch and Fructan in Barley.

Sequential carbohydrate synthesis is important for plant survival because it guarantees energy supplies for growth and development during plant ontogeny and reproduction. Starch and fructan are two important carbohydrates in many flowering plants and in human diets. Understanding this coordinated starch and fructan synthesis and unraveling how plants allocate photosynthates and prioritize different carbohydrate synthesis for survival could lead to improvements to cereals in agriculture for the purposes of greater food security and production quality. Here, we report a system from a single gene in barley employing two alternative promoters, one intronic/exonic, to generate two sequence-overlapping but functionally opposing transcription factors, in sensing sucrose, potentially via sucrose/glucose/fructose/trehalose 6-phosphate signaling. The system employs an autoregulatory mechanism in perceiving a sucrose-controlled trans activity on one promoter and orchestrating the coordinated starch and fructan synthesis by competitive transcription factor binding on the other promoter. As a case in point for the physiological roles of the system, we have demonstrated that this multitasking system can be exploited in breeding barley with tailored amounts of fructan to produce healthy food ingredients. The identification of an intron/exon-spanning promoter in a hosting gene, resulting in proteins with distinct functions, adds to the complexity of plant genomes.

1D NMR methods for determination of degree of cross-linking and BDDE substitution positions in HA hydrogels.

Hyaluronic acid polymers cross-linked with BDDE are today among the most used hydrogels for biomedical applications. The physical properties of the hydrogels depend, among other parameters, on the degree of cross-linking of HA. Another parameter likely to affect the physical properties is the substitution position of the linker on the HA functional groups. A NMR-based method for the determination of these parameters in hyaluronic acid hydrogels is presented. The method is based on the degradation of HA cross linked hydrogels by chondroitinase ABC followed by one-dimensional 1H and 13C NMR analysis. The necessary structural information to obtain both the degree of cross-linking and the substitution positions can be obtained from the same NMR sample and no chromatographic separation step is required prior to NMR analysis.

Determination of substitution positions in hyaluronic acid hydrogels using NMR and MS based methods.

In hydrogels of cross-linked polysaccharides, the total amount of cross-linker and the degree of cross-linking influence the properties of the hydrogel. The substitution position of the cross-linker on the polysaccharide is another parameter that can influence hydrogel properties; hence methods for detailed structural analysis of the substitution pattern are required. NMR and LC-MS methods were developed to determine the positions and amounts of substitution of 1,4-butanediol diglycidyl ether (BDDE) on hyaluronic acid (HA), and for the first time it is shown that BDDE can react with any of the four available hydroxyl groups of the HA disaccharide repeating unit. This was achieved by studying di-, tetra-, and hexasaccharides obtained from degradation of BDDE cross-linked HA hydrogel by chondroitinase. Furthermore, amount of linker substitution at each position was shown to be dependent on the size of the oligosaccharides. For the disaccharide, substitutions were predominantly at ΔGlcA-OH2 and GlcNAc-OH6 while in the tetra- and hexasaccharides, it was mainly at the reducing end GlcNAc-OH4. In the disaccharide there was no substitution at this position. Since chondroitinase is able to completely hydrolyse non-substituted HA into unsaturated disaccharides, these results indicate that the enzyme is prevented to cleave on the non-reducing side of an oligosaccharide substituted at the reducing end GlcNAc-OH4. The procedure can be adopted for the determination of substitution positions in other types of polymers.

Modification and cross-linking parameters in hyaluronic acid hydrogels--definitions and analytical methods.

Definitions and methods for the quantification of degree of modification and cross-linking in cross-linked hyaluronic acid (HA) hydrogels are outlined. A novel method is presented in which the HA hydrogel is degraded by the enzyme chondroitinase AC and the digest product analyzed by size exclusion chromatography combined with electrospray ionization mass spectrometry (SEC-ESI-MS). This method allows for the determination of effective cross-linker ratio (CrR) which together with the degree of modification (MoD), determined by, e.g. (1)H NMR spectroscopy, enables the calculation of the degree of substitution (DS) and degree of cross-linking (CrD). The method, could be applicable to the major cross-linked HA hydrogels currently on the market, and is exemplified here by application to two HA hydrogels. The definitions and methods presented are important contributions in attempts to find relationships between MoD, DS and CrD to mechanical properties as well as to biocompatibility of HA hydrogels.

On the reliability of heteronuclear precursors-ligand effects in the Li-MOCVD synthesis of SrTiO3 films.

Strontium titanate SrTiO3 thin films are highly perspective as gate dielectric material. Difference in volatility of the common homometallic precursors-strontium beta-diketonates and titanium alkoxides remains major hinder for preparation of high quality coatings based on this phase. An attractive alternative in its synthesis by MOCVD is provided by application of heterometallic mixed-ligand complexes, Sr2Ti2(beta-diket)4(OR)8(ROH)x. Mass-spectrometric study reveals, however, that none of these species can be considered a true single-source precursor. The relative stability of the molecules in solution and the congruence of in-situ release of homometallic species on evaporation are, on the other hand, crucial for the quality of the produced films and are strongly influenced by the nature of alkoxide ligands, OR. The historically first discovered representative of this heterometallic family, a sec-alkoxide derivative Sr2Ti2(thd)4(O(i)Pr)8, is in fact unexpectedly unstable, transforming in solution into Sr2Ti(thd)4(O(i)Pr)4((i)PrOH), which explains difficulties in keeping the correct stoichiometry using isopropoxide precursor. The primary alkoxide complexes, Sr2Ti2(thd)4(OR)8(ROH)2, R = Et, (n)Pr are also unstable yielding Sr4Ti2(thd)4(OR)8(ROH)2 on decomposition. The best solution stability and most uniform evaporation was observed for the iso-derivative, Sr2Ti2(thd)4(O(i)Bu)8, permitting to apply it in long term experiments under industrial process conditions. Present contribution provides detailed experimental comparison between and sec-and iso-alkoxide derivatives and sheds light on the influence of the ligand on molecular stability of a precursor and how it influences the quality of the derived oxide film, especially in relation to its electrophysical properties.

Simple and efficient synthesis of a Nd:LaAlO3 NIR nanophosphor from rare earth alkoxo-monoaluminates Ln2Al2(O(i)Pr)12((i)PrOH)2 single source precursors by Bradley reaction.

Nanoparticles of a Nd-doped LaAlO(3) perovskite can be obtained rapidly and with quantitative yield using the Bradley (ether elimination) treatment of a mixture of individual Ln(2)Al(2)(O(i)Pr)(12)((i)PrOH)(2), Ln = La, Nd, in acetophenone. The initially produced particles are poorly crystalline, but their crystallinity improves strongly on heating to 800 degrees C, which leads also to a controllable aggregation. The prepared nanoparticles are rather solution stable and can easily be surface-modified, which opens prospects for their use as phosphors in bioimaging applications. The precursors, bimetallic isopropoxides of rare earth elements and aluminum with a 1:1 composition, Ln(2)Al(2)(O(i)Pr)(12)((i)PrOH)(2), can be prepared with high yields via direct dissolution of metallic lanthanoids in a solution of aluminum isopropoxide in a toluene-isopropanol medium or through a short time reflux of "Ln(O(i)Pr)(3)" with 1 equiv of Al(O(i)Pr)(3) in toluene. In spite of good volatility and their proper composition, the Ln(2)Al(2)(O(i)Pr)(12)((i)PrOH)(2), Ln = La, Nd, do not act as single-source precursors in MOCVD, because of their quantitative transformation into LnAl(3)(O(i)Pr)(12) together with Ln(5)O(O(i)Pr)(13) on evaporation. These molecules are, however, present intact in solution according to variable temperature NMR studies, which permits application of them successfully as single source precursors in the synthesis of Ln:LaAlO(3) perovskite nanopowders with compositions thoroughly controlled through the conditions of the synthesis. Luminescent properties of the Nd:LaAlO(3) were examined and discussed in detail. The thermal population of the (4)F(5/2) and (2)H(9/2) states was found as a consequence of the grain size effect causing difficulties in heat dissipation. Moreover, luminescence behavior of the powder annealed at a lowest temperature shows well-defined short-range order.

Methodical thermolysis of [Ba2Ti2(thd)4(OnPr)8(nPrOH)2] under autogenous pressure followed by combustion for the synthesis of dielectric tetragonal BaTiO3 nanopowder.

The tetragonal BaTiO(3) nanopowder is synthesized in a solvent-less, efficient process by the thermolysis of a single [Ba(2)Ti(2)(thd)(4)(OnPr)(8)(nPrOH)(2)] precursor in a closed reactor at 700 degrees C under autogenous pressure, followed by combustion. This paper compiles the synthesis of the [Ba(2)Ti(2)(thd)(4)(OnPr)(8)(nPrOH)(2)] precursor, its analysis by mass spectrometry, and implementation for the fabrication of dielectric tetragonal BaTiO(3) nanopowder by controlled efficient thermal decomposition. The as-prepared, intermediate, and final forms of the obtained nanomaterials are systematically analysed by XRD, Raman, and EDS measurements to gain structural and compositional information. Employing HR-SEM, TEM, and HR-TEM techniques, the morphological changes during the structural evolution of all the phases are pursued. The mechanistic elucidation for the fabrication of BaTiO(3) nanopowder is developed on the basis of TGA and DTA data obtained for the initial [Ba(2)Ti(2)(thd)(4)(OnPr)(8)(nPrOH)(2)] reactant as well as the as-prepared BaCO(3) with amorphous Ti phase.

Antifungal compounds from cultures of dairy propionibacteria type strains.

Antifungal compounds from cultures of five type strains of dairy propionibacteria, as well as from the cultivation medium, were studied. Cell-free supernatants and medium were fractionated by C(18) solid phase extraction. The aqueous 95% acetonitrile fractions were analyzed by GC-MS or subjected to reversed-phase HPLC, to identify, quantify or isolate antifungal substances. The resulting HPLC fractions were screened for antifungal activity against the mold Aspergillus fumigatus and the yeast Rhodotorula mucilaginosa. Active fractions were further separated by HPLC and the structures of the compounds were determined by spectroscopic and chromatographic methods. All five strains produced 3-phenyllactic acid, at concentrations ranging from 1.0 microg mL(-1) (Propionibacterium freudenreichii ssp. shermanii) to 15.1 microg mL(-1) (Propionibacterium thoenii), and at L/D -ratios ranging from 2 : 3 (Propionibacterium acidipropionici) to 9 : 1 (Propionibacterium freudenreichii). A number of active compounds found in cultures of propionibacteria were also present in noninoculated growth medium: two antifungal diketopiperazines, cyclo(L-Phe-L-Pro) and cyclo(L-Ile-L-Pro), and seven antifungal linear peptides. Three of the linear peptides corresponded to sequences found in the medium component casein, suggesting their origin from this component, whereas the diketopiperazines were suggested to be formed from medium peptides by heat treatment.

Asymmetric hydrogenation of enol phosphinates by iridium catalysts having N,P ligands.

[reaction: see text] Enol phosphinates, which are structural analogues of enol acetates, have for the first time been employed as substrates for Ir-catalyzed asymmetric hydrogenation. A number of enol phosphinates have been synthesized and reduced successfully with up to and above 99% ee.

Evidence of the presence of 2-O-beta-D-xylopyranosyl-alpha-l-arabinofuranose side chains in barley husk arabinoxylan.

(Glucurono)arabinoxylans were extracted from barley husks and degraded with endo-beta-xylanase or subjected to periodate oxidation. The released oligosaccharide fragments were separated and isolated on Biogel-P2, and their structures were determined by NMR spectroscopy. The oligosaccharides identified consisted of beta-d-(1-->4)-linked xylopyranosyl residues, of which some were substituted at O-3 with alpha-l-arabinofuranosyl groups or at O-2 with 4-O-methylglucuronic acid. In addition to these substituents, a disaccharide side chain, 2-O-beta-d-xylopyranosyl-alpha-l-arabinofuranose, attached at position O-3 of the main chain, was proved to exist in arabinoxylan from barley husks. The compound was fully characterized with NMR, and all (1)H and (13)C NMR signals were assigned. The arabinose to xylose ratio was low (approximately 0.2) and no 2,3-disubstitution existed. No blocks of substituted xylose residues could be observed along the main chain.

Synthesis, physicochemical and biochemical studies of 1',2'-oxetane constrained adenosine and guanosine modified oligonucleotides, and their comparison with those of the corresponding cytidine and thymidine analogues.

We have earlier reported the synthesis and antisense properties of the conformationally constrained oxetane-C and -T containing oligonucleotides, which have shown effective down-regulation of the proto-oncogene c-myb mRNA in the K562 human leukemia cells. Here we report on the straightforward syntheses of the oxetane-A and oxetane-G nucleosides as well as their incorporations into antisense oligonucleotides (AONs), and compare their structural and antisense properties with those of the T and C modified AONs (including the thermostability and RNase H recruitment capability of the AON/RNA hybrid duplex by Michaelis-Menten kinetic analyses, their resistance in the human serum, as well as in the presence of exo and endonucleases).

Synthesis of the DNA-[Ru(tpy)(dppz)(CH(3)CN)](2+) conjugates and their photo cross-linking studies with the complementary DNA strand.

We here report our studies on the conjugation of photoreactive Ru(2+) complex to oligonucleotides (ODNs), which give a stable duplex with the complementary target DNA strand. These functionalized DNA duplexes bearing photoreactive Ru(2+) complex can be specifically photolyzed to give the reactive aqua derivative, [Ru(tpy)(dppz)(H(2)O)](2+)-ODN (tpy = 2,2':6',2' '-terpyridine; dppz = dipyrido[3,2-a:2',3'-c]phenazine), in situ, which successfully cross-links to give photoproduct(s) in the duplex form with the target complementary DNA strand. Thus, the stable precursor of the aquaruthenium complex, the monofunctional polypyridyl ruthenium complex [Ru(tpy)(dppz)(CH(3)CN)](2+), has been site-specifically tethered to ODN, for the first time, by both solid-phase synthesis and postsynthetic modifications. (i) In the first approach, pure 3'-[Ru(tpy)(dppz)(CH(3)CN)](2+)-ODN conjugate has been obtained in 42% overall yield (from the monomer blocks) by the automated solid-phase synthesis on a support labeled with [Ru(tpy)(dppz)Cl](+) complex with subsequent liberation of the crude conjugate from the support under mild conditions and displacement of the Cl(-) ligand by acetonitrile in the coordination sphere of the Ru(2+) label. (ii) In the second approach, the single-modified (3'- or 5'- or middle-modified) or 3',5'-bis-modified Ru(2+)-ODN conjugates were prepared in 28-50% yield by an amide bond formation between an active ester of the metal complex and the ODNs conjugated with an amino linker. The pure conjugates were characterized unambiguously by ultraviolet-visible (UV-vis) absorption spectroscopy, enzymatic digestion followed by HPLC quantitation, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry (MALDI-TOF as well as by ESI). [Ru(tpy)(dppz)(CH(3)CN)](2+)-ODNs form highly stabilized ODN.DNA duplexes compared to the unlabeled counterpart (DeltaT(m) varies from 8.4 to 23.6 degrees C) as a result of intercalation of the dppz moiety; they undergo clean and selective photodissociation of the CH(3)CN ligand to give the corresponding aqua complex, [Ru(tpy)(dppz)(H(2)O)](2+)-ODNs (in the aqueous medium), which is evidenced from the change of their UV-vis absorption properties and the detection of the naked Ru(2+)-ODN ions generated in the course of the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis. Thus, when [Ru(tpy)(dppz)(CH(3)CN)](2+)-ODN conjugate was hybridized to the complementary guanine (G)-rich target strand (T), and photolyzed in a buffer (pH 6.8), the corresponding aqua complex formed in situ immediately reacted with the G residue of the opposite strand, giving the cross-linked product. The highest yield (34%) of the photo cross-linked product obtained was with the ODN carrying two reactive Ru(2+) centers at both 3'- and 5'-ends. For ODNs carrying only one Ru(2+) complex, the yield of the cross-linked adduct in the corresponding duplex is found to decrease in the following order: 3'-Ru(2+)-ODN (22%) > 5'-Ru(2+)-ODN (9%) > middle-Ru(2+)-ODN (7%). It was also found that the photo cross-coupling efficiency of the tethered Ru(2+) complex with the target T strand decreased as the stabilization of the resulting duplex increased: 3'-Ru(2+)-ODN (VI.T) (DeltaT(m)(b) = 7 degrees C) < 5'-Ru(2+)-ODN (V.T) (DeltaT(m)(b) = 16 degrees C) < middle-Ru(2+)-ODN (VII.T) (DeltaT(m)(b) = 24.3 degrees C, Table 2). This shows that, with the rigidly packed structure, as in the duplex with middle-Ru(2+)-ODN, the metal center flexibility is considerably reduced, and consequently the accessibility of target G residue by the aquaruthunium moiety becomes severely restricted, which results in a poor yield in the cross-coupling reaction. The cross-linked product was characterized by PAGE, followed by MALDI-TOF MS.