Sequence-dependent separation of trinucleotides by ion-interaction reversed-phase liquid chromatography-A structure-retention study assisted by soft-modelling and molecular dynamics.

We studied sequence-dependent retention properties of synthetic 5'-terminal phosphate absent trinucleotides containing adenine, guanine and thymine through reversed-phase liquid chromatography (RPLC) and QSRR modelling. We investigated the influence of separation conditions, namely mobile phase composition (ion interaction agent content, pH and organic constituent content), on sequence-dependent separation by means of ion-interaction RPLC (II-RPLC) using two types of models: experimental design-artificial neural networks (ED-ANN), and linear regression based on molecular dynamics data. The aim was to determine those properties of the above-mentioned analytes responsible for the retention dependence of the sequence. Our results show that there is a deterministic relation between sequence and II-RPLC retention properties of the studied trinucleotides. Further, we can conclude that the higher the content of ion-interaction agent in the mobile phase, the more prominent these properties are. We also show that if we approximate the polar component of solvation energy in QSRR by the electrostatic work in transferring molecules from vacuum to water, and the non-polar component by the solvent accessible surface area, these parameters best describe the retention properties of trinucleotides. There are some exceptions to this finding, namely sequences 5'-NAN-3', 5'-ANN-3', 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' (N stands for generic nucleotide). Their role is still unknown, but since linear regression including these specific constellations showed a higher observable variance coverage than the model with only the basic descriptors, we may assume that solvent-analyte interactions are responsible for the exceptional behaviour of 5'-NAN-3' & 5'-ANN-3' trinucleotides and some intramolecular interactions of neighbouring nucleobases for 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' trinucleotides.

Poly (9-(2-diallylaminoethyl)adenine HCl-co-sulfur dioxide) deposited on silica nanoparticles constructs hierarchically ordered nanocapsules: curcumin conjugated nanocapsules as a novel strategy to amplify guanine selectivity among nucleobases.

Poly (9-(2-diallylaminoethyl)adenine HCl-co-sulfur dioxide) (Poly A) deposited on silica nanoparticles self-assembles to form hierarchically ordered nanocapsules. These nanocapsules can be conjugated with curcumin. The curcumin-conjugated nanocapsules are found to be spherical in size and their size ranges between 200 and 600 nm. We found that curcumin conjugated with silica nanoparticles marginally shows a selectivity (∼20%) for guanine over adenine, cytosine, thymine and uracil, but this selectivity is extraordinarily amplified to more than 500% in curcumin-conjugated nanocapsules prepared from the above procedure. FT-IR spectra along with lifetime measurements suggest that specific interaction between adenine moieties of Poly A nanocapsules and thymine/uracil does not affect the fluorescence of poly A nanocapsules. Thus, the sensitivity and selectivity for guanine estimation is due to hydrophobic interactions, which are assisted by the low water solubility of guanine as compared to the other nucleobases. The present method illustrates a wider linear dynamic range in the higher concentration range as compared to the reported methods. Finally, the degradation study proves that stability of curcumin is improved dramatically in such nanocapsules demonstrating that nanotechnology could be a viable method to improve selectivity of specific analyte and robustness of probe molecule during fluorescence based bio-sensing.

Real-time analysis of self-assembled nucleobases by Venturi easy ambient sonic-spray ionization mass spectrometry.

In this study, the real-time analysis of self-assembled nucleobases was employed by Venturi easy ambient sonic-spray ionization mass spectrometry (V-EASI-MS). With the analysis of three nucleobases including 6-methyluracil (6MU), uracil (U) and thymine (T) as examples, different orders of clusters centered with different metal ions were recorded in both positive and negative modes. Compared with the results obtained by traditional electrospray ionization mass spectrometry (ESI-MS) under the same condition, more clusters with high orders, such as [6MU7+Na](+), [6MU15+2NH4](2+), [6MU10+Na](+), [T7+Na](+), and [T15+2NH4](2+) were detected by V-EASI-MS, which demonstrated the soft ionization ability of V-EASI for studying the non-covalent interaction in a self-assembly process. Furthermore, with the injection of K(+) to the system by a syringe pumping, the real-time monitoring of the formation of nucleobases clusters was achieved by the direct extraction of samples from the system under the Venturi effect. Therefore, the effect of cations on the formation of clusters during self-assembly of nucleobases was demonstrated, which was in accordance with the reports. Free of high voltage, heating or radiation during the ionization, this technique is much soft and suitable for obtaining the real-time information of the self-assembly system, which also makes it quite convenient for extraction samples from the reaction system. This "easy and soft" ionization technique has provided a potential pathway for monitoring and controlling the self-assembly processes.

Sensitive naked-eye detection of Hg2+ based on the aggregation and filtration of thymine functionalized vesicles caused by selective interaction between thymine and Hg2+.

We report a sensitive, selective and low-cost method for the naked-eye detection of Hg(2+). The principle is based on rapid interaction between functionalized PDA vesicles and Hg(2+), which leads to obvious aggregation of vesicles. Furthermore, using only a simple filtration process, without using any other color indicator or specialized equipment, a higher detection sensitivity for Hg(2+) (0.1 µM) than chromophoric colorimetric sensors (approximately 1-100 µM) was obtained.

[Separation of bases, phenols and pharmaceuticals on ionic liquid-modified silica stationary phase with pure water as mobile phase].

N-methylimidazolium ionic liquid (IL) -modified silica was prepared with the reaction of 3-chloropropyl modified silica and N-methylimidazole using toluene as solvent. Based on the multiple interactions between N-methylimidazolium IL-modified silica and analytes such as hydrophobic interaction, electrostatic attraction, repulsion interaction, hydrogen-bonding, etc., the bases (cytosine, thymine, 2-aminopyrimidine and 6-chloroguanine), phenols (m-aminophenol, resorcinol and m-nitrophenol) and three pharmaceuticals (moroxydine hydrochloride, acyclovir and cephalexin hydrate) were separated successfully with only pure water as the mobile phase. These chromatographic separations are environmental friendly, economical and convenient, without any organic solvent or buffer additive. The retention mechanism of these samples on the stationary phase was also investigated.

Multi-wall carbon nanotubes bonding on silica-hydride surfaces for open-tubular capillary electrochromatography.

Prepared multi-wall carbon nanotube (MWNT) materials, including untreated MWNT, HNO(3)-treated MWNT and HNO(3)-HCl-treated MWNT were covalently attached onto a silica-hydride-modified capillary by hydrosilation, using the abundant double bonds between the pentagon carbons in the MWNT structure. These MWNT-incorporated capillaries were characterized by SEM, ATR-IR and electroosmotic flow (EOF) measurements in phosphate buffers with a pH range of 3.7-9.3 and in the mixtures of acetonitrile modifier. The untreated capillary was assumed to carry some carboxylate groups formed on the non-acid-treated MWNTs, as it had higher EOF values than the hydride capillary. As the MWNTs were treated with HNO(3) and HCl solutions, the capillaries had increasingly higher EOF values. To examine the existence of an electrochromatography mechanism in the modified capillaries, a mixture of nucleosides and thymine was probed to check the velocity factor and retention factor. In addition to the pi-pi interaction between the probe solutes and the MWNT immobilized stationary phases; a reversed-phase mechanism could contribute to the chromatographic retention. For acidic tetracyclines, increasing the loadability of MWNTs resulted in a high retention factor and improved the separation resolution.

Etched succinate-functionalized silica hydride stationary phase for open-tubular CEC.

An open-tubular (OT) CEC column was designed to anchor ionizable succinate-functionalized ligands onto a silica hydride-based stationary phase through surface etching, silanization, and hydrosilation reactions beginning from a bare fused-silica tube. The modified columns that were produced in each step were monitored by analysis of the effect of performance of EOF on the changes of pH values, concentrations, and the amount of ACN added in the running buffers. By tracking the EOF patterns between columns, the author determined that the surface composition of the final product column was a combination of silanols, silica hydrides, and succinate ligands. Furthermore, lower loading volumes of the succinate ligands prepared for the hydrosilation reaction served to complete the mixed-mode OT-CEC columns, and subsequently to carry out the separation of six phenyl alcohols. Studies on the elution order of these alcohols identified the presence of chromatographic interactions in addition to electrophoresis. Based on the employment of a solvation parameter model, these interactions likely included dispersion interactions, dipole-type interactions, and interactions arising through the polarizable electrons in the solute. The optimum buffer conditions for CEC separations of phenyl alcohols, carbonyl-substituted phenols, and a mixture of nucleosides and thymine were a phosphate buffer (50 mM, pH 10.51), a borate buffer (50 mM, pH 8.62), and a borate buffer (50 mM, pH 9.50), respectively. Overall, the hydride-based stationary phases with ionizable ligands were successfully applied to the OT-CEC separations, and these results confidently propose an ideal route to the synthesis of novel OT-CEC columns.

A marine sulfate-reducing bacterium producing multiple antibiotics: biological and chemical investigation.

A marine sulfate-reducing bacterium SRB-22 was isolated by means of the agar shake dilution method and identified as Desulfovibrio desulfuricans by morphological, physiological and biochemical characteristics and 16S rDNA analysis. In the bioassay, its extract showed broad-spectrum antimicrobial activity using the paper disc agar diffusion method. This isolate showed a different antimicrobial profile than either ampicillin or nystatin and was found to produce at least eight antimicrobial components by bioautography. Suitable fermentation conditions for production of the active constituents were determined to be 28 day cultivation at 25 degrees C to 30 degrees C with a 10% inoculation ratio. Under these conditions, the SRB-22 was fermented, extracted and chemically investigated. So far an antimicrobial compound, mono-n-butyl phthalate, and an inactive compound, thymine, have been isolated and characterized.

[Chemical constituents from sea cucumber Holothuria nobilis].

OBJECTIVE: To study the chemical constituents of sea cucumber Holothuria hilla L.. METHODS: Compounds were isolated from the sea cucumber by multi-chromatography on Si gel, ODS Rp C-18 gel, DA101 resin, Sephadex LH-20 and HPLC. The structures were elucidated according to their spectral data and chemical character. RESULTS: Four compounds were isolated from the sea cucumber, and were identified as: holothuria A (I), thymine (II), uracil (III), cholesterol (IV). CONCLUSION: Compounds I IV are obtained from this kind of sea cucumber for the first time.

Identification of cytotoxic constituent of Indonesian sponge Kaliapsis sp. (Bowerbank).

Identification of cytotoxic constituent of Indonesian sponge Kaliapsis sp. has been conducted. The structure identification was judged based on the spectroscopic data, namely, ultraviolet, MS, one and two-dimensional 1H-NMR and 13C-NMR methods. The cytotoxic constituent was identified as 1-(tetrahydro-4-hydroxy-5-(hydroxymethyl)furan-2-yl)-5-methyl pyrimidine-2,4(1H,3H)-dione. This constituent hasn't been isolated from sponges as natural product.

[Studies on chemical constituents of marine bryozoan Bugula neritina L].

Seven compounds were isolated from the marine bryozoan Bugula neritina L. Their chemical structures were identified by NMR and MS spectroscopies as follows: cholesterol (I), cholest-4-en-3-one (II), cholesteryl myristate (III), 3beta,5alpha,9alpha-trihydrox-y-(22E,24R)-ergosta-,22-dien-6-one (IV), 3beta,5alpha,6beta-trihydroxy-(22E,24R)-ergosta-7,22-dien (V), uracil (VI), thymine (VII). Compounds II-VII were isolated from Bugula neritina L. for the first time.

[Studies on chemical constitutes of Acantophora spicifera].

OBJECTIVE: To study the chemical constitutes of Acantophora spicifera. METHOD: Compounds were isolated by normal phase silica gel and Sephadex LH-20 gel column chromatography, and reverse-phase HPLC, as well as recrystallization. Their structures were elucidated by spectroscopic methods. RESULT: Seven compounds were isolated from A. spicifera and their structures were identified as aplysin (1), loloilide (2), (R)-(-)-dehydrovomifoliol (3), uracil (4), thymine (5), 1-methoxy-4-(1-propenyl) benzene (6). CONCLUSION: The compounds were obtained from this genus for the first time. Compound 6 was firstly obtained from marine organisms.

[Chemical constituents from the south China sea gorgonian coral Subergorgia reticulata].

Nine compounds, cholesterol (1), ergostra-7,22-diene-3beta, 5alpha, 6beta-triol (2), cholesta-7,22-diene-3beta, 5alpha, 6beta-triol (3),5,8-epidioxycampesta-6,22-dien-3-o1 (4), batyl alcohol (5), theine (6), thymine (7), uracil (8), guanine (9), were isolated from the South China Sea gorgonian coral Subergorgia reticulata and their structures elucidated on the basis of spectral data. All of these compounds were isolated for the first time from this gorgonian coral.

Nonlinear adsorption isotherm as a tool for understanding and characterizing molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) have frequently been characterized by quantities which are easily determined from experiments but have no theoretical foundation. This makes it difficult to compare different MIP preparations or to transfer MIP based methods to different experimental conditions. Since the adsorption isotherms of MIPs are markedly nonlinear, one can build a better characterization strategy on isotherms as shown by examples in this paper.

HPLC isolation and mass spectrometric characterization of two isomers of thymine glycols in oligodeoxynucleotides.

Thymine glycol, or 5,6-dihydroxy-5,6-dihydrothymine, is the major oxidation product of thymine. Herein we report the isolation of both the (5S, 6R) and (5R, 6S) isomers of cis thymine glycols from several synthetic oligodeoxynucleotides (ODNs) upon oxidation with osmium tetraoxide. Our results show that tandem mass spectrometry can determine the sites of thymine glycol in ODNs by producing characteristic fragment ions, [a - 143] and its complementary w ions at the modification site. We further demonstrate that the [M + H]+ and [M + Na]+ ions of the two cis stereoisomers of thymine glycol in the dinucleotides, which are extricated from the ODNs by nuclease P1, gave distinctive product-ion spectra.

[Separation and determination of purine bases and pyrimidine bases from nucleic acid hydrolysis by HPLC on BDS column].

The hydrolysates of nucleic acid, six purine bases and pyrimidine bases (cytosine, uracil, guanine, hypoxanthine, adenine and thymine) were separated and determined by using HPLC. It is discussed how the column and mobile phase affect the separation. The peaks of cytosine and adenine are tailed on ordinary C18 column, and they are very good on BDS-C18 column. The KH2PO4-H3PO4 buffer can be used in separation the hydrolysates of RNA and DNA, and the NaAc-HAc buffer is only used in DNA. In addition, pH value is a very important factor for separation. With pH value of mobile phase increasing, the retention times of guanine, hypoxanthine and thymine were first increased and then decreased, adenine was increased, and cytosine and uracil were almost constant. The chosen mobile phase was 0.1 mol/L KH2PO4-H3PO4 buffer, with a pH value of 4.05. It was detected at UV 260 nm. The determination was completed within 10 min. The RSDs were all less than 3% and the recoveries were in the range of 82%-114%. The method has been applied to the detection of yeast hydrolysates.

Reduced formation of bipyrimidine photoproducts in DNA UV irradiated at high intensity.

DNA was irradiated using an excimer laser (248 nm) at low intensity (3.15 x 10(7) watts/m2) or high intensity (1.25 x 10(11) watts/m2). Fluences up to 30 kJ/m2 were delivered at either intensity. Following irradiation, DNA damage products were measured, yielding the following findings: 1) the rate of formation of thymine-thymine and thymine-cytosine cyclobutane dimers and the bipyrimidine photoadduct 6-4'-[pyrimidine-2'-one]thymine were reduced at high intensity by about 2-fold and 2) extensive release of free thymine and thymine decomposition fragments occurred at high intensity, but not at low intensity. The effects of high intensity UV are due to promotion of low-lying excited state(s) by absorption of a second photon, producing higher excited state(s) with consequent ionization and base loss. Possible excited state intermediates in this process are the lowest triplet state of DNA bases and prolonged singlet states associated with excimer formation. The depletion of these excited states via promotion may be the cause of the diminished yield of bimolecular pyrimidine photoproducts, suggesting that these photoproducts are formed at low UV intensity in part from long-lived excited states. Long-lived excited states present at conventional UV intensities may contribute to formation of some photoproducts that occur rarely, but are of potential biologic importance, such as dimers between nonadjacent pyrimidines on the same strand and interstrand dimers forming DNA cross-links.

Catabolism of exogenously supplied thymidine to thymine and dihydrothymine by platelets in human peripheral blood.

The interference of platelets with the estimation of unscheduled DNA synthesis in human peripheral mononuclear leukocytes following genotoxic exposure was studied. A 96% reduction in the unscheduled DNA synthesis value was achieved by incubating [3H]thymidine with platelet-rich plasma for 5 hr at 37 degrees. Using radioactive thymine-containing compounds, together with quantitative analyses based on thin-layer and ion-exchange chromatographies, we have shown that thymidine was converted to thymine which, in turn, was converted to dihydrothymine in platelet-rich plasma. The enzymes responsible were separated from platelet lysates by gel filtration and were identified as thymidine phosphorylase and dihydrothymine dehydrogenase. The phosphorylase reversibly catalyzed the formation of thymine from thymidine and converted bromodeoxyuridine to bromouracil. The dehydrogenase reversibly catalyzed the interconversion of thymine and dihydrothymine in a reaction dependent on NADP(H), and it was inhibited by diazouracil and by thymine. Nearly all the thymidine-catabolizing activity found in whole blood samples supplied exogenously with thymidine was accounted for by the platelets. Since most genetic toxicological tests that use blood samples do not involve removing platelets from the blood cell cultures, then it is concluded that precautions should be taken in the future to determine the influence of platelets on these test systems. This is particularly true for methods dependent on thymidine pulses such as unscheduled DNA synthesis, or those dependent on bromodeoxyuridine, such as sister chromatid exchanges, since this nucleoside is also a substrate for thymidine phosphorylase.

Identification of the cis-thymine glycol moiety in chemically oxidized and gamma-irradiated deoxyribonucleic acid by high-pressure liquid chromatography analysis.

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) is formed in DNA by chemical oxidants and ionizing radiation. We describe the separation of thymine glycol, 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol), thymine, and thymidine by high-pressure liquid chromatography (HPLC). Enzymatic hydrolysates of chemically oxidized or gamma-irradiated single-stranded DNA were cochromatographed with 14C-containing marker compounds. In chemically oxidized DNA, thymidine glycol was the major derivative formed. In addition, there were four rapidly eluting thymine-derived components. In irradiated DNA, thymidine glycol constituted about 5% of the modified thymines, and the rapidly eluting fractions were proportionately increased. DNA isolated from gamma-irradiated and nonirradiated HeLa cells grown in the presence of [3H]thymidine was subjected to enzymatic hydrolysis and HPLC analysis. In control DNA, 0.3% of the thymines were modified. Thirty-six kilorads of gamma radiation caused a 30% increase in thymine damage. Thus, most of the base damage was due to internal beta radiation from incorporated [3H]thymidine. The chromatographic patterns of irradiated and nonirradiated samples were qualitatively the same, but the yields of some products increased 2-fold, while others remained unchanged. A comparison of the HPLC profiles of hydrolysates of in vitro oxidized and irradiated DNA with those of the cellular DNA revealed one fast eluting peak to be absent in cellular DNA, suggesting that it was formed only in single-stranded DNA. In cellular DNA, the major modified thymine was a more hydrophobic derivative not formed by in vitro radiation nor chemical oxidation. As in in vitro irradiated DNA, thymidine glycol constituted 5% of the modified thymines. The presence of cis-thymidine glycol in hydrolysates was confirmed by chromatography on Sephadex LH-20 using water and borate as eluants.