A novel amide stationary phase for hydrophilic interaction liquid chromatography and ion chromatography.

A novel amide stationary phase (ASP) for hydrophilic interaction liquid chromatography (HILIC) has been prepared via the Click chemistry method. It was based on the strategy that the amino group of Asparagine was easily transferred to the corresponding azido group and then clicked onto terminal alkyne-silica gel in the presence of Cu(I)-based catalyst. For the tested polar compounds including nucleosides and nucleic acid bases, ASP-based column has demonstrated good performance in terms of separation efficiency and column stability, and the retention mechanism was found to match well the typical HILIC retention. In addition, the ASP described here showed much better selectivity in separation of inorganic anions under ion chromatography mode relative to other kinds of commercial ASP.

Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column.

A stationary phase composed of silica-bonded cyclofructan 6 (FRULIC-N) was evaluated for the separation of four cyclic nucleotides, six nucleoside monophosphates, four nucleoside diphosphates, and five nucleoside triphosphates via hydrophilic interaction chromatography (HILIC) in both isocratic and gradient conditions. The gradient conditions gave significantly better separations by narrowing peak widths. Sixteen out of nineteen nucleotides were baseline separated on the FRULIC-N column in one run. Unlike other known HILIC stationary phases, there can be dual-retention mechanisms unique to this media. Traditional hydrogen bonding/dipolar interactions can be supplemented by dynamic ion interaction effects for anionic analytes. This occurs because the FRULIC-N stationary phase is able to bind certain buffer cations. The extent of the ion interaction is tunable, in comparison to stationary phases with embedded charged groups, where the inherent ionic properties are fixed. The best mobile phase conditions were determined by varying the organic modifier (acetonitrile) content, as well as salt type/concentration and electrolyte pH. The thermodynamic characteristic of the FRULIC-N column was investigated by evaluating the column temperature effect on retention and utilizing van't Hoff plots. This study shows that there is a greater entropic contribution for the retention of nucleotide di and triphosphates, whereas there is a greater enthalphic contribution for the cyclic nucleotides with the FRULIC-N column.

Determination of acid-base dissociation constants of amino- and guanidinopurine nucleotide analogs and related compounds by capillary zone electrophoresis.

CZE has been applied for determination of acid-base dissociation constants (pKa) of ionogenic groups of newly synthesized amino- and (amino)guanidinopurine nucleotide analogs, such as acyclic nucleoside phosphonate, acyclic nucleoside phosphonate diesters and other related compounds. These compounds bear characteristic pharmacophores contained in various important biologically active substances, such as cytostatics and antivirals. The pKa values of ionogenic groups of the above compounds were determined by nonlinear regression analysis of the experimentally measured pH dependence of their effective electrophoretic mobilities. The effective mobilities were measured by CZE performed in series of BGEs in a broad pH range (3.50-11.25), at constant ionic strength (25 mM) and temperature (25 degrees C). pKa values were determined for the protonated guanidinyl group in (amino)guanidino 9-alkylpurines and in (amino)guanidinopurine nucleotide analogs, such as acyclic nucleoside phosphonates and acyclic nucleoside phosphonate diesters, for phosphonic acid to the second dissociation degree (-2) in acyclic nucleoside phosphonates of amino and (amino)guanidino 9-alkylpurines, and for protonated nitrogen in position 1 (N1) of purine moiety in acyclic nucleoside phosphonates of amino 9-alkylpurines. Thermodynamic pKa of protonated guanidinyl group was estimated to be in the range of 7.75-10.32, pKa of phosphonic acid to the second dissociation degree achieved values of 6.64-7.46, and pKa of protonated nitrogen in position 1 of purine was in the range of 4.13-4.89, depending on the structure of the analyzed compounds.

Morphology reversion activity of phosmidosine and phosmidosine B, a newly isolated derivative, on src transformed NRK cells.

An antifungal antibiotic, phosmidosine (1) was previously isolated (J. Antibiotics 44: 375 approximately 381, 1991). Phosmidosine derivatives, phosmidosines B (2) and C (3) were newly isolated as detransforming compounds from the fermentation broth of Streptomyces sp. strain RK-16 which is a producer strain of phosmidosine (1). The structures of 2 and 3 were established by spectroscopic methods, including UV, HRFAB-MS, and NMR. 1 and 2 showed the inhibitory activity of the cell cycle progression and the morphological reversion activity on srcts-NRK cells. On the other hand, 3 had no activity. These results indicate that the prolyl group in phosmidosine derivatives plays an important role in the inhibitory activity against the cell cycle progression and the morphological reversion activity on srcts-NRK cells.

Importance of ribonucleotide availability to proliferating T-lymphocytes from healthy humans. Disproportionate expansion of pyrimidine pools and contrasting effects of de novo synthesis inhibitors.

Sensitive high performance liquid chromatography techniques, which differentiate between purine and pyrimidine ribonucleoside and deoxyribonucleoside triphosphates, were used to quantify pools in phytohemagglutinin-stimulated T-lymphocytes (98% CD4+ and CD8+) from healthy volunteers. The importance of de novo synthesis and salvage was evaluated by incubating the cells with 14C-radiolabeled precursors (40 microM), azaserine (20 microM; a glutamine antagonist), and ribavirin (50 microM; an IMP dehydrogenase inhibitor). We confirmed that resting T-lymphocytes meet their metabolic requirements by salvage. Noteworthy observations were as follows. First, nucleotide pool expansion over 72 h is disproportionate, with that for purines (ATP and GTP) being 2-fold compared with up to 8-fold for pyridine (NAD) or pyrimidine (UTP, UDP-Glc, and CTP) pools. This supports an additional role for the latter in membrane lipid biosynthesis, protein glycosylation, and strand break repair. Second, intact de novo pathways are essential for such expansion. Azaserine not only inhibited purine synthesis (confirmed by N-formylglycinamide polyphosphate accumulation), but also reduced expansion of pyrimidine and NAD pools by 70%. Ribavirin depleted GTP pools by 40% and reduced pyrimidine pool expansion by 40% at 72 h. These findings underline the importance of pyrimidine ribonucleotide availability as well as GTP synthesis de novo to proliferating T-lymphocytes. They also demonstrate an absence of coordinate regulation between de novo purine and pyrimidine biosynthesis.

T-lymphocytes from AIDS patients are unable to synthesize ribonucleotides de novo in response to mitogenic stimulation. Impaired pyrimidine responses are already evident at early stages of HIV-1 infection.

Proliferative defects have been reported at the level of DNA synthesis, even in T-lymphocytes from asymptomatic human immunodeficiency virus type-1+ (HIV-1+) patients. Since purine and pyrimidine ribonucleotide availability is crucial for proliferation, we compared the ability of HIV-1- and HIV-1+ T-lymphocytes (> 95% CD4+ and CD8+) to activate de novo biosynthetic and salvage pathways following phytohemagglutinin stimulation using 14C-labeled precursors. The striking abnormality already detectable in asymptomatic patients' cells was the impaired ability of CTP, UDP-Glc, and UTP pools to expand over 72 h (44-70% of control), although ATP and GTP pools and responses were normal. In symptomatic patients, resting T-cells showed markedly reduced pyrimidine pools (53-74% of control) with no change following activation. Relatively normal ATP, GTP, and NAD pools masked the same impaired response of de novo synthesis to activation, with ATP and GTP being reduced by 50% at 48 h. Purine salvage was more active than the control in unstimulated HIV-1+ cells. This impaired de novo synthesis in HIV-1+ T-lymphocytes severely restricts the availability of ribonucleotides for vital growth-related activities such as membrane expansion and strand break repair as well as DNA and RNA synthesis. The data indicate that resting T-lymphocytes from symptomatic patients survive through enhanced salvage, but the stimulation induces metabolic cell death, and provide an explanation for the activation-associated lymphocyte death seen in HIV-1+ T-lymphocytes.

Extraction and assay of creatine phosphate, purine, and pyridine nucleotides in cardiac tissue by reversed-phase high-performance liquid chromatography.

The levels of creatine phosphate, purine, and pyridine nucleotides in tissues provide important information on energetic and oxidative cellular states. Nevertheless, technical, theoretical, and methodological difficulties in extraction and quantification procedures have so far limited our understanding of the exact role that these substances play in metabolic processes which take place in cells. The objective of our study was to find an easy and rapid method for extracting, separating, and quantifying creatine phosphate, purine, and pyridine nucleotides in solid tissues. We adapted the classic acid-extraction procedure with HClO4 for purine and oxidized pyridine nucleotides and then developed a new alkaline extraction with phenol in a phosphate buffer solution (pH 7.8) for reduced pyridine nucleotides. Biopsies of myocardial tissue were frozen and ground at -180 degrees C using the appropriate extraction procedure. The separation and quantification of the metabolites were performed using a reversed-phase 3-microns Supelchem C18 column, with the addition of tetrabutylammonium as an ion-pair agent to the buffer solution, by ultraviolet detection. The recovery of the external and internal standards always exceeded 90%. The autooxidation or interconversion processes were almost insignificant for each reduced form. This technique allowed us to avoid complex enzymatic procedures and difficulties in the selective assay of pyridine nucleotides with chemiluminescence and surface spectroscopy.

Purine nucleotide levels in host tissues of Ehrlich ascites tumor-bearing mice in different growth phases of the tumor.

The pool sizes of purine nucleotides, nucleosides, and nucleobases were measured in the host tissues liver, skeletal muscle, and blood of Ehrlich ascites tumor-bearing mice during the different periods of tumor growth. There were large differences of tissue concentrations of these metabolites between control animals, animals in the logarithmic growth period of the Ehrlich ascites tumor, and animals in the resting phase of tumor growth. The ATP concentrations in liver, muscle, and erythrocytes were higher during the proliferating phase of the tumor compared with the ATP levels of these organs in healthy animals. In liver and skeletal muscle the ATP concentration decreased during the transition from proliferating into resting phase of tumor growth. The concentrations of nucleosides and nucleobases within the RBC and blood plasma deceased during the logarithmic growth phase but restored during the plateau period. As well as in the organs/cells investigated and in the body fluids (plasma, ascites fluid) a tremendous increase of adenosine concentration during the resting phase of tumor growth was observed. From changes of total purine ribonucleotide pattern an activation period in the nucleotide metabolic pathways of liver and skeletal muscle during the proliferating phase of tumor growth is postulated.

Optimized separation of purine bases and nucleosides in human cord plasma by capillary zone electrophoresis.

An optimized separation of the main purine compounds of human serum by capillary zone electrophoresis is presented. Separations were performed in an uncoated silica capillary (44 cm x 75 microns I.D., 37 cm to window) on a SpectraPhoresis 1000 system with UV detection. The separation of adenine (Ade), adenosine (Ado), guanine (Gua), guanosine (Guo), hypoxanthine (Hyp), inosine (Ino), xanthine (Xan) and uric acid (UA) was optimized with respect to pH, temperature, applied potential and hydrodynamic injection time. Optimum conditions were 20 mM borate buffer (pH 9.4), 37 degrees C, 20 kV and 9 s load and detection at 260 nm. Linearity extended from 1 to 125 microM. The sensitivity of the method was 0.5 microM, which is adequate for measuring Ade, Gua, Hyp and UA in plasma samples. Plasma samples from newborns were precipitated with an equal volume of perchloric acid (7%, v/v), the supernatant was adjusted to neutral pH with potassium carbonate and, before injection, the sample was alkalized with sodium hydroxide. The method presented here allows the determination of Ade, Guo, Hyp and UA. The levels of the determined purines were compared in samples from control newborns, preterm babies and newborns with asphyxia or acidic serum pH values.

Isolation and characterization of phosmidosine. A new antifungal nucleotide antibiotic.

A new nucleotide antibiotic, phosmidosine was isolated from a culture filtrate of a newly isolated streptomycete identified as Streptomyces sp. RK-16. HRFAB-MS and elemental analysis established the molecular formula of C16H24N7O8P. 1H, 13C and 31P NMR indicated the presence of a methyl phosphate group and UV spectra were similar to those of 8-hydroxyadenosine. The antibiotic inhibited spore formation of Botrytis cinerea at the concentration of 0.25 micrograms/ml.

A high-performance liquid chromatography method for separation of purine bases, nucleosides and ureides: application to studies on purine catabolism in higher plants.

Mixtures of purine nucleotides, nucleosides, nucleobases, uric acid, allantoin and allantoic acid were fractionated by high-performance liquid chromatography on a polyvinyl alcohol gel column, Asahipak GS-320H, with isocratic elution by sodium phosphate. Application of this system to the determination of the sizes of cellular pools of purine derivatives in plant cells and of the activity of related enzymes, as well as to the purification of enzymatically synthesized radioactive compounds, is described.

DNA methylation in Trypanosoma cruzi.

DNA isolated from the protozoan Trypanosoma cruzi has been found to contain 5-methylcytosine. Analysis of T. cruzi DNA by both HpaII and MspI restriction endonucleases suggests that the sequence -CCGG- is not methylated. Probably T. cruzi DNA also contains N6-methyladenine. This report constitutes the first clear demonstration of the presence of methylated bases in the nuclear DNA from trypanosomes.

Regulation of 5-phosphoribosyl 1-pyrophosphate and of hypoxanthine uptake and release in human erythrocytes by oxypurine cycling.

Uptake and release of purines by red blood cells has been shown to be markedly sensitive to changes in pH, inorganic phosphate (Pi), and oxygen concentration (Berman, P., Black, D., Human, L., and Harley, E. (1988) J. Clin. Invest. 82, 980-986). The mechanism of this regulation has been further studied. We have shown that incubation of red cells in medium containing xanthine oxidase rapidly and completely depletes intracellular hypoxanthine and causes accumulation of 5-phosphoribosyl 1-pyrophosphate (PRPP) at physiological Pi concentrations. Hypoxanthine release from intracellular IMP is strictly dependent on PRPP depletion, induced by either alkalinizing the cells or by adding excess adenine. Xanthine oxidase abolishes this dependence. Oxygen depletion enhances adenine uptake and prevents hypoxanthine release. The results suggest that hypoxanthine release is governed by PRPP-dependent recycling of hypoxanthine to IMP. We propose that PRPP accumulation in red cells is regulated by a substrate cycle, comprising hypoxanthine, IMP, and inosine. Cycle flux is controlled by Pi inhibition and 2,3-bisphosphoglycerate activation of purine-5'-nucleotidase, which converts IMP to inosine. Oxypurine cycling may account for the sensitive control of purine uptake and release by changes in pH and oxygen tension that occur physiologically.