Synthetic Strategies for Dinucleotides Synthesis.

Dinucleoside 5',5'-polyphosphates (DNPs) are endogenous substances that play important intra- and extracellular roles in various biological processes, such as cell proliferation, regulation of enzymes, neurotransmission, platelet disaggregation and modulation of vascular tone. Various methodologies have been developed over the past fifty years to access these compounds, involving enzymatic processes or chemical procedures based either on P(III) or P(V) chemistry. Both solution-phase and solid-support strategies have been developed and are reported here. Recently, green chemistry approaches have emerged, offering attracting alternatives. This review outlines the main synthetic pathways for the preparation of dinucleoside 5',5'-polyphosphates, focusing on pharmacologically relevant compounds, and highlighting recent advances.

A Symmetric Molecule Produced by Mycobacteria Generates Cell-Length Asymmetry during Cell-Division and Thereby Cell-Length Heterogeneity.

Diadenosine polyphosphates, Ap(2-7)A, which contain two adenosines in a 5',5' linkage through phosphodiester bonds involving 2-7 phosphates, regulate diverse cellular functions in all organisms, from bacteria to humans, under normal and stress conditions. We had earlier reported consistent occurrence of asymmetric constriction during division (ACD) in 20-30% of dividing mycobacterial cells in culture, irrespective of different growth media, implying exogenous action of some factor of mycobacterial origin. Consistent with this premise, concentrated culture supernatant (CCS), but not the equivalent volume-wise concentrated unused medium, dramatically enhanced the ACD proportion to 70-90%. Mass spectrometry and biochemical analyses of the bioactive fraction from CCS revealed the ACD-effecting factor to be Ap6A. Synthetic Ap6A showed a mass spectrometry profile, biochemical characteristics, and bioactivity identical to native Ap6A in the CCS. Thus, the present work reveals a novel role for Ap6A in generating cell-length asymmetry during mycobacterial cell-division and thereby cell-length heterogeneity in the population.

Tumor necrosis factor-alpha gene promoter methylation in Japanese adults with chronic periodontitis and rheumatoid arthritis.

BACKGROUND AND OBJECTIVE: Over-expression of tumor necrosis factor-alpha (TNF-α) plays a pathological role in chronic periodontitis (CP) and rheumatoid arthritis (RA), which might be regulated by the epigenetic mechanism. The aim of the present study was to evaluate whether there is a unique methylation profile of the TNF-α gene promoter in blood cells of individuals with CP and RA. MATERIAL AND METHODS: The study participants consisted of 30 Japanese adults with RA (RA group), 30 race-matched adults with CP only (CP group) and 30 race-matched healthy controls (H group). Genomic DNA isolated from peripheral blood was modified by sodium bisulfite and analyzed, by direct sequencing, to investigate DNA methylation of the TNF-α gene promoter region. The level of TNF-α produced in mononuclear cells stimulated with Porphyromonas gingivalis lipopolysaccharide was determined using ELISA. RESULTS: Twelve cytosine-guanine dinucleotide (CpG) motifs were identified in the TNF-α promoter fragment from -343 to +57 bp. The CP group showed a significantly higher methylation rate and frequency at -72 bp than the H group (p < 0.01). The RA group exhibited significantly higher methylation rates at seven CpG motifs (-302, -163, -119, -72, -49, -38 and +10 bp), and significantly higher methylation frequencies at six CpG motifs (-163, -119, -72, -49, -38 and +10 bp), than the H group (p < 0.01 for all comparisons). The levels of TNF-α produced were significantly different between individuals with and without methylation at -163 bp (p = 0.03). CONCLUSION: These results suggest that the hypermethylated status of CpG motifs in the TNF-α gene promoter in blood cells may be unique to Japanese adults with CP and RA.

Bifunctional inhibition of human immunodeficiency virus type 1 reverse transcriptase: mechanism and proof-of-concept as a novel therapeutic design strategy.

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a major target for currently approved anti-HIV drugs. These drugs are divided into two classes: nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). This study illustrates the synthesis and biochemical evaluation of a novel bifunctional RT inhibitor utilizing d4T (NRTI) and a TMC-derivative (a diarylpyrimidine NNRTI) linked via a poly(ethylene glycol) (PEG) linker. HIV-1 RT successfully incorporates the triphosphate of d4T-4PEG-TMC bifunctional inhibitor in a base-specific manner. Moreover, this inhibitor demonstrates low nanomolar potency that has 4.3-fold and 4300-fold enhancement of polymerization inhibition in vitro relative to the parent TMC-derivative and d4T, respectively. This study serves as a proof-of-concept for the development and optimization of bifunctional RT inhibitors as potent inhibitors of HIV-1 viral replication.

Highly efficient enzymatic preparation of c-di-AMP using the diadenylate cyclase DisA from Bacillus thuringiensis.

Cyclic 3',5'-diadenosine monophosphate (c-di-AMP) is a newly recognized bacterial nucleotide second messenger molecule. In addition, it has been shown to be a potential vaccine adjuvant. Although multiple methods are available for c-di-AMP synthesis, the yields are low and the purification procedures are laborious. Here, we report an enzymatic method for more efficient and economical c-di-AMP synthesis using a diadenylate cyclase DisA from Bacillus thuringiensis BMB 171 (btDisA). After overexpression and purification of btDisA, the enzyme-catalyzed reaction conditions were further investigated. Under the optimum conditions, in which 100mM CHES (pH 9.5) containing 2µM btDisA, 10mM ATP, and 10mM MgCl2 was incubated at 50°C for 4h, a high conversion rate of c-di-AMP was obtained. Coupling this process with HPLC purification and lyophilization yielded 100mg of highly pure c-di-AMP that was harvested in white powder form from a 50mL enzyme-catalyzed reaction system. The protocol is not only directly applicable for preparing abundant amounts of c-di-AMP for extensive biochemical and immunological use, but can also be scaled up to meet the requirements for medical applications.

Isolation and identification of diadenosine 5',5'''-P1,P4-tetraphosphate binding proteins using magnetic bio-panning.

We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap(4)A)-'molecular hook' attached to magnetic beads enabling the isolation of Ap(4)A-binding proteins from bacterial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is performed sequentially by mass spectrometry. The observation of positive controls suggests that these newly observed proteins are putative Ap(4)A-binding partners, and we have expectations that others can be found with further technical improvements in our methods.

Separation and characterization of oxaliplatin dinucleotides from DNA using HPLC-ESI ion trap mass spectrometry.

Oxaliplatin is a third-generation platinum complex, and has a broad spectrum of antitumor activity. Such platinum complexes with the DACH carrier ligand have recently received increasing attention since they show efficacy against cisplatin-resistant cell lines. As the foremost indication of antitumor activity of platinum drugs is the formation of adducts with genomic DNA, calf thymus DNA-oxaliplatin adducts were the major target in this study. Calf thymus DNA was incubated with oxaliplatin, resulting in the formation of a large number of platinum-DNA adducts. Treated DNA was digested into the dinucleotides with a combination of enzymes, namely, benzonase, alkaline phosphatase, and nuclease S1. Using a high-performance liquid chromatography, we carried out the separation of individual platinum-DNA adducts which were concurrently identified using electrospray ionization ion trap mass spectrometry (MS). Both 1,2-intrastrand and 1,2-interstrand cross-linked adducts were found; however, those of the intrastrand nature have a considerably higher abundance than those of the interstrand cross-links. Among them, d(GpG)-oxaliplatin was the most abundant bifuctional adduct. To a lesser extent, a few monofunctional adducts were detected as well. MS(n) experiments served to ascertain the detailed structures of oxaliplatin adducts of dinucleoside monophosphates and of dinucleotides.

Synthesis and purification of fluorinated benzimidazole and benzene nucleoside-5'-triphosphates.

The expression "universal base" is very often used to express hybridization properties and recognition patterns of nucleosides. Their behaviour in biological applications, however, is of great interest regarding, e.g.,' their incorporation by polymerases. The 4,6-difluorobenzimidazole and the 2,4-difluorobenzene nucleoside analogues have proven to be universal bases that do not discriminate between the four natural nucleobases in RNA duplexes. Therefore, we synthesized the corresponding triphosphates to evaluate their behavior in polymerase catalyzed reactions and to investigate their ability to serve as substrates for the T7 RNA polymerase.

Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns.

In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the micromolar range. The aim of this study was both to describe a chromatographic assay with an increased efficiency of the dinucleoside separation, which enables the reduction of analytical run times, and to establish a chromatographic assay using conditions, which allow MALDI-mass spectrometric analysis of the resulting fractions. We compared the performance of conventional silica reversed phase chromatography columns, a perfusion chromatography column and a monolithic reversed-phase C18 chromatography column. The effects of different ion-pair reagents, flow-rates and gradients on the separation of synthetic diadenosine polyphosphates as well as of diadenosine polyphosphates isolated from human platelets were analysed. Sensitivity and resolution of the monolithic reversed-phase chromatography column were both higher than that of the perfusion chromatography and the conventional reversed phase chromatography columns. Using a monolithic reversed-phase C18 chromatography column, diadenosine polyphosphates were separable baseline not only in the presence of tetrabutylammonium hydrogensulfate (TBA) but also in the presence of triethylammonium acetate (TEAA) as ion-pair reagent. The later reagent is useful because, in contrast to TBA, it is compatible with MALDI mass-spectrometric methods. This makes TEAA particularly suitable for identification of unknown nucleoside polyphosphates. Furthermore, because of the lower backpressure of monolithic reversed-phase chromatography columns, we were able to significantly increase the flow rate, decreasing the amount of time for the analysis close to 50%, especially using TBA as ion-pair reagent. In summary, monolithic reversed phase C18 columns markedly increase the sensitivity and resolution of dinucleoside polyphosphate analysis in a time-efficient manner compared to reversed-phase perfusion chromatography columns or conventional reversed-phase columns. Therefore, further dinucleoside polyphosphate analytic assays should be based on monolithic silica C18 columns instead of perfusion chromatography or conventional silica reversed phase chromatography columns. In conclusion, the use of monolithic silica C18 columns will lead to isolation and quantification of up to now unknown dinucleoside polyphosphates. These chromatography columns may facilitate further research on the biological roles of dinucleoside polyphosphates.

Isolation and characterization of a novel cross-link lesion in d(CpC) induced by one-electron photooxidation.

Recently we reported the identification and characterization of a novel cross-link lesion formed between two adjacent cytosines in d(CpC), which is the major product formed upon 365 nm photoirradiation of d(CpC) in the presence of 2-methyl-1,4-naphthoquinone. In this study we discuss the isolation and structure characterization of another cross-link lesion formed under the same irradiation condition. Electrospray ionization mass spectroscopy, tandem mass spectrometry and two-dimensional nuclear Overhauser effect spectroscopy results demonstrate that the C6 carbon atom of the 5' cytosine and the N3 nitrogen atom of the 3' cytosine are covalently bonded. In addition, the 5' cytosine moiety is deaminated and the C5 carbon atom in this cytosine is oxidized to a carbonyl group.

Identification of dinucleoside polyphosphates in adrenal glands.

Dinucleoside polyphosphates have been characterised as extracellular mediators controlling numerous physiological functions like vascular tone or cell proliferation. Here we describe the isolation and identification of dinucleoside polyphosphates Ap(n)A (with n=2-3), Ap(n)G (with n=2-6) as well as Gp(n)G (with n=2-6) from adrenal glands. These dinucleoside polyphosphates are localised in granules of the adrenal glands. The dinucleoside polyphosphates diadenosine diphosphate (Ap(2)A), diadenosine triphosphate (Ap(3)A), adenosine guanosine polyphosphates (Ap(n)G) and diguanosine polyphosphates (Gp(n)G), both with phosphate group (p) numbers (n) ranging from 2 to 6, were identified by fractionating them to homogeneity by preparative size-exclusion- and affinity-chromatography as well as analytical anion-exchange and reversed-phase-chromatography from deproteinised adrenal glands and by analysis of the homogeneous dinucleoside polyphosphates containing fractions with post-source-decay (PSD) matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). The identity of the dinucleoside polyphosphates was confirmed by retention time comparison with authentic dinucleoside polyphosphates. Enzymatic analysis demonstrated an interconnection of the phosphate groups with the adenosines in the 5(')-positions of the riboses in all dinucleoside polyphosphates purified from adrenal glands. In conclusion, the identification of these dinucleoside polyphosphates in adrenal gland granules emphasises that these dinucleoside polyphosphates can be released from the adrenal glands upon stimulation into the circulation.

Separation of diadenosine polyphosphates by capillary electrophoresis.

The influence of buffer composition and pH on the electrophoretic behavior of diadenosine polyphosphates with a phosphate chain ranging from two to five phosphate groups has been examined. The electrophoretic mobility in carbonate buffer increases according to the number of phosphates, whereas in borate buffer the mobility changes in an irregular way as a function of pH. This finding can be rationalized by a well-known interaction of borate with ribose rings, which modifies the charge and the hydrodynamic radius of each diadenosine polyphosphate in a different way. Our study shows that the best separation of diadenosine polyphosphates can be achieved at the highest pH values of the range examined both in borate and carbonate buffers.

Identification and characterization of P(1), P(7)-Di(adenosine-5')-heptaphosphate from human platelets.

Diadenosine pentaphosphate and diadenosine hexaphosphate have been isolated in human platelets and have been postulated to play an important role in the control of vascular tone. Here we describe the isolation and identification of diadenosine heptaphosphate from human platelets. Dinucleoside polyphosphates were concentrated by affinity chromatography from a nucleotide-containing fraction from deproteinated human platelets. Dinucleoside polyphosphates were purified by anion-exchange and reversed phase high performance liquid chromatography to homogeneity. Analysis of one of these fractions with matrix-assisted laser desorption/ionization mass spectrometry revealed a molecular mass of 1076.4 (1077.4 = [M + H](+)) Da. UV spectroscopic analysis of this fraction showed the spectrum of an adenosine derivative. Comparison of the postsource decay matrix-assisted laser desorption/ionization mass spectrum of the fraction minus that of diadenosine heptaphosphate (Ap(7)A) demonstrated that the isolated substance was identical to Ap(7)A. The identity of the retention times of the authentic and the isolated compound confirmed this result. Enzymatic analysis demonstrated an interconnection of the phosphate groups with the adenosines in the 5'-positions of the riboses. With thrombin-induced platelet aggregation, Ap(7)A is released from the platelets into the extracellular space. The vasoconstrictive action of Ap(7)A on the vasculature of the isolated perfused rat kidney Ap(7)A was slightly less than that of Ap(6)A. The threshold of the vasoconstrictive action of Ap(7)A was 10(-5) mol/liter. The vasoconstrictive effect was abolished by suramin and pyridoxal phosphate 6-azophenyl-2', 4'-disulfonic acid, suggesting an activation of P(2x) receptors. Furthermore, Ap(7)A inhibits ADP-induced platelet aggregation. Thus, the potent vasoconstrictor Ap(7)A derived from human platelets, like other diadenosine polyphosphates, may play a role in the regulation of vascular tone and hemostasis.

Purification of chemically synthesised dinucleoside(5',5') polyphosphates by displacement chromatography.

Dinucleoside(5',5') polyphosphates (ApnA, ApnG, GpnG, n=3-6) are new group of hormones controlling important biological processes. Because some of the dinucleoside(5',5') polyphosphates are commercially not available purification of chemical synthesised dinucleoside(5',5') polyphosphates became necessary in order to test their physiological and pharmacological properties. It was the aim of this study to find a method which allows purification of 0.1-0.2 g quantities of dinucleoside polyphosphates by analytical HPLC columns yielding products with impurities lower than 1.0%. Adenosine(5')-polyphospho-(5')guanosines were synthesised by mixing the corresponding mononucleotides. The reaction results in a complex mixture of ApnA, ApnG and GpnG (with n=3-6 in all cases). The reaction mixture was concentrated on a preparative C18 reversed-phase column. The concentrate was displaced on a reversed-phase stationary. As a result of displacement chromatography, anion-exchange chromatography in gradient modus yielded baseline separated dinucleoside polyphosphates (homogeneity of the fractions>99%). The identity of the substances were determined by matrix assisted laser desorption ionisation mass spectrometry.

T4 DNA ligase synthesizes dinucleoside polyphosphates.

T4 DNA ligase (EC 6.5.1.1), one of the most widely used enzymes in genetic engineering, transfers AMP from the E-AMP complex to tripolyphosphate, ADP, ATP, GTP or dATP producing p4A, Ap3A, Ap4A, Ap4G and Ap4dA, respectively. Nicked DNA competes very effectively with GTP for the synthesis of Ap4G and, conversely, tripolyphosphate (or GTP) inhibits the ligation of DNA by the ligase. As T4 DNA ligase has similar requirements for ATP as the mammalian DNA ligase(s), the latter enzyme(s) could also synthesize dinucleoside polyphosphates. The present report may be related to the recent finding that human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5''-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29).

Adenosine(5') oligophospho-(5') guanosines and guanosine(5') oligophospho-(5') guanosines in human platelets.

We isolated and identified nucleoside(5') oligophospho-(5') nucleosides containing adenosine and guanosine (ApnG; n = 3-6) as well as diguanosine polyphosphates (GpnG; n = 3-6) in human platelets. For identification, UV spectrometry, matrix-assisted laser desorption/ionization, postsource decay matrix-assisted laser desorption/ionization mass spectrometry, and enzymatic cleavage experiments were used. The adenosine(5') oligophospho-(5') guanosines act as vasoconstrictors and growth factors. The diguanosine polyphosphates are potent modulators of growth in vascular smooth muscle cells, but do not affect vascular tone.

Distribution of [3H]diadenosine tetraphosphate binding sites in rat brain.

The distribution of the diadenosine tetraphosphate high-affinity binding sites has been studied in rat brain by an autoradiographic method using [3H]diadenosine tetraphosphate as the ligand. The binding characteristics are comparable to those described in studies performed on rat brain synaptosomes. White matter is devoid of specific binding. The range of binding site densities in gray matter varies from 3 to 15 fmol/mg of tissue, exhibiting a widespread but heterogeneous distribution. The highest densities correspond to the seventh cranial nerve, medial superior olive, pontine nuclei, glomerular and external plexiform layers of the olfactory bulb, and the granule cell layer of the cerebellar cortex. Intermediate density levels of binding correspond to different cortical areas, several nuclei of the amygdala, and the oriens and pyramidal layers of the hippocampal formation. The localization of diadenosine tetraphosphate binding sites in the brain may provide information on the places where diadenosine polyphosphate compounds can be expected to function in the central nervous system.

Diadenosine polyphosphates. A novel class of glucose-induced intracellular messengers in the pancreatic beta-cell.

Diadenosine polyphosphates are a group of low-weight compounds that increase after exposure to a wide variety of oxidants and have been suggested to act as "alarmones," alerting the cell to the onset of metabolic stress. We demonstrate here that glucose at concentrations that induce insulin release produce a 30- to 70-fold increase in the concentration of diadenosine triphosphate (Ap3A) and tetraphosphate (Ap4A) in beta-cells. Furthermore, Ap3A and Ap4A, at the concentrations found in glucose-stimulated cells, are effective inhibitors of the ATP-regulated K+ channels when applied to the intracellular side of excised membrane patches from cultured beta-cells. We suggest that Ap3A and Ap4A act as second messengers mediating a glucose-induced blockade of the pancreatic beta-cell ATP-regulated potassium channel.