Palladium(II) complexes containing pyrazole-derived ligands of biological interest. Four,five and six coordinate palladium(II) complexes with 5(3)-methylpyrazole-3(5)-carboxamide and 5(3)-methylpyrazole-3(5)-carbohydrazide

Neutral bis-complexes of palladium(II) with 5(3)- methylpyrazole-3(5)-carboxamide (mpa) and 5(3)- methylpyrazole-3(5)carbohydrazide (mph) of the type PdX₂·2L (where L = mpa, mph; X = Cl, Br, I, SCN, NO₃, ClO₄) have been synthesised and spectroscopically characterised. Available physico-chemical data indicate the mpa complexes of Pd(II) to be trans-square planar species with the primary ligand molecule showing monodentate behavior through the pyrazolyl ring nitrogen only. The Pd(II)—thiocyanate complex of mpa is proposed to have a pseudo-square pyramidal geometry attained through bridging anionic component. The complexes PdX₂·2mph (X = Cl, NO₃) appear to be six-coordinate species at least in the solid state; the corresponding thiocyanato variety has in all probability a five coordinate structure. The species, Pd(ClO₄)₂·2mph is a usual four coordinate one with non-coordinating perchlorate group. The monochelates, PdX₂·mph (X = Br, I), are cis-square planar varieties. The ligand molecule, mph, in all cases is believed to function as (N,N) bidentate ligand in its ‘imidol’ form except in the thiocyanate complex where unusually it exhibits both the monodentate and bidentate functions in the same species.     

Specific phosphorylase from Euglena gracilis splits diadenosine 5',5''-P(1),P(4)-tetraphosphate (Ap(4)A) and diadenosine 5',5''-P(1),P(3)-triphosphate (Ap(3)A)

1. Phosphorolytic cleavage of Ap(4),A was demonstrated in cell-free extracts from two protozoan organisms, Euglena gracilis and Acanthamoeba castellanii. 2. A specific dinucleoside oligophosphate (DNOP) alpha, beta-phosphorylase which degrades substrates with formation of corresponding nucleoside 5'-diphosphate (NDP) as one of the reaction products was purified 625-fold from Euglena gracilis cells. 3. In addition to Ap(4)A, the phosphorylase degrades AP(3)A, Ap(5)A, Gp(4)G and one of phosphonate analogs, ApppCH(2)pA. The K(m) values for Ap(4), A and Ap(3) A are 27 and 25 micron, and relative velocities 100 and 14, respectively. The K(m) for phosphate is 0.5 mM. 4. Some anions (arsenate, chromate, molybdate and vanadate) can substitute for phosphate in the catalyzed reactions and in their presence the DNOPs yield corresponding nucleoside 5'-monophosphate as one of the reactions' product. The enzyme supports also an anion-dependent dephosphorylation of NDPs. 5. Molecular weight of the native Euglena phosphorylase is 30,000. Optimum pH for its activity is at 8.0 Divalent metal cations are essential for the phosphorolysis of DNOPs but are not for the NDP dephosphorylation mentioned.     

Deamination and reductive deamination of (2-amino-5, 6-dimethylbenzimidazolyl)cobamide. Preparation of (2-hydroxy-5, 6-dimethylbenzimidazolyl)cobamide and (5, 6-dimethyl-[2(-14)C]-benzimidazolyl)cobamide

(2-Amino-5, 6-dimethylbenzimidazolyl)-cobamide (III) is transformed to (2-hydroxy-5, 6-dimethylbenzimidazolyl) cobamide (IV) by nitrous acid. Exchange of the NH2-group by hydrogen with nitrous acid/hypophosphorous acid yields vitamin B12 (I). This reaction completes a cycle vitamin B12 (I)----[carboxy(2-cyanoamino-4,5-dimethylphenyl)amino]cobamide+ ++ (II)----(2-amino-5,6-dimethylbenzimidazolyl)cobamide (III)----vitamin B12 (I), which allows chemical 14C-labelling of vitamin B12. In this procedure cyanogen bromide, which is necessary for the first step, was labelled with [14C] cyanide. By the following reactions a vitamin B12 was formed in which C-2 of the 5, 6-dimethylbenzimidazole moiety is labelled.     

Tissue-Specific Differences in DNA Modifications (5-Hydroxymethylcytosine, 5-Formylcytosine, 5-Carboxylcytosine and 5-Hydroxymethyluracil) and Their Interrelationships

Background

Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions.

Results

Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine, as well as 5-hydroxymethyl-2′-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2′-deoxycytidine in both porcine (R² = 0.88) and rat tissues (R² = 0.83); no such relationship was observed for 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2′-deoxycytidine and its product 5-formyl-2′-deoxycytidine, as well as between 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine (R² = 0.60 and R² = 0.71, respectively).

Conclusions

Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2′-deoxycytidine and/or 5-carboxyl-2′-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2′-deoxycytidine which defines the active demethylation process.     

Infrared CD of deoxy oligonucleotides. Conformational studies of 5''d(GCGC)3'', 5''d(CGCG)3'', 5''d(CCGG)3'', and 5''d(GGCC)3'' in low and high salt aqueous solution.

Infrared (vibrational) circular dichroism (VCD) spectra have been obtained for the self-complementary tetranucleotides, 5'd(CGCG)3', 5'd(GCGC)3', 5'd(CCGG)3', and 5'd(GGCC)3'. In buffered aqueous solution at low salt concentration, these tetramers exhibit spectra associated with right-handed polymers, although the spectra differ significantly for the four species. In high salt solution, a B-->Z transition occurs in 5'd(CGCG)3', while the other tetranucleotides appear only slightly altered. Temperature dependent studies of these oligonucleotides reveal a greater amount of thermal stability for the tetramers in low salt than for the high salt solutions. VCD intensities computed via the exciton formalism are compared with observed results.     

Activation of 2-5A-dependent RNase by analogs of 2-5A (5'-O-triphosphoryladenylyl(2'----5')adenylyl(2'----5')adenosine ) using 2',5'-tetraadenylate (core)-cellulose

A variety of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) analogs were assayed for their abilities to activate murine 2-5A-dependent RNase (subsequently "the nuclease") using a recently developed method. This technique consists of immobilizing and partially purifying the nuclease using core-cellulose [A2'p)3A-cellulose) and then monitoring the breakdown of poly(U)-3'-[32P]Cp into acid-soluble fragments. Several 5'-adenosinecapped analogs of 2-5A (containing a tetra-, tri-, or diphosphate) were analyzed, and it was found that reducing the number of phosphoryl groups between the 5' to 5'-diadenosine linkages resulted in a progressive loss of activity. Because A5' pppp(A2'p)3A was a potent activator of the nuclease yet stable during the assay these results suggested that a free 5'-phosphoryl group may not be required for the activation of the nuclease. A number of 8-bromoadenosine-substituted analogs of 2-5A were also studied. Curiously, the brominations decreased the activities of the 5'-di- and triphosphorylated molecules while substantially increasing the activities of the 5'-monophosphorylated species. The results indicated that a tri- or diphosphate moiety on the 5'-end of 2-5A or the presence of ATP is not absolutely required for the nuclease to be active. Furthermore, the ATP analog, beta, gamma-methylene ATP, did not inhibit the activity of the nuclease. Finally, a 3',5'-phosphodiester linkage isomer of 2-5A and a 3'-deoxy (cordycepin) analog of 2-5A were tested, and both were found to be completely without activity.     

Nucleoside 5'-(beta, gamma-peroxytriphosphates)

Procedures for the synthesis, purification, and characterization of beta, gamma-peroxy analogues of the eight common ribo- and deoxyribonucleoside triphosphates have been developed. Although adenosine 5'-(beta, gamma-peroxytriphosphate) was stable to conditions in most biochemical systems, incubation of a solution of the analogue at 100 degrees C led to formation of AMP and ATP, as well as ADP. NAD+ pyrophosphorylase was the only enzyme among 13 tested for which adenosine 5'-(beta, gamma-peroxytriphosphate) was a good substrate, but the analogue was an effective inhibitor for a number of kinases. The peroxy compounds tested inactive with Escherichia coli RNA polymerase and DNA polymerase I, as well as with wheat germ RNA polymerase II.     

Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioates from adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate)

The chiral and achiral phosphorothioate analogues of 2',5'-oligoadenylates (2-5A) have been enzymatically synthesized from the Sp and Rp isomers of adenosine 5'-O-(2-thiotriphosphate) [(Sp)-ATP beta S and (Rp)-ATP beta S, respectively] and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) by 2-5A synthetase from L929 cells and lysed rabbit reticulocytes. These 2',5'-phosphorothioate analogues were separated, purified, and structurally characterized. While ATP gamma S and (Sp)-ATP beta S were as efficient substrates for the 2-5A synthetase as was ATP, (Rp)-ATP beta S was more than 50-fold less efficient a substrate. The beta- and gamma-phosphorothioates were more resistant to enzymatic hydrolysis than was authentic 2-5A. Compared to 2-5A, there were marked differences in the biological activities of the 2',5'-phosphorothioates as determined by (i) binding to 2-5A-dependent endoribonuclease (RNase L), (ii) activation of RNase L to hydrolyze RNA, and (iii) inhibition of protein synthesis in intact L929 cells. These studies extend previous reports on the elucidation of the stereochemical requirements of 2-5A synthetase and RNase L [Karikó, K., Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (first of three papers in this issue); Karikó, K., Li, S. W., Sobol, R. W., Jr., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (second of three papers in this issue)] with the phosphorothioate analogues of 2-5A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and antiviral activity assay of novel (E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine

(E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) selectivity of COX inhibitors

In vitro evaluations of the selectivity of COX inhibitors are based on a great variety of experimental protocols. As a result, data available on cyclooxygenase (COX)-1/COX-2/5- lipoxygenase (LOX) selectivity of COX inhibitors lack consistency. We, therefore, performed a systematic analysis of the COX-1/COX-2/5-LOX selectivity of 14 compounds with selective COX inhibitory activity (Coxibs). The compounds belonged to different structural classes and were analyzed employing the well-recognized whole-blood assay. 5-LOX activity was also tested on isolated human polymorphonuclear leukocytes. Among COX inhibitors, celecoxib and ML-3000 (licofelone) inhibited 5-LOX in human neutrophils at micromolar ranges. Surprisingly, ML-3000 had no effect on 5-LOX product synthesis in whole-blood assay. In addition, we could show that inhibition of COX pathways did not increase the transformation of arachidonic acid by the 5-LOX pathway.