Nucleic acid components and their analogues: new synthesis of bicyclic thiopyrimidine nucleosides

A novel synthesis of condensed bicyclic thiopyrimidine glycosides utilising 1H-cyclopentapyrimidine-2(3H)-thiones and alpha-bromoglucose or alpha-bromogalactose tetraacetate as starting components is described.     

Pulse radiolysis studies of some aza analogues of nucleic acid components

The technique of pulse radiolysis has been utilized to study the reactions of some aza analogues of nucleic acid components with hydrated electrons and OH radicals. The absorption spectra of the transient free radical adducts which result from these reactions and their decay kinetics were determined. The 5-aza analogues gave similar results to those of pyrimidine bases. The 6-aza analogues also showed similar kinetics, however, transient spectra were different. The presence of the sugar moiety in these aza analogues changed the rate law of the OH adduct transient decay from second order to first order kinetics. This finding may have implications for the understanding of the radiation chemistry of DNA.     

Investigation of biologically active components in ginkgo leaf products on the Japanese market

The rapid and highly separative ultra high-performance liquid chromatography (UHPLC)-UV method was adopted and validated to investigate the flavonol glycoside compositions in ginkgo leaf products on the Japanese market. The result indicates that certain products contained amounts of flavonol glycosides approximately equivalent to the medicinal product. Additionally, we examined the correlations between the total amount of flavonol glycosides and of terpene lactones in various ginkgo leaf products.     

N-hydroxybenzenecarboximidic acid derivatives: a new class of nitroxyl-generating prodrugs.

On the basis of the propensity of Piloty's acid to generate nitroxyl (HNO), we previously prepared a number of N,O-bisacylated Piloty's acid derivatives and showed that such prodrugs underwent a disproportionation reaction following ester hydrolysis to give an unstable intermediate that hydrolyzed to nitroxyl. To expand the versatility of this series, we desired some mixed N,O-diacylated Piloty's acid derivatives and devised a synthetic route to them. Such efforts led us, serendipitously, to a new series of heretofore unreported nitroxyl-generating compounds. Thus, benzohydroxamic acid was acylated on the hydroxylamino oxygen and the resulting product converted to its sodium salt. Treatment of this salt with arenesulfonyl chorides would be expected to give the mixed N,O-diacylated derivatives of Piloty's acid. However, the products obtained were the isomeric carboximidic acid derivatives whose structures were deduced from the IR and (13)C NMR spectral frequencies associated with the sp(2) carbons. The structures were verified by analysis of the X-ray crystal structure of a prototype compound of this series. When incubated with porcine liver esterase or mouse plasma, these N-acyloxy-O-arenesulfonylated benzenecarboximidic acid derivatives liberated HNO, measured as N(2)O, as well as the expected arenesulfinic acid and benzoic acid. Alkaline hydrolysis also produced N(2)O, but the major products were the arenesulfonic acid and benzohydroxamic acid. Thus, these N-hydroxybenzenecarboximidic acid derivatives represent a new series of nitroxyl prodrugs that require enzymatic bioactivation before nitroxyl can be liberated.     

Inhibition of glutamine synthetase activity by biologically active derivatives of glutamic acid

The inhibition of activity of glutamine synthetase from Chlorella and porcine brain by 4-hydroxy-D-4-fluoro-D,L- and 4-amino-D,L-glutamic acids diastereoisomers was studied. Each compound was shown to exert the same inhibiting effect on glutamine synthetase from both sources. In case of threo-4-hydroxy-D-glutamic acid the inhibition of the Chlorella enzyme was of a competitive and of a completely mixed type. The enzyme inhibition by 4-fluoro-D, L-glutamic acids seemed to be of a completely non-competitive type. The Ki values for all inhibition reactions were determined. A comparison of biochemical parameters and biological activity revealed that the most effective inhibitors of the enzyme exert a most potent antitumour and antiviral action.     

Amino acid conjugated sophorolipids: A new family of biologically active functionalized glycolipids

Sophorolipids (SLs) are extra cellular glycolipids produced by Candida bombicola ATCC 22214 when grown in the presence of glucose and fatty acids. These compounds have a disaccharide head group connected to a long-chain hydroxyl-fatty acid by a glycosidic bond. To explore structure-activity of modified SLs, a new family of amino acid-SL derivatives was prepared. Synthesized analogs consist of amino acids linked by amide bonds formed between their alpha-amino moiety and the carboxyl group of ring-opened SL fatty acids. Their preparation involved the following: (i) hydrolysis of a natural SL mixture with aqueous alkali to give SL free acids, (ii) coupling of free acids to protected amino acids using dicarbodiimide, and (iii) removing amino acid carboxyl protecting groups. These conjugates were evaluated for their antibacterial, anti-HIV, and spermicidal activity. All tested analogs showed antibacterial activity against both gram +ve and gram -ve organisms. Leucine-conjugated SL was most efficient. For example, the minimum inhibitory concentrations (MIC) for Moraxella sp. and E. coli were 0.83 and 1.67 mg/mL, respectively. Among the alkyl esters of amino acid conjugated SLs, the ethyl ester of leucine-SLs was most active. Against Moraxella sp., S. sanguinis, and M. imperiale, MIC values are 7.62 x 10(-4), 2.28 x 10-(3) and 1.67 mg/mL, respectively. All compounds displayed virus-inactivating activity with 50% effective concentrations (EC50) below 200 microg/mL. The EC50 of leucine-SL ethyl ester was 24.1 microg/mL, showing that it is more potent than commercial spermicide nonoxynol-9 (EC50 approximately 65 microg/mL).     

Permanganate oxidation of nucleic acid components: a reinvestigation.

KMnO4 consumption in solution by nucleoside added at an excess was measured to explore any possible oxidative interactions between these molecules. Thymidine, 5-methyldeoxycytidine and deoxycytidine rapidly decomposed KMnO4 with the pseudo-first-order kinetics (thymidine greater than 5-methyl-deoxycytidine greater than deoxycytidine), while deoxyguanosine showed only a very slow consumption and deoxyadenosine did not decompose KMnO4 at all under the conditions employed (0.16 mM KMnO4, 0.80 mM nucleoside, at 27 degrees C and pH 4.3, 7.4 and 8.6, up to 60 min). UV absorption spectra of KMnO4-treated nucleosides also indicated modification of the pyrimidine nucleosides but not of the purine nucleosides. These results are consistent with the original report of Hayatsu and Ukita published in 1967 on the KMnO4 oxidation of nucleosides, and provide difficulty in understanding the mechanism of recently reported formation of 8-hydroxypurines on treatment of DNA with KMnO4.     

Synthesis of biologically active tritiated amylin and salmon calcitonin analogues

Amylin is a hormone belonging to the calcitonin protein family of peptides. To facilitate receptor screening studies, alternatively radiolabeled and biologically active amylin and salmon calcitonin analogues were synthesized by reductive methylation. Free amino groups of amylin and salmon calcitonin were methylated by reaction of peptides with formaldehyde and sodium [(3)H]borohydride. Radioactively labeled peptides were purified by size exclusion chromatography followed by HPLC. Analysis by MALDI-TOF mass spectrometry of purified amylin and salmon calcitonin peptides revealed incorporation of both two and four tritiated methyl groups per peptide molecule. Specific activities of 22.6 and 23.2 GBq/mmol were measured for amylin and salmon calcitonin, respectively. Methylation of rat amylin and salmon calcitonin did not affect their biological activities as both retained their potency to inhibit insulin-stimulated glycogen synthesis in isolated rat soleus muscle. The synthesis of these tritiated analogues provides an alternative chemically stable radiolabeled ligand which may be useful in exploring receptor interactions within the calcitonin peptide family.     

Nucleic acid components and their analogues: a novel and efficient method for the synthesis of a new class of bipyridyl and biheterocyclic-nitrogen thioglycosides from pyridine-2(1H)-thiones

A novel synthesis of a new class of bipyridyl and biheterocyclic-nitrogen thioglycosides utilizing the reactions of heterocyclic substituted pyridine-2(1H)-thiones and alpha-bromoglucose or alpha-bromogalactose tetraacetate as starting components is described.     

Anthranilic acid derivatives: a new class of non-peptide CCK1 receptor antagonists

Having successfully obtained new CCK(1) ligands holding appropriate groups on the anthranilic acid dimer used as molecular scaffold we were interested in increasing their micromolar affinity for the CCK(1) receptors by modifying the spatial relationship of the main pharmacophoric groups. Since, we have proposed simplified analogues reducing the anthranilic acid dimer to a monomer. In this stage of our research program we have prepared and tested on CCK receptors a series of N-substituted anthranilic acid derivatives keeping a Phe residue at the C-terminal site. The indole-2-carbonyl group imparts the best CCK(1) receptor binding affinity (compound 1: IC(50)=197.5 nM) while a sharp decrease in binding affinity is observed for the other indole containing derivatives. Moreover, in order to support the different binding behaviour observed for the synthesized compounds, a conformational investigation was carried out. Finally, on the basis of the main pharmacophoric groups of the obtained new lead compound (1) (coded VL-0395) a receptor binding hypothesis has been provided.     

Rigid spin-labeled nucleoside C: a nonperturbing EPR probe of nucleic acid conformation

Rigid spin-labeled nucleoside C, an analog of deoxycytidine that base-pairs with deoxyguanosine, was incorporated into DNA oligomers by chemical synthesis. Thermal denaturation experiments and circular dichroism (CD) measurements showed that C has a negligible effect on DNA duplex stability and conformation. Nucleoside C was incorporated into several positions within single-stranded DNA oligomers that can adopt two hairpin conformations of similar energy, each of which contains a four-base loop. The relative mobility of nucleotides in the alternating C/G hairpin loops, 5'-d(GCGC) and 5'-d(CGCG), was determined by electron paramagnetic resonance (EPR) spectroscopy. The most mobile nucleotide in the loop is the second one from the 5'-end, followed by the third, first and fourth nucleotides, consistent with previous NMR studies of DNA hairpin loops of different sequences. The EPR hairpin data were also corroborated by fluorescence spectroscopy using oligomers containing reduced C (C(f)), which is fluorescent. Furthermore, EPR spectra of duplex DNAs that contained C at the end of the helix showed features that indicated dipolar coupling between two spins. These data are consistent with end-to-end duplex stacking in solution, which was only observed when G was paired to C, but not when C was paired with A, C or T.     

Fluorosubstitution and 7-alkylation as prospective modifications of biologically active 6-aryl derivatives of tricyclic acyclovir and ganciclovir analogues

A series of fluorine containing tricyclic analogues of acyclovir (ACV, 1) and ganciclovir (GCV, 2) were synthesized and evaluated for their activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and cytostatic activity against HSV-1 thymidine kinase (TK) gene-transduced human osteosarcoma tumour cells. It was found that fluorine substitution reduced the antiviral activity, but most of the new compounds were pronounced cytostatic agents with potency and selectivity similar to those of parental ACV and GCV. Compounds 12, 13 and 16 seem to be promising as labeled substrates for (19)F NMR studies of the HSV TK-ligand interaction and/or monitoring of their metabolites in cells expressing HSV TK.     

Synthesis of a new disulfide Fmoc monomer for creating biologically susceptible linkages in peptide nucleic acid oligomers

Peptide nucleic acids (PNA) are one of many synthetic mimics of DNA and RNA that have found applications as biological probes, as nano-scaffold components, and in diagnostics. In an effort to use PNA as constructs for cellular delivery we investigated the possibility of installing a biologically susceptible disulfide bond in the backbone of a PNA oligomer. Here we report the synthesis of a new abasic Fmoc monomer containing a disulfide bond that can be incorporated into a PNA oligomer (DS-PNA) using standard solid phase peptide synthesis. The disulfide bond survives cleavage from the resin and DS-PNA forms duplexes with complementary PNA oligomers. Initial studies aimed at determining if the disulfide bond is cleavable to reducing agents while in a duplex are explored using UV thermal analysis and HPLC.