Human kanadaptin and kidney anion exchanger 1 (kAE1) do not interact in transfected HEK 293 cells

Kanadaptin (kidney anion exchanger adaptor protein) is a widely expressed protein, shown previously to interact with the cytosolic domain of mouse Cl-/HCO3- anion exchanger 1 (kAE1) but not erythroid AE1 (eAE1) by a yeast-two hybrid assay. Kanadaptin was co-localized with kAE1 in intracellular membranes but not at the plasma membrane in alpha-intercalated cells of rabbit kidney. It was suggested that kanadaptin is an adaptor protein or chaperone involved in targeting kAE1 to the plasma membrane. To test this hypothesis, the interaction of human kanadaptin with human kAE1 was studied in co-transfected HEK293 cells. Human kanadaptin contains 796 amino acids and was immuno-detected as a 90 kDa protein in transfected cells. Pulse-chase experiments showed that it has a half-life (t1/2) of 7 h. Human kanadaptin was localized predominantly to the nucleus, whereas kAE1 was present intracellularly and at the plasma membrane. Trafficking of kAE1 from its site of synthesis in the endoplasmic reticulum to the plasma membrane was unaffected by co-expression of human kanadaptin. Moreover, we found that no interaction between human kanadaptin and kAE1 or eAE1 could be detected in co-transfected cells either by co-immunoprecipitation or by histidine6-tagged co-purification. Taken together, we found that human kanadaptin did not interact with kAE1 and had no effect on trafficking of kAE1 to the plasma membrane in transfected cells. Kanadaptin may not be involved in the biosynthesis and targeting of kAE1. As such, defects in kanadaptin and its interaction with kAE1 are unlikely to be involved in the pathogenesis of the inherited kidney disease, distal renal tubular acidosis (dRTA).     

Nucleosides,XLVI1 Syntheses and Reactions of 6- and 7-p-Chlorophenyllumazine Nucleosides

Abstract

The syntheses of 6-(4) and 7-p-chlorphenyl-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-lumazine (6), was well as the debenzoylation to the corresponding free nucleosides 5 and 7, were improved. Thiation of 4 and 6 by P₄S₁₀ led in excellent yields to 4-thiolumazine nucleosides (8, 10) which could be deblocked to 9 and 11 and converted on treatment with ammonia into the isopterin-N-1- ribofuranosides 13 and 14. 2,2′-Anhydro-nucleoside formation worked well with 5 and 7 respectively to give 15 and 16, which formed on acid hydrolysis the 6- and 7-substituted 1-β-D-arabinofuranosyl-lumazines 18 and 19. The new nucleosides have been characterized by UV and ¹H-NMR spectra.     

Synthesis of Some Novel Acyclolumazine N-1 Nucleosides

Abstract

Reactjon of (2-acetoxyethoxy)methyl bromide with the silylated lumazine bases (1-6) in the presence of n-Bu₄NI leads to the formation of the nucleosides 8, 10, 12, 14, 16 and 18 respectively. Deacetylation with methanolic ammonia afforded the free nucleosides 9, 11, 13, 15, 17 and 19, respectively, in good yields. Structural proofs of the newly synthesized compounds are based on elemental analyses, UV and ¹H-NMR spactra. None of the acyclic nucleosides exhibited antiviral activity against HSV-1 in vitro.     

Pyrazolo[3,4-d)Pyrimidine 2′-Deoxyribo and 2′,3′-Dideoxyribo-Furanosides: Synthesis and Application to Oligonucleotide Chemistry

Abstract

The synthesis of pyrazolo[3,4-d]pyrimidine 2′-deoxyribo-nucleosides with various substituents at C-4 and C-6 (1 4) is described employing either liquid-liquid or solid-liquid phase-transfer glycosylation. From 1a (Z⁸C⁷A_d) and 2b (Z⁸C⁷G_d) the phosphoramidites 12a, b and 15a, b were synthesized. They were used in automated solid-phase synthesis resulting in the oligonucleotides 16 - 25. Deoxygenation (3′-OH) of 1a and 2b yielded pyrazolo[3,4-d]-pyrimidine 2′,3′-dideoxynucleosides isosteric to ddA, ddG, and ddI.     

P1-(Thymidine 5′-)P1-amino-triphosphate: Synthesis,Hydrolysis and Reaction with Cytidine in Alkali1

Abstract

The title compound 1 is prepared from thymidine 5′-phos-phorodiamidate (2) and inorganic pyrophosphate (3) in anhydrous DMF, at 30–32°C. The products of alkaline hydrolysis of 1, at room temperature, are: thymidine 5′-phosphoramidate (4), thymidine 3′-phosphoramidate (8) and thymidine (9) as well as 3 and inorganic trimetaphosphate (10). In 1 N NH₄OH, 1 reacts with cytidine (15) to form cytidylyl-/2^T(3′)-5′/-thymidine (16) and a mixture of cytidine 2′,3′-cyclic phosphate (17) and 9.     

Synthesis and X-Ray Crystal Structure of 3-Amino-1-β-D-Ribofuranosyl-s-Triazolo[5, 1-c]-s-Triazole

Abstract

The first chemical synthesis of 3-amino-1-β-D-ribofuranosyl-s-triazolo[5,1-c]-s-triazole (6) is described. Direct glycosylation of 3-amino-5(7)H-s-triazolo[5,1-c]-s-triazole (2) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose (3) in the presence of TMS-triflate gave 3-amino-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-s-triazolo[5, 1-c]-s-triazole (4) which, on ammonolysis, gave 6. The absolute structure of 6 is determined by X-ray diffraction techniques employing Mo Kα radiation. The structure is solved by direct methods and refined to the R value of 0.044 by using a full-matrix least-squares method. The sugar of 6 has a ³T₂ configuration. The torsion angles about the C5′–C4′ bond are both gauche and the torsion angle about the glycosidic bond is in the anti range. Each azole ring of the aglycon is planar and the dihedral angle between the planes of the rings is 3.6°.     

THE NMR CONFORMATION STUDY OF THE COMPLEXES OF DEOXYCYTIDINE KINASE (dCK) AND 2′-DEOXYCYTIDINE/2′-DEOXYADENOSINE

The structures of the bound ¹³C/²H double-labelled 2′(R/S), 5′(R/S)-²H₂-1′,2′,3′,4′,5′-¹³C₅-2′-deoxyadenosine and the corresponding 2′-deoxycytidine moieties in the complexes with human deoxycytidine kinase (dCK) have been characterized for the first time by the solution NMR spectroscopy, using Transferred Dipole-Dipole Cross-correlated Relaxation and Transferred nOe experiments. It has been shown that the ligand adopts a South-type sugar conformation when bound to dCK.     

Studies on the Dehydration of New 2- (Pentitol-1-YL) Pyridines Having D-Gulo,D-IDO,and L-Manno Configurations

Abstract

Fusion of 2-trimethylsilylpyridine and tetra-O-acetyl-aldehydo-D-xylose or 2,3:4,5-di-O-isopropylidene-aldehydo-L-arabinose led, after removing of the protecting groups, to 2-(pentitol-1-yl)pyridines of D-gulo and D-ido or L-manno configurations. Dehydration of the sugar-chain with D-gulo and D-ido configurations gave the corresponding 2′,5′-anhydro derivatives, whereas 2-(5-O-isopropyl-L-manno-pentitol-1-yl)-pyridine was the only compound formed by dehydration of the sugar-chain with L-manno configuration. Structural proofs are based on ¹H and ¹³C NMR spectra.     

Cyclic AMP Diphenyl Phosphoric Mixed Anhydride: Synthesis,P NMR Characterization and Reaction with Dimethylamine

Abstract

Phosphorus diastereoisomers, R _p and S _p of p¹-adenosine cyclic 3′, 5′ P² -diphenylpyrophosphate (cyclic AMP diphenylphosphoric mixed anhydride) (1) were prepared from adenosine cyclic 3′, 5′-monophosphate (cyclic AMP) and diphenyl phosphorochloridate and characterized by ³¹p NMR. The synthesis preferentially gave R _p-1. Reaction of 1 with dimethylamine resulted in the formation of a (～ 3:1) mixture of adenosine cyclic 3′,5′-N, N-dimethylphosphoramidate and diphenyl-N, N-dimethylphosphoramidate and occurred with inversion of configuration at cyclic AMP phosphorus.     

Synthesis of Pyrazolo[3, 4-d]Pyrimidine Ribonucleoside 3′, 5′-Cyclic Phosphates Related to cAMP,cIMP and cGMP1

Abstract

The synthesis of pyrazolo[3,4-d]pyrimidine ribonucleoside 3′, 5′-cyclic phosphates related to cAMP, cIMP and cGMP has been achieved for the first time. Phosphorylation of 4-amino-6-methylthio-1-β-D-ribo-furanosylpyrazolo[3,4-d]pyrimidine (1) with POCl₃ in trimethyl phosphate gave the corresponding 5′-phosphate (2a). DCC mediated intramolecular cyclization of 2a gave the corresponding 3′, 5′-cyclic phosphate (3a), which on subsequent dethiation provided the cAMP analog 4-amino-1-β-D-ribofuranosylpyrazolo[3, 4-d]pyrimidine 3′, 5′-cyclic phosphate (3b). A similar phosphorylation of 6-methylthio-1-β-D-ribofuranosylpyrazolo[3, 4-d]pyrimidin-4(5H)-one (5), followed by cyclization with DCC gave the 3′, 5′-cyclic phosphate of 5 (9a). Dethiation of 9a with Raney nickel gave the cIMP analog 1-β-D-ribofuranosylpyrazolo[3, 4-d]pyrimidin-4(5H)-one 3′, 5′-cyclic phosphate (9b). Oxidation of 9a with m-chloroperoxy benzoic acid, followed by ammonolysis provided the cGMP analog 6-amino-1-β-D-ribofuranosylpyrazolo [3, 4-d] pyrimidin-4(5H)-one 3′, 5′-cyclic phosphate (7). The structural assignment of these cyclic nucleotides was made by UV and H NMR spectroscopic studies.     

Multilevel regulation of autophagosome content by ethanol oxidation in HepG2 cells

Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental (nonmetabolizing) and recombinant (ethanol-metabolizing) Hep G2 cells to identify the ethanol metabolite that causes AV enhancement by quantifying AVs or their marker protein, microtubule-associated protein 1 light chain 3-II (LC3-II). The ethanol-elicited rise in LC3-II was dependent on ethanol dose, was seen only in cells that expressed alcohol dehydrogenase (ADH) and was augmented in cells that coexpressed cytochrome CYP2E1 (P450 2E1). Furthermore, the rise in LC3-II was inversely related to a decline in proteasome activity. AV flux measurements and colocalization of AVs with lysosomes or their marker protein Lysosomal-Associated Membrane Protein 1 (LAMP1) in ethanol-metabolizing VL-17A cells (ADH⁺/CYP2E1⁺) revealed that ethanol exposure not only enhanced LC3-II synthesis but also decreased its degradation. Ethanol-induced accumulation of LC3-II in these cells was similar to that induced by the microtubule inhibitor, nocodazole. After we treated cells with either 4-methylpyrazole to block ethanol oxidation or GSH-EE to scavenge reactive species, there was no enhancement of LC3-II by ethanol. Furthermore, regardless of their ethanol-metabolizing capacity, direct exposure of cells to acetaldehyde enhanced LC3-II content. We conclude that both ADH-generated acetaldehyde and CYP2E1-generated primary and secondary oxidants caused LC3-II accumulation, which rose not only from enhanced AV biogenesis, but also from decreased LC3 degradation by the proteasome and by lysosomes.     

31P-15N One-Bond Couplings of Diastereoisomeric Adenosine Cyclic 3′,5′-Phosphoramidates

Abstract

¹J(³¹P¹⁵N) coupling constants of R _p and S _p adeno-sine cyclic 3′,5′-phosphoramidates (1), -N-methylphosphor-amidates (2) and -N,N-dimethylphosphoramidates (3) increase in the order of 1<2<3 and obey the Stec rule (J(R _p)< J(S _p)). A possible interpretation of coupling constant differences based on differences in substituent electronegativities and variation in hybridization at nitrogen atom, is suggested.     

3′-3′,3′-5′ and 5′-5′ TpT-Amides: Synthesis,Characterization and Alkaline Hydrolysis

Abstract

A simple procedure is described for the preparation of the title compounds 1, 8 and 9. 3′-3′ or 3′-5′ or 5′-5′ TpT was reacted with a twofold molar excess of TPS in anhydrous DMF, at room temperature, for 5 min, followed by a 1 min in situ treatment of the reaction mixture with excess 7.0 N NH₄OH, at 0°C. The alkaline hydrolysis of 1, 8 and 9 proceeds without the assistance of 3′- and 5′-hydroxyl groups resulting in equimolar mixtures of thymidine (4) and thymidine 3′-phosphoramidate (6) (for the 3′-3′ isomer) or thymidine 5′-phosphoramidate (7) (for the 5′-5′ isomer) or 6 and 7 in equal quantities (for the 3′-5′ isomer).     

Synthesis of 2H,13C-Labelled 2′-Deoxynucleosides and Their Site Specific Incorporation into Oligo-DNA for Structural Studies via Relaxation Time Measurements

Abstract

We have recently shown¹ the usefulness of ²H, ¹³C-labelled 2′-deoxynu-cleoside building blocks for structural studies via relaxation time measurements. The synthesis of phosphoramidite blocks 11 and 12 for their site-specific incorporation (indicated by underlines) into the d⁵′(¹C²G³ A ⁴ T ⁵ T ⁶ A ⁷ A ⁸ T ⁹C¹⁰G)₂ ³′ is briefly described for studying the T₁ and T_1[sgrave] relaxations of ²H and ¹³C at specific deuterated carbons in a large molecule.     

Conformation and Antiherpes Activity of 3′- and 5′-Azido and Amino Analogs of 5-Methoxymethyl-2′-Deoxyuridine

Abstract

The molecular conformations of 3′- and 5′-azido and amino derivatives of 5-methoxymethyl-2′-deoxyuridine, 1, were investigated by nmr. The glycosidic conformation of 5-methoxymethyl-5′-amino-2′,5′-dideoxy-uridine, 5 had a considerable population of the syn form. The 5′-derivatives show a preference for the S conformation of the furanose ring as in 1. In contrast, the 3′-derivatives show preference for the N conformation. For 5-methoxymethyl-3′-amino-2′,3′-dideoxyuridine, 3, the shift towards the N state is pH dependent. The preferred conformation for the exocyclic (C4′,C5′) side chain is g⁺ for all compounds except 5 which has a strong preference for the t rotamer (79%). Compounds 1, 3 and 5 inhibited growth of HSV-1 by 50% at 2, 18 and 70 μg/ml respectively, whereas 2 and 4 were not active up to 256 μg/ml (highest concentration tested). The compounds were not cytotoxic up to 3,000 μM.     

Nucleosides,XL1 Synthesis and Properties of lin-Naphthamidazole-Ribonucleosides

Abstract

The fusion reaction between 1-trimethylsilyl-naphth[2,3-d]imidazole (3) and its 2-methyl derivative (4) with 2, 3, 5-tri-O-benzoyl-1-bromo-D-ribofuranose (6) leads to anomeric mixtures of the corresponding 2', 3', 5'-tri-O-benzoyl-1α- and β-D-ribofuranosylnaphth[2,3-d]imidazoles (7, 11 and 13). Separation of the anomers was achieved by chromatographical means and debenzoylation yielded the corresponding nucleosides (8, 12 and 10, 14). Structural proofs are based on elementary analysis, UV- and ¹H-NMR spectra.     

9-(6-Deoxy- β -D-Allofuranosyl)Adenine Cyclic 3′,5′ -Phosphor-Amidate: A New Cyclic AMP Amide Derivative Containing an Equatorial Methyl Group at the 5′-Position

Abstract

R _P and S _P phosphorus diastereoisomers of the title compound (2) are prepared from the corresponding cyclic monophosphate. Solution conformation of the dioxaphosphor-inane ring and hydrolysis of R _p-2 and S _p-2 are studied and compared with those of the phosphorus diastereoisomers of the isomeric compound that contains the 5′-methyl group in the axial position.     

Nucleosides,III1 Synthesis and Properties of 2-Trifluoromethyl-Naphthimidazole-Ribonucleoside

Abstract

The fusion reaction between 2-trifluoromethylnaphth[2,3-d]imidazole (1) and 1-0-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose (2) leads to 2,3′,5′-tri-O-benzoyl-1-β-D-ribofuranosylnaphth[2,3-d]imidazole (3). Debenzoylation of (3) gives the corresponding nucleoside 1-β-D-ribofuranosyl -2-trifluoromethylnaphth[2,3-d]imidazole (4). Structural proofs are based on elementary analysis, UV-and ¹H-NMR spectra.     

Structural Features of 2′,3′-Dideoxy-2′,3′-Didehydrocytidine,A Potent Inhibitor of the HIV (AIDS) Virus

Abstract

The structure and conformation of 2′,3′-dideoxy-2′,3′-didehydrocytidine (2′,3′-dideoxycytidin-2′-ene, d4C), a potent inhibitor of the human immunodeficiency virus, was determined by X-ray crystallography. The nucleoside crystallizes in the orthorhombic space group P2₁2₁2₁ with cell dimensions a = 8.603(1), b = 9.038(1), c = 25.831(2) A and with two independent molecules in the asymmetric unit (Z = 8). Atomic parameters were refined by full-matrix least squares to a final value of R = 0.033 for 2258 observed reflections. The molecules are quite flexible: in molecule A the glycosyl torsion angle (X_CN) is 61.3° and the -CH₂OH side chain is in the gauche ⁺ orientation while in molecule B X_CN = 19.8° and the side chain is trans. The five-membered rings are slightly puckered (～0.1 Å), 04′ being endo in molecule A and exo in molecule B. A mechanism is proposed for the known instability of 2′,3′-unsaturated nucleosides.