An Improved Method for Large Scale Synthesis of Oligonucleotides Applying the NPE/NPEOC-Strategy

Abstract

Several oligonucleotides were synthesized in scales up to 60 μmol in a standard 10 μmol cartridge on a standard DNA synthesizer. The advantage of a special phosphoramidite approach using only β-eliminating protecting groups over the commonly practised automated oligonucleotide synthesis using ammonia-labile blocking groups could be demonstrated by ¹H-NMR-spectroscopy.     

Amide derivatives with pyrazole carboxylic acids of 5-amino-1,3,4-thiadiazole 2-sulfonamide as new carbonic anhydrase inhibitors: Synthesis and investigation of inhibitory effects

Pyrazole carboxylic acid amides of 5-amino-1,3,4-thiadiazole-2-sulfonamide were synthesized from 4-benzoyl-1,5-diphenyl-1H-pyrazole-3-carbonyl chloride and 4-benzoyl-1-(3-nitrophenyl)-5-phenyl-1H-pyrazole-3-carbonyl chloride. Carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from human erythrocyte cells by the affinity chromatography method. The inhibitory effects of 5-amino-1,3,4-thiadiazole-2-sulfonamide 1, acetazolamide 2 and new synthesized amides on these isozymes have been studied in vitro. The I₅₀ concentrations (the concentration of inhibitor producing a 50% inhibition of CA activity) against hydratase activity ranged from 1.2 to 2.2 nM for hCA-I and from 0.4 to 2 nM for hCA-II. The I₅₀ values against esterase activity ranged from 1.4 to 8 nM for hCA-I and from 1.3 to 6 nM for hCA-II. The K_i values were observed between 8.2·10^{? 5} to 6.2·10^{? 4} M for hCA-I and between 2.9·10^{? 4} to 8.2·10^{? 4} M for hCA-II. The comparison of new synthesized amides to 5-amino-1,3,4-thiadiazole-2-sulfonamide 1, acetazolamide 2 indicated that the new synthesized compounds (18–23) inhibit CA activity more potently than the parent compounds.     

The Effect of Universal Fluorinated Nucleobases on the Catalytic Activity of Ribozymes

Abstract

Four fluoro modified universal nucleobases have been synthesized. The universal nucleobases 1 and 2 , containing a 2,4-difluorobenzene as nucleobase and a 4,6-difluorobenzimidazole, respectively, were chemically incorporated into a selected hammerhead ribozyme sequence which has already been retrovirally expressed as an anti-HIV ribozyme to investigate their effect on the catalytic activity of the ribozymes. The substitution of the natural nucleosides with either 1 or 2 results only in a small decrease of the catalytic activity. The K_m value for the monosubstituted ribozyme with a 2,4-difluorobenzene is 309 nM^?1, the corresponding k_cat is 2.91 · 10^?3 min^?1. A disubstituted hammerhead ribozyme carrying one of each modification has also been synthesized. For a further stabilization of the ribozyme/substrate complex 2′-(β-aminoethoxy) modified fluorinated nucleosides 15 and 16 have been developed.     

Design,synthesis, and biological properties of triazole derived compounds and their transition metal complexes

Triazole derived Schiff bases and their metal complexes (cobalt(II), copper(II), nickel(II), and zinc(II)) have been prepared and characterized using IR, ¹H and ¹³C NMR, mass spectrometry, magnetic susceptibility and conductivity measurements, and CHN analysis data. The structure of L², N-[(5-methylthiophen-2-yl)methylidene]-1H-1,2,4-triazol-3-amine, has also been determined by the X-ray diffraction method. All the metal(II) complexes showed octahedral geometry except the copper(II) complexes, which showed distorted octahedral geometry. The triazole ligands and their metal complexes have been screened for their in vitro antibacterial, antifungal, and cytotoxic activity. All the synthesized compounds showed moderate to significant antibacterial activity against one or more bacterial strains. It is revealed that all the synthesized complexes showed better activity than the ligands, due to coordination.     

Large-Scale Solid-Phase Preparation of 3′-Unprotected Trinucleotide Phosphotriesters - Precursors for Synthesis of Trinucleotide Phosphoramidites

Abstract

The approach to large-scale solid-phase synthesis of 3′-unprotected trinucleotide phosphotriesters has been developed. The trinucleotides have been synthesized in 5 g scale by phosphotriester approach using CPG with pore size 70A. Total yield of target products was 75–90%. The molar extinctions of trinucleotides at various wavelengths were calculated; the experimental UV-spectra of trinucleotides show a good agreement with theoretical ones. The trinucleotides synthesized were used for synthesis of trinucleotide phosphoramidites - synthons for generation of DNA/peptide libraries.     

Spectroscopic investigations on DNA binding profile of two new naphthyridine carboxamides and their application as turn-on fluorescent DNA staining probes

Abstract

Two new 10-methoxydibenzo[b,h][1,6]naphthyridine-2-carboxamide derivatives (R1 and R2) have been synthesized and characterized using different spectral techniques. The binding of these probes with DNA was investigated using spectral (Electronic, fluorescence, ¹H NMR and circular dichroism) and molecular docking studies. These probes exhibited a strong fluorescence around 440?nm upon excitation around 380?nm. Electronic and competitive fluorescence titration studies, in HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)] buffer/dimethyl sulfoxide (pH 7.4) medium, suggest that these probes bind strongly to DNA, which is substantiated by ¹H NMR study. The binding constants are calculated to be 5.3?×?10⁷ and 6.8?×?10⁶ M^?1 for R1 and R2, respectively. From the results of spectral studies, it is proposed that the mechanism of binding of these probes with DNA is through minor groove binding mode, which is further confirmed by circular dichroism and molecular docking studies. Initial cell viability screening using MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay shows that normal Vero cells are viable towards these probes at nano molar concentration, which is the concentration range employed in the present study for DNA staining (IC₅₀ in the order of 0.023?mM). The enhancement in fluorescence intensity of these probes upon binding with DNA enables the staining of DNA in agarose gel in gel electrophoresis experiment. The sensitivity of these probes is comparable with that of ethidium bromide and DNA amounts as low as 4 nano gram are detectable.

Communicated by Ramaswamy H. Sarma     

Nucleosides,XLIV1 Synthesis,Properties and Biological Activity of Indazole Nucleosides

Abstract

Various new haloindazole-1-β-D-ribofuranosides (10-17,20,21) and a 2-β-D-ribofuranoside (18) have been synthesized by the fusion method and by direct halogenations, respectively. The new nucleosides have been characterized by UV and ¹H NMR spectra as well as pK_a determinations. Indazole ribofuranosides behave in aqueous acid like purine and benzimidazole nucleosides showing the same mechanism of cleavage of the glycosidic bonds. Toxicity studies against various cell populations indicate only little biological activities.     

Synthesis,DNA Binding,and Oxidative Cleavage Studies of Fe(II) and Co(III) Complexes Containing Bioactive Ligands

The two complexes containing bioactive ligands of the type and [Fe(L)] (PF₆)₂ (1) (where L = [1-{[2-{[2-hydroxynaphthalen-1-yl)methylidine]amino}phenyl)imino] methyl}naphthalene-2-ol]) and [Co(L₁L₂)] (PF₆)₃ (2) (where L₁L₂ = mixed ligand of 2-seleno-4-methylquinoline and 1,10-phenanthroline in the ratio 1:2, respectively) were synthesized and structurally characterized. The DNA binding property of the complexes with calf thymus DNA has been investigated using absorption spectra, viscosity measurements, and thermal denaturation experiments. Intrinsic binding constant K_b has been estimated at room temperature. The absorption spectral studies indicate that the complexes intercalate between the base pairs of the CT-DNA tightly with intrinsic DNA binding constant of 2.8 × 10⁵ M^?1 for (1) and 4.8 × 10⁵ M^?1 for (2) in 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2, respectively. The oxidative cleavage activity of (1) and (2) were studied by using gel electrophoresis and the results show that complexes have potent nuclease activity.     

Synthesis and DNA Binding Studies of Novel Heterocyclic Substituted Quinoline Schiff Bases: A Potent Antimicrobial Agent

The present article deals with the synthesis of 2-chloroquinoline-3-carbaldehyde [(2-hydroxy-1-naphthyl) methylene] hydrazone (CQCMH) (2a-c) and 2-chloroquinoline-3-carbaldehyde [4-(dimethylamino) benzylidene] hydrazone (CQCDBH) (3a-c) from quinoline derivatives under suitable experimental conditions. The synthesized compounds were characterized by elemental analysis, FTIR, ¹HNMR, and mass spectral data. The selected compounds were studied for interaction with calf thymus-DNA (CT-DNA) by electronic spectra, viscosity measurements as well as thermal denaturation studies. On binding to DNA, the absorption spectrum underwent bathochromic and hypochromic shifts. The binding constant (K_b) had value of 2.3×10³ M^?1 for (2a) and 2.5×10⁴ M^?1 for (3a). The viscosity measurements indicated that the viscosity of sonicated rod like DNA fragments increased. The synthesized derivatives have been screened for antibacterial and antifungal activities.     

Asymmetric Synthesis of Cyclopropyl Carbocyclic Nucleosides

Abstract

A number of nucleosides have been synthesized as potential antiviral and antitumor agents.¹ More recently, various dideoxynucleosides have been synthesized and found to be potent anti-HIV agents.² As a part of our drug discovery program for the treatment of HIV and HBV, we have initiated to synthesize cyclopropyl carbocyclic nucleosides as potential antiviral agents. Several papers regarding the synthesis of cyclopropyl carbocyclic nucleosides have appeared in the literature.^3–5 However, they are all reported as racemic mixtures. In this abstract, we wish to report the asymmetric synthesis of cylopropyl carbocyclic nucleosides from optically active common intermediates, 6 and 11.     

Synthesis and Antiviral Evaluation of 2-Mercapto-5,6-dichlorobenzimidazole-β-D-ribofuranonucleoside Derivatives

Abstract

2-Mercapto-5,6-dichlorobenzimidazole β-D-ribofuranonucleoside derivatives 8–10 have been synthesized and their antiviral properties examined. According to the glycosylation procedure used, the β-D-N-1 isomer (and the N, N-bis-riboside) or the β-D-S²-isomer have been obtained. All the prepared compounds were tested for their activity against a variety of RNA and DNA viruses, but they did not show significant antiviral activity.     

New dinuclear copper(II) and zinc(II) complexes for the investigation of sugar–metal ion interactions

We have studied the binding interactions of biologically important carbohydrates (d-glucose, d-xylose and d-mannose) with the newly synthesized five-coordinate dinuclear copper(II) complex, [Cu₂(hpnbpda)(μ-OAc)] (1) and zinc(II) complex, [Zn₂(hpnbpda)(μ-OAc)] (2) [H₃hpnbpda = N,N′-bis(2-pyridylmethyl)-2-hydroxy-1,3-propanediamine-N,N′-diacetic acid] in aqueous alkaline solution. The complexes 1 and 2 are fully characterized both in solid and solution using different analytical techniques. A geometrical optimization was made of the ligand H₃hpnbpda and the complexes 1 and 2 by molecular mechanics (MM+) method in order to establish the stable conformations. All carbohydrates bind to the metal complexes in a 1:1 molar ratio. The binding events have been investigated by a combined approach of FTIR, UV–vis and ¹³C NMR spectroscopic techniques. UV–vis spectra indicate a significant blue shift of the absorption maximum of complex 1 during carbohydrate coordination highlighting the sugar binding ability of complex 1. The apparent binding constants of the substrate-bound copper(II) complexes have been determined from the UV–vis titration experiments. The binding ability and mode of binding of these sugar substrates with complex 2 are indicated by their characteristic coordination induced shift (CIS) values in ¹³C NMR spectra for carbon atoms C1, C2, and C3 of sugar substrates.     

Bacteriophage T4 rIIB protein synthesis with a temperature-sensitive mutation in the rIIB initiation codon.

Summary In protein synthesis, the incorporation of an N-terminal formylmethionine residue is directed by an initiation codon. The most frequently used codon is AUG, although initiation at GUG and UUG codons has also been observed. The HD263 mutation is an AUG to AUA change in the rIIB initiation codon. Evidence is presented here that wild type and HD263 rIIB proteins, whether synthesized in vivo or in vitro, have identical fmet peptides. It is concluded that translation began at the AUA mutant initiation codon in vitro and in phage T4 infected cells.In the in vitro translation system used in these studies, the rIIB protein synthesized at 25° no longer contains the N-terminal formyl group whereas a large proportion of the formyl group is retained at 37°.Abbreviations used tss-mutation temperature-sensitive, synthesis mutation - PrIIB protein product of gene rIIB - PrIIB⁺ PHD263 and PHE122, rIIB proteins synthesized by rIIB⁺ phage, tss-mutant HD263 and amber mutant HE122 - fmet-tRNA N-formylmethionyl-tRNA _inf ^met     

Synthesis and cardioprotective properties of apelin-12 and its structural analogues

Apelin-12 and a number of its analogues (Nle¹⁰-, MeArg¹, Nle¹⁰, MeArg¹, Nle¹⁰, Phe¹²-NH₂-, Arg¹(NO₂), Nle¹⁰, Phe¹²-NH₂-), resistant to the degradation of proteases, were synthesized by the Fmocmethod of SPPS. By-products of synthesis were examined. It was found that the serine hydroxyl group was sulfating during the final deprotection of apelin-12 and its analogues. The sulfate moiety of the Arg-protecting group transfers into the hydroxyl group of Ser. The amount of by-product depends on the water presence in cleavage mixture. Furthermore, the final deprotection of amide analogues of apelin-12 was accompanied by the formation of a by-product — 4-hydroxybenzylamide; its amount ranged from 20% to 8% in the reaction mixture (according to HPLC data) and also depended on the composition of the cleavage mixture. Effects of the synthesized peptides on recovery of the cardiac function after ischemia were examined in a model of isolated perfused rat heart. Infusions of any of the peptides (I–V) before ischemia resulted in a significant improvement of contractile and pump function recovery compared with the control. Cardioprotective efficacy of the peptides increased in the following rank (I) < (II) = (III) < (IV) = (V).     

RNase H Activation by Stereoregular Boranophosphate Oligonucleotide

Abstract

A stereoregular all-(S _p)-boranophosphate oligodeoxyribonucleotide (BH₃ ^?-ODN) 15-mer was synthesized using an enzymatic approach. The BH₃ ^?-ODN formed a hybrid with the complementary RNA 15-mer and induced RNase H hydrolysis of the RNA strand at ODN concentrations as low as 10 nM at 37°C, but with a lower efficiency than that of its natural phosphodiester analogue.     

Synthesis of Uridylyl (3′-5′) Uridine Derivatives Containing 5-(Methylamino-Methyl) Uridine as A Modified Nucleoside Found from E. COLI Minor tRNAArg

Abstract

5-(Methylaminomethyl)uridine-containing uridylyl (3′-5′) uridine derivatives (14, 26, and 29), which were the original and modified sequences corresponding to the first letter (position 34) and the 5′-upper ribonucleoside (position 33) in the anticodon loop of minor tRNA^Arg, have been synthesized via 5-(methylaminomethyl)uridine derivatives (4 and 24).     

Synthesis of the conjugation ready, downstream disaccharide fragment of the O-PS of Vibrio cholerae O:139

The linker-equipped disaccharide, 8-amino-3,6-dioxaoctyl 2,6-dideoxy-2-acetamido-3-O-β-d-galactopyranosyluronate-β-d-glucopyranoside (10), was synthesized in eight steps from acetobromogalactose and ethyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-1-thio-β-d-glucopyranoside. The hydroxyl group present at C-4^II in the last intermediate, 8-azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2-trichloroacetamido-3-O-(benzyl 2,3-di-O-benzyl-β-d-galactopyranosyluronate)-β-d-glucopyranoside (9), is positioned to allow further build-up of the molecule and, eventually, construction of the complete hexasaccharide. Global deprotection (9→10) was done in one step by catalytic hydrogenolysis over palladium-on-charcoal.     

DNA groove-binding and acid-base properties of a Ru(II) complex containing anthryl moieties

Abstract

DNA groove binders have been poorly studied as compared to the intercalators. A novel Ru(II) complex of [Ru(aeip)₂(Haip)](PF₆)₂ {Haip?=?2-(9-anthryl)-1H-imidazo[4,5-f][1,10]phenanthroline and aeip = 2-(anthracen-9-yl)-1-ethyl-imidazo[4,5-f][1, 10]phenanthroline} is synthesized and characterized by elemental analysis, ¹H NMR spectroscopy and mass spectrometry. The complex is evidenced to be a calf-thymus DNA groove binder with a large intrinsic binding constant of 10⁶ M^?1 order of magnitude as supported by UV–visible absorption spectral titrations, salt effects, DNA competitive binding with ethidium bromide, DNA melting experiment, DNA viscosity measurements and density functional theory calculations. The acid-base properties of the complex studied by UV–Vis spectrophotometric titrations are reported as well.     

Synthesis of 4-(2-substituted hydrazinyl)benzenesulfonamides and their carbonic anhydrase inhibitory effects

In this study, 4-(2-substituted hydrazinyl)benzenesulfonamides were synthesized by microwave irradiation and their chemical structures were confirmed by ¹H NMR, ¹³CNMR, and HRMS. Ketones used were: Acetophenone (S1), 4-methylacetophenone (S2), 4-chloroacetophenone (S3), 4-fluoroacetophenone (S4), 4-bromoacetophenone (S5), 4-methoxyacetophenone (S6), 4-nitroacetophenone (S7), 2-acetylthiophene (S8), 2-acetylfuran (S9), 1-indanone (S10), 2-indanone (S11). The compounds S9, S10 and S11 were reported for the first time, while S1–S8 was synthesized by different method than literature reported using microwave irradiation method instead of conventional heating in this study. The inhibitory effects of 4-(2-substituted hydrazinyl)benzenesulfonamide derivatives (S1–S11) against hCA I and II were studied. Cytosolic hCA I and II isoenzymes were potently inhibited by new synthesized sulphonamide derivatives with K_is in the range of 1.79?±?0.22–2.73?±?0.08?nM against hCA I and in the range of 1.72?±?0.58–11.64?±?5.21?nM against hCA II, respectively.     

31P NMR Studies on the Putative Chemoselective Activation of Nucleoside-3′-thiophosphate-O-esters

Abstract

P-diastereomerically pure O-esters of N^Bz-5′-DMT-dA-3′-monothiophosphate, having charged S?P?O^? moiety, have been synthesized. Chemoselectivity of their activations by formation of different mixed anhydrides, followed by couplings with N^Bz-3′-levulinyl-dA, were studied by ³¹P NMR spectroscopy.