Effect of microwave irradiation on phosphoramidite couplings on controlled pore glass

The chain extension step in the synthesis of DNA oligomers on controlled pore glass was shown to be higher yielding when the reaction mixture is irradiated with microwaves. Both a commercial thymidine 3'-phosphoramidite building block and a 3'-phosphoramidite of protected 1'-aminomethylthymidine were coupled using dilute solutions that give only partial conversion. In either case, higher coupling yields were observed when microwaves were used. The results of our exploratory experiments suggest that microwave-assisted DNA syntheses might require fewer equivalents of phosphoramidites and/or shorter coupling times than those performed at room temperature.     

Stereocontrolled Facile Synthesis and Biological Evaluation of (3′S) and (3′R)-3′-Amino (and Azido)-3′-Deoxy Pyranonucleosides

This article describes the synthesis of (3 ′S) and (3 ′R)-3 ′-amino-3 ′-deoxy pyranonucleosides and their precursors (3 ′S) and (3 ′R)-3 ′-azido-3 ′-deoxy pyranonucleosides. Azidation of 1,2:5,6-di-O-isopropylidene-3-O-toluenesulfonyl-α-D-allofuranose followed by hydrolysis and subsequent acetylation afforded 3-azido-3-deoxy-1,2,4,6-tetra-O-acetyl-D-glucopyranose, which upon coupling with the proper silylated bases, deacetylation, and catalytic hydrogenation, obtained the target 3 ′-amino-3 ′-deoxy-β-D-glucopyranonucleosides. The desired 1-(3 ′-amino-3 ′-deoxy-β-D-allopyranosyl)5-fluorouracil was readily prepared from the suitable imidazylate sugar after azidation followed by a protection/deprotection sequence and reduction of the unprotected azido precursor. No antiviral activity was observed for the novel nucleosides. Moderate cytostatic activity was recorded for the 5-fluorouracil derivatives.     

Versatile Solid Supports for Oligonucleotide Synthesis that Incorporate Urea Bridge

The universal solid support, USIII, representing a new and improved version of commercial USII, as well as 2 ′-deoxynucleoside and 2 ′-deoxy-2 ′-fluoronucleoside bound supports, incorporating a labile phenoxyacetyl fragment, was synthesized by an aminomethyl polystyrene carbamoylation with corresponding azides in the presence of aqueous triethylammonium bicarbonate. All three solid phases incorporate a stable urea tether, thus bridging the polymer and functional linker. These new matrices proved to be potent solid phases for the synthesis of DNA, RNA, or modified oligonucleotides as well as randomized mixed 2 ′-ribo/2 ′-deoxy-2 ′-fluoro-RNA libraries and/or DNA libraries, randomized with trinucleotides (codons).     

Characterization of the Aldehyde Reactive Probe Reaction with AP-Sites in DNA: Influence of AP-Lyase on Adduct Stability

Alkoxyamines react with the open-chain aldehyde form of AP-sites in DNA to produce open-chain aldehyde oximes. Here we characterize the effect of AP-site cleavage by yeast AP-endonuclease 1 (APN1) or T4 pyrimidine dimer DNA glycosylase/AP-lyase (T4 Pdg) on the efficiency and stability of the alkoxyamine aldehyde reactive probe (ARP) condensation reaction with AP-sites. The results indicate that (1) reaction of ARP with the open-chain aldehyde equilibrium form of the AP-site was less efficient than with the 3 ′-α,β-unsaturated aldehyde produced by T4 Pdg; (2) the dRP moiety was least reactive with ARP; (3) both the AP-site and 3 ′-α,β-unsaturated aldehyde were stable with regard to reaction with ARP over a 30-min incubation period at 37°C; and (4) ARP adducted to the open-chain aldehyde form of the AP-site could be replaced by methoxyamine, but the 3 ′-α,β-unsaturated aldehyde ARP oxime was stable against methoxyamine attack.     

Selective Biocatalytic Acylation Studies on 5′-O-(4,4′-Dimethoxytrityl)-2′,3′-Secouridine: An Efficient Synthesis of UNA Monomer

Lipozyme® TL IM (Theremomyces lanuginosus lipase immobilized on silica) in toluene catalyzes the acylation of the 2 ′-OH over the 3 ′-OH group in 5 ′-O-(4,4 ′-dimethoxytrityl)-2 ′,3 ′-secouridine (5 ′-O-DMT-2 ′,3 ′-secouridine) in a highly selective fashion in moderate to almost quantitative yields. The turn over during benzoyl transfer reactions mediated by vinyl benzoate or benzoic anhydride was faster than in acyl transfer reactions with vinyl acetate or C₁ to C₅ acid anhydrides; except in the case of butanoic anhydride. The 2 ′-O-benzoyl-5 ′-O-DMT-2 ′,3 ′-secouridine obtained by Lipozyme® TL IM catalyzed benzoylation of 5 ′-O-DMT-2 ′,3 ′-secouridine was successfully converted into its 3 ′-O-phosphoramidite derivative in satisfactory yield, which is a building block for the preparation of oligonucleotides containing the uracil monomer of UNA (unlocked nucleic acid).     

Synthesis and Biological Evaluation of (3′S)-3′-Deoxy-3′-Fluoro-3′-C-Ethynylcytidine

The novel pyrimidine nucleoside, (3 ′S)-3 ′-deoxy-3 ′-fluoro-3 ′-C-ethynylcytidine (1) was synthesized from cytidine in seven steps. The key step in the synthesis was the introduction of the tertiary fluorine at the 3 ′-position. Compound 1 was evaluated in vitro against several RNA viruses.     

Synthesis and Properties of ApA Analogues with Shortened Phosphonate Internucleotide Linkage

A complete series of the 2 ′–5 ′ and 3 ′–5 ′ regioisomeric types of r(ApA) and 2 ′-d(ApA) analogues with the α-hydroxy-phosphonate C3 ′-O-P-CH(OH)-C4 ″ internucleotide linkage, isopolar but non-isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU studied. Due to the chirality on the 5 ′-carbon atom of the modified internucleotide linkage bearing phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs. To examine the role of the 5 ′-hydroxyl of the α-hydroxy-phosphonate moiety during hybridization, the appropriate r(ApA) analogues with 3 ′(2 ′)-O-P-CH₂-C4 ″ linkage lacking the 5 ′-hydroxyl were synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the modified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable differences in the stability of complexes with polyU, depending on the 5 ′-carbon atom configuration. Potential usefulness of the α-hydroxy-phosphonate linkage in modified oligoribonucleotides is discussed.     

A simple and efficient synthesis of puromycin, 2,2′-anhydro-pyrimidine nucleosides,cytidines and 2′,3′-anhydroadenosine from 3′,5′-O-sulfinyl Xylo-nucleosides

Synthesis of antibiotics, puromycin and 3 ′-amino-3 ′-deoxy-N ⁶,N ⁶-dimethyladenosine 11 was achieved by utilizing the cyclic sulfite 6a of the xylo-3 ′,5 ′-dihydroxy group as a new protective group. The key synthetic step is the deprotection of the sulfite moiety through the intramolecular cyclization of 2-α-carbamate 7. In a similar manner, 2,2 ′-anhydro-pyrimidine nucleosides 15, ribo-cytidines 17 and 2 ′,3 ′-anhydroadenosine 14 were prepared in high yields from the corresponding sulfites 4, 5, and 6b, respectively.     

DDX Profiles in Agricultural Fields Used for Cucurbit Production in Sakarya,Turkey

DDT (2,2-bis(chlorophenyl)-1,1,1- trichloroethane) and its metabolites DDD (2,2-bis(chlorophenyl)-1,1 -dichloroethane) and DDE (2,2-bis(chloraphenyl)-1,1 –dichloroethylene) have half-lives in soil measured in years or decades and are classified as Persistent Organic Pollutants (POPs). In this study, p,p′-DDT, p,p′-DDD, and p,p′-DDE residues were investigated in select agricultural fields of Sakarya Province, Turkey, where Cucurbitaceae have been grown for many years. Total squash and pumpkin production in Sakarya is approximately 3% of total cucurbit production of Turkey but little is known about the concentrations of DDT, DDD, and DDE in these agricultural soils. Thirty-three soil samples were collected from agricultural fields in different counties of Sakarya. p,p′-DDT was detected in all soil samples, with concentrations ranging from 0.23 ng/g to 123 ng/g soil (dry weight). The concentrations of p,p′-DDT metabolites ranged from nondetectable (<0.06 ng/g) to 120 ng/g for p,p′-DDD and from nondetectable (<0.03 ng/g) to 294 ng/g for p,p′-DDE. The highest total DDX (sum of p,p′-DDT, p,p′-DDD, and p,p′-DDE) concentrations among the soil samples was 428 ng/g in a sample collected from Karasu County. Further research in this field was conducted to measure p,p′-DDT, p,p′-DDE, and p,p′-DDD concentrations at multiple locations as a function of soil depth. p,p′-DDT concentrations were measured from 52 ng/g to 1935 ng/g at 0–60 cm depth. The highest DDX concentration was observed at a location where plants have been actively grown since 1987. The lowest DDX concentrations were observed where crops have not been grown since 1987. Our data proved that soil DDX levels at the field gradually increased as a function of how extensively the field has been used for cucurbits production. However, it is not certain whether the application of p,p′-DDT was terminated or if there may still be illegal usage in agricultural soils.     

An Improved Protection-Free One-Pot Chemical Synthesis of 2′-Deoxynucleoside-5′-Triphosphates

A facile, straightforward, reliable, and an efficient method for the gram-scale chemical synthesis of both purine deoxynucleotides such as 2 ′-deoxyguanosine-5 ′-triphosphate (dGTP) and 2 ′-deoxyadenosine-5 ′-triphosphate (dATP) and pyrimidine deoxynucleotides such as 2 ′-deoxycytidine-5 ′-triphosphate (dCTP), thymidine-5 ′-triphosphate (TTP), and 2 ′-deoxyuridine-5 ′-triphosphate (dUTP) starting from the corresponding nucleoside is described. This improved “one-pot, three step” Ludwig synthetic strategy involves the monophosphorylation of nucleoside followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65%–70%).     

Design and Synthesis of Novel 1′,3′-Dioxolane 5′-Deoxyphosphonic Acid Purine Analogues as Potent Antiviral Agents

Electronic parameters of 1′,3 ′-oxygen play significant roles in steering the conformation of nucleoside phosphonic acid analogues. To investigate the relationship of two oxygen atoms with antiviral enhancement, novel 1′,3 ′-dioxolane 5 ′-deoxyphosphonic acid purine analogues were synthesized via de novo acyclic stereoselective route from acrolein and glycolic acid. The synthesized nucleoside phosphonic acid analogues 14 and 19 were subjected to antiviral screening against several viruses, such as HIV-1, HSV-1, HSV-2, and HCMV. The guanine analogue 19 exhibits in vitro anti-HIV-1 activity similar to that of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) in MT-4 cells.     

Molecular Structure of the Core-Modified siRNA Duplexes Containing Diastereomeric Pair of [C6′(R)-OH]- versus [C6′(S)-OH]-carba-LNAs Suggests a Model for RNAi Action

Molecular structures of native and a pair of modified small interfering RNA–RNA duplexes containing carbocyclic [6 ′-(R)-OH/7 ′-(S)-methyl]- and [6 ′-(S)-OH/7 ′-(S)-methyl]-carba-LNA-thymine nucleotides, which are two diastereomeric analogs of the native T nucleotide, incorporated at position 13 in the antisense (AS) strand of siRNA, have been simulated using molecular mechanics/dynamics techniques. The main aim of the project has been to find a plausible structural explanation of why modification of siRNA at T¹³ position by the [6 ′(R)-O-(p-Toluoyl)-7 ′(S)-methyl]-carba-LNA-Thymine [IC₅₀ of 3.32 ± 0.17 nM] is ca 24 times more active as an RNA silencing agent against the target HIV-1 TAR RNA than the [6 ′(S)-O-(p-Toluoyl)-7 ′(S)-methyl]-counterpart [IC₅₀ of 79.8 ± 17 nM] [1]. The simulations reveal that introduction of both C6 ′(R)-OH and C6 ′(S)-OH stereoisomers does not lead even to local perturbation of the siRNA–RNA duplex structures compared to the native, and the only significant difference between 6 ′(S)- and 6 ′(R)-diastereomers found is the exposure of the 6 ′-OH group of the 6 ′(R)-diastereoisomer toward the edge of the duplex while the 6 ′-hydroxyl group of the 6 ′(S)-diastereoisomer is somewhat buried in the minor groove of the duplex. This rules out a hypothesis about any possible local distortion by the nature of chemical modification of the siRNA-target the RNA duplex, which might have influenced the formation of the effective RNA silencing complex (RISC) and puts some weight on the hypothesis about the 6 ′-hydroxy group being directly involved with most probably Ago protein, since it is known from exhaustive X-ray studies [2, 3] that the core residues are indeed involved with hydrogen bonding with the internucleotidyl phosphates. Further systematic investigation is in progress to map the position-dependent functional and nonfunctional interactions of the modified [6 ′(R or S)-O-(p-Toluoyl)-7 ′(S)-methyl]-carba-LNA-T with the Ago2 protein of the RISC.     

A new insect cell line from the pupal ovary of the Asian corn borer moth Ostrinia furnacalis

Ovaries were removed from Ostrinia furnacalis (Guenée) pupae and were placed in a flask containing TNM-FH medium with 10% inactivated fetal bovine serum. Cell migration occurred after about 1 wk of the initiation in June 2011. The migrated cells were distributed over most of the flask and were treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a chemical carcinogen, after about 1 mo of initiation for 26 d. Cells were first subcultured successfully 12 d after the MNNG was removed, followed by subculturing for 30 passages. The established cell line, designated IOZCAS-Osfu-1, were analyzed by DNA fingerprinting–PCR (DAF-PCR) to confirm that it originated from O. furnacalis.     

Study on injury effect of food additive citric acid on liver tissue in mice

To investigate the damaging effect and action mechanism of the food additive citric acid (CA) on mouse liver, 40 healthy male Kunming mice were randomly divided into control group (0.9 % saline), low CA dose (120 mg/kg), middle dose (240 mg/kg) and high dose groups (480 mg/kg). All experimental mice have received peritoneal injection of the corresponding reagent each week for 3 weeks. After 7 days since the third injection, morphological changes were observed by light microscope; activities of T-SOD, glutathione peroxidase (GSH-Px), caspase-3, and contents of hydrogen peroxide (H₂O₂) and malonyldialdehyde (MDA) in the liver were evaluated using the corresponding assay kits; DNA fragmentation was assayed using agarose gel electrophoresis. Microscopical detection showed a series of hispathological changes in mouse livers treated with CA, such as indiscriminate liver cell cord, blood clot in central veins, and lymphocyte infiltrating. Biochemical examination suggested the gradually but moderately reduced T-SOD activity and elevated H₂O₂ level with the increase of CA dose (P > 0.05), and the gradually reduced GSH-Px activity and increased MDA content depending on graded doses with a significant difference (P < 0.05) between the high dose group and the control group. According to cell apoptosis assays, caspase-3 activity were significantly higher in all treatment groups than in the control (P < 0.05) in a dose-dependent manner. Contrasting to the control, characteristic DNA laddering was observed when injected with any of the three graded doses. It can be concluded that certain concentrations of CA cause oxidative damage of the liver by means of the decrease of antioxidative enzyme activities, thus resulting in MDA level elevation and DNA fragmentation inducing active caspase-3.     

Global DNA methylation profiles of somatic embryos of peach palm (Bactris gasipaes Kunth) are influenced by cryoprotectants and droplet-vitrification cryopreservation

Regrowth capacity and genetic stability of plants recovered following cryopreservation are associated with changes in DNA epigenetics, particularly in DNA methylation levels. In this study, global DNA methylation profiles associated with frequency of regrowth of peach palm (Bactris gasipaes) somatic embryos following cryopreservation using droplet-vitrification were investigated. Somatic embryo clusters (SEC) subjected to plant vitrification solution 3 (PVS3) for different durations (0, 60, 120, 180, and 240 min) were evaluated for regrowth capacity. The highest frequency of regrowth (52.4 %) was obtained when SEC were incubated in PVS3 for 120 min prior to droplet-vitrification cryopreservation. Global DNA methylation profiles were influenced by both cryoprotectants and droplet-vitrification cryopreservation. Incubation of SEC in PVS3 for limited durations not only reduced frequency of regrowth, but also increased DNA methylations levels when compared with proliferating SEC grown in a temporary immersion system. Although SEC subjected to cryopreservation exhibited the highest DNA methylation variation, 120 min SEC incubation in a PVS3 solution resulted in the recovery of initial global methylation profiles after 24 weeks of regrowth.     

Effect of various microwave frequencies on the physiology of a cyanobacterium, Schizothrix mexicana

The effect of microwave modulated with square waves of different pulse repetition frequencies was studied on the physiologic behavior of the cyanobacterium Schizothrix mexicana Gomont (Oscillatorials). The organism was exposed for 2 h to microwave (9.685 GHz) modulated with square wave pulse repetition frequencies of 900, 720, 540, 360 and 180 Hz at a fixed incident power density (0.768 mW/cm²). Results revealed that growth rate, dry weight and photosynthetic pigments increased significantly especially in frequency of 540 Hz. Total carbohydrate and protein contents, and photosynthesis and nitrogenase activity showed drastic increase in 180, 360 and 540 Hz and dropped down when treated with 720 and 900 Hz frequency. This study revealed that the increase effect of microwave radiation on metabolism of Schizothrix mexicana was found to be frequency dependent. So, electrostimulation of Schizothrix mexicana provides a new extended domain of disciplines and methodologies for cultivation, processing of biomass and biofuels, biofertilizers and biotechnology applications. This study reveals that microwaves athermally induce different biologic effects.     

Acute low-intensity microwave exposure increases DNA single-strand breaks in rat brain cells

Levels of DNA single-strand break were assayed in brain cells from rats acutely exposed to low-intensity 2450 MHz microwaves using an alkaline microgel electrophoresis method. Immediately after 2 h of exposure to pulsed (2 μs width, 500 pulses/s) microwaves, no significant effect was observed, whereas a dose rate-dependent [0.6 and 1.2 W/kg whole body specific absorption rate (SAR)] increase in DNA single-strand breaks was found in brain cells of rats at 4 h postexposure. Furthermore, in rats exposed for 2 h to continuous-wave 2450 MHz microwaves (SAR 1.2 W/kg), increases in brain cell DNA single-strand breaks were observed immediately as well as at 4 h postexposure. © 1995 Wiley-Liss, Inc.     

Screening of lipid high producing mutant from rhodotorula glutinis by low ion implantation and study on optimization of fermentation medium

In order to obtain lipid producing strain with high-yield, the wild type stain Rhodotorula glutinis was treated by low ion implantation, and optimization of fermentation medium for higher lipid yield was carried out using mutant strain. It was found that the strain had a higher positive mutation rate when the output power was 10 keV and the dose of N⁺ implantation was 80 × 2.6 × 10¹³ ions/cm². Then a high-yield mutant strain D30 was obtained through cid-heating coupling ultrasonic method and lipid yield was 3.10 g/L. Additionally, the surface response method was used to optimize fermentation medium. The three significant factors (glucose, peptone, KH₂PO₄) were optimized using response surface methodology (RSM), and the optimized parameters of fermentation medium were as follows: glucose 73.40 g/L, peptone 1.06 g/L and KH₂PO₄ 3.56 g/L. Finally the fermentation characteristic of high-yield mutation strain D30 was studied, when fermentation time was 10 days, which lipid yield increased to 7.81 g/L. Fatty acid composition of the lipid was determined by GC, and the most represented fatty acids of mutant D30 were C16:0 (11.4 %), C16:1 (5.66 %), C18:1 (49.3 %), and C18:2 (27.0 %).     

Measurements of alkali-labile DNA damage and protein-DNA crosslinks after 2450 MHz microwave and low-dose gamma irradiation in vitro

In vitro experiments were performed to determine whether 2450 MHz microwave radiation induces alkali-labile DNA damage and/or DNA-protein or DNA-DNA crosslinks in C3H 10T(1/2) cells. After a 2-h exposure to either 2450 MHz continuous-wave (CW) microwaves at an SAR of 1.9 W/kg or 1 mM cisplatinum (CDDP, a positive control for DNA crosslinks), C3H 10T(1/2) cells were irradiated with 4 Gy of gamma rays ((137)Cs). Immediately after gamma irradiation, the single-cell gel electrophoresis assay was performed to detect DNA damage. For each exposure condition, one set of samples was treated with proteinase K (1 mg/ml) to remove any possible DNA-protein crosslinks. To measure DNA-protein crosslinks independent of DNA-DNA crosslinks, we quantified the proteins that were recovered with DNA after microwave exposure, using CDDP and gamma irradiation, positive controls for DNA-protein crosslinks. Ionizing radiation (4 Gy) induced significant DNA damage. However, no DNA damage could be detected after exposure to 2450 MHz CW microwaves alone. The crosslinking agent CDDP significantly reduced both the comet length and the normalized comet moment in C3H 10T(1/2) cells irradiated with 4 Gy gamma rays. In contrast, 2450 MHz microwaves did not impede the DNA migration induced by gamma rays. When control cells were treated with proteinase K, both parameters increased in the absence of any DNA damage. However, no additional effect of proteinase K was seen in samples exposed to 2450 MHz microwaves or in samples treated with the combination of microwaves and radiation. On the other hand, proteinase K treatment was ineffective in restoring any migration of the DNA in cells pretreated with CDDP and irradiated with gamma rays. When DNA-protein crosslinks were specifically measured, we found no evidence for the induction of DNA-protein crosslinks or changes in amount of the protein associated with DNA by 2450 MHz CW microwave exposure. Thus 2-h exposures to 1.9 W/ kg of 2450 MHz CW microwaves did not induce measurable alkali-labile DNA damage or DNA-DNA or DNA-protein crosslinks.     

Exposure to non-ionizing electromagnetic fields emitted from mobile phones induced DNA damage in human ear canal hair follicle cells

The aim of this study was to investigate effect of radiofrequency radiation (RFR) emitted from mobile phones on DNA damage in follicle cells of hair in the ear canal. The study was carried out on 56 men (age range: 30–60 years old)in four treatment groups with n = 14 in each group. The groups were defined as follows: people who did not use a mobile phone (Control), people use mobile phones for 0–30 min/day (second group), people use mobile phones for 30–60 min/day (third group) and people use mobile phones for more than 60 min/day (fourth group). Ear canal hair follicle cells taken from the subjects were analyzed by the Comet Assay to determine DNA damages. The Comet Assay parameters measured were head length, tail length, comet length, percentage of head DNA, tail DNA percentage, tail moment, and Olive tail moment. Results of the study showed that DNA damage indicators were higher in the RFR exposure groups than in the control subjects. In addition, DNA damage increased with the daily duration of exposure. In conclusion, RFR emitted from mobile phones has a potential to produce DNA damage in follicle cells of hair in the ear canal. Therefore, mobile phone users have to pay more attention when using wireless phones.