Nucleic-Acid Analogs with Restricted Conformational Flexibility in the Sugar-Phosphate Backbone (‘Bicyclo-DNA’). Part 5. Synthesis,Characterization, and Pairing Properties of Oligo-α-D-(bicyclodeoxynucleotides) of the Bases Adenine and Thymine (α-Bicyclo-DNA)

A conformational analysis of the (3′S,5′R)-2′-deoxy-3′,5′-ethano-α-D -ribonucleosides (a-D-bicyclodeoxynucleosides) based on the X-ray analysis of N⁴-benzoyl-α-D -(bicyclodeoxycytidine) 6 and on ¹H-NMR analysis of the α-D -bicyclodeoxynucleoside derivatives 1 - 7 reveals a rigid sugar structure with the furanose units in the l′-exo/2′-endo conformation and the secondary OH groups on the carbocyclic ring in the pseudoequatorial orientation. Oligonucleotides consisting of α-D -bicyclothymidine and α-D -bicyclodeoxyadenosine were successfully synthesized from the corresponding nucleosides by phosphoramidite methodology on a DNA synthesizer. An evaluation of their pairing properties with complementary natural RNA and DNA by means of UV/melting curves and CD spectroscopy show the following characteristics: i) α-bcd(A₁₀) and α-bcd(T₁₀) (α = short form of α-D )efficiently form complexes with complementary natural DNA and RNA. The stability of these hybrids is comparable or slightly lower as those with natural β-d(A₁₀) or β-d(T₁₀)( β = short form ofβ-D ). ii) The strand orientation in α-bicyclo-DNA/β-DNA duplexes is parallel as was deduced from UV/melting curves of decamers with nonsymmetric base sequences. iii) CD Spectroscopy shows significant structural differences between α-bicyclo-DNA/β-DNA duplexes compared to α-DNA/β-DNA duplexes. Furthermore, α-bicyclo-DNA is ca. 100-fold more resistant to the enzyme snake-venom phosphodiesterase with respect to β-DNA and about equally resistant as α-DNA.     

Berberrubine Phosphate: A Selective Fluorescent Probe for Quadruplex DNA

A phosphate-substituted, zwitterionic berberine derivative was synthesized and its binding properties with duplex DNA and G4-DNA were studied using photometric, fluorimetric and polarimetric titrations and thermal DNA denaturation experiments. The ligand binds with high affinity toward both DNA forms (K_b = 2–7 × 10⁵ M⁻¹) and induces a slight stabilization of G4-DNA toward thermally induced unfolding, mostly pronounced for the telomeric quadruplex 22AG. The ligand likely binds by aggregation and intercalation with ct DNA and by terminal stacking with G4-DNA. Thus, this compound represents one of the rare examples of phosphate-substituted DNA binders. In an aqueous solution, the title compound has a very weak fluorescence intensity (Φ_fl < 0.01) that increases significantly upon binding to G4-DNA (Φ_fl = 0.01). In contrast, the association with duplex DNA was not accompanied by such a strong fluorescence light-up effect (Φ_fl < 0.01). These different fluorimetric responses upon binding to particular DNA forms are proposed to be caused by the different binding modes and may be used for the selective fluorimetric detection of G4-DNA.     

ED-XRF spectrometry-based trace element composition of genetically engineered rhizoclones vis-à-vis natural roots of a multi-medicinal plant, butterfly pea (Clitoria ternatea L.)

The energy dispersive X-ray fluorescence set-up incorporating a molybdenum secondary exciter was used for quantitative determination of major and minor elements in genetically transformed root somaclones (rhizoclones) of butterfly pea (Clitoria ternatea L.) which had been established via explant co-cultivation with Agrobacterium rhizogenes. The multi-elemental composition of these transformed rhizoclones was compared with that of the naturally grown in vivo donor plant. Trace elements namely Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Rb, Sr and Pb in addition to two macro-elements K and Ca were identified and quantified in root tissues of both sources. The elemental content of transformed root cultures was found to be at par with that of the natural roots of in vivo grown plants of the same species. These findings are implicated on the context of utilization of such Agrobacterium-mediated genetically transformed root cultures as a viable alternative to natural roots, the former being a fast-proliferating renewable resource of medicinally useful minerals essential for designing of effective drugs, besides providing an ex situ means for plant conservation.     

Identification of p-Coumaric Acid and Ethyl p-Coumarate as the Main Phenolic Components of Hemp (Cannabis sativa L.) Roots

Hemp (Cannabis sativa L.) contains a variety of secondary metabolites, including cannabinoids, such as psychoactive (−)-trans-Δ⁹-tetrahydrocannabinol. The present study was conducted to identify the major phenolic components contained in hemp root, which has been relatively under-researched compared to other parts of hemp. The aqueous ethanol extract of hemp roots was fractionated into methylene chloride (MC), ethyl acetate (EA), and water (WT) fractions, and high-performance liquid chromatography with photodiode array detection (HPLC-DAD) analysis was performed. The main ultraviolet (UV)-absorbing phenolic compound contained in the EA fraction was identified as p-coumaric acid by comparing the retention time and UV absorption spectrum with a standard. Silica gel column chromatography was performed to isolate a hydrophobic derivative of p-coumaric acid contained in the MC fraction. Nuclear magnetic resonance (NMR) analysis identified the isolated compound as ethyl p-coumarate. For comparative purposes, ethyl p-coumarate was also chemically synthesized by the esterification reaction of p-coumaric acid. The content of p-coumaric acid and ethyl p-coumarate in the total extract of hemp root was estimated to be 2.61 mg g⁻¹ and 6.47 mg g⁻¹, respectively, by HPLC-DAD analysis. These values correspond to 84 mg Kg⁻¹ dry root and 216 mg Kg⁻¹ dry root, respectively. In conclusion, this study identified p-coumaric acid and ethyl p-coumarate as the main phenolic compounds contained in the hemp roots.     

Effects of Phytohormones and Jasmonic Acid on Glucosinolate Content in Hairy Root Cultures of Sinapis alba and Brassica rapa

Although some study have established hairy root cultures from brassicaceous plants with glucosinolates (GS) as characteristic secondary metabolite, studies are missing which compare hairy roots with the corresponding mother plants. Therefore, two different plant species—Sinapis alba and Brassica rapa subsp. rapa pygmeae teltoviensis—were transformed with the Agrobacterium rhizogenes strain A4. Aliphatic and indolyl GS were present in B. rapa, exhibiting larger quantities in leaves than in roots. Aromatic p-hydroxybenzyl GS were found particularly in the leaves of S. alba. However, the proportion of indolyl GS increased suddenly in transformed hairy roots of S. alba and B. rapa. Cultivation with the phytohormone kinetin (0.5 mg?L^?1) enhanced GS accumulation in B. rapa hairy roots, however not in S. alba, but 2,4-D (0.4 mg?L^?1) induced de-differentiation of roots in both species and reduced GS levels. GS levels especially of 1-methoxyindol-3ylmethyl GS increased in hairy roots in response to JA, but root growth was inhibited. While 2 weeks of cultivation in 100 to 200 μM JA were determined at optimum for maximum GS yield in S. alba hairy root cultures, 4 weeks of cultivation in 50 to 100 μM JA was the optimum for B. rapa.     

Thermodynamic characteristics of triphenylantimony diacrylate

The heat capacity of triphenylantimony diacrylate Ph₃Sb(O₂CCH=CH₂)₂ was studied in an adiabatic vacuum calorimeter at 6?C350 K and differential scanning calorimeter at 330?C450 K. Melting was revealed at these temperatures; the melting point was estimated at 428.4 ± 0.5 K. It was accompanied by the partial decomposition of the substance. The low-temperature (20 K ?? T ?? 50 K) heat capacity was treated using the Debye theory of the heat capacity of solids and its multifractal model. The type of the structure topology was determined. The standard thermodynamic functions C _p ^o (T), H ^o(T) ? H ^o(0), S ^o(T), and G ^o(T) ? H ^o(0) of the compound in the crystal state were calculated from the obtained experimental data in the range from T ?? 0 to 428 K. The standard entropy of the formation of the crystalline compound Ph₃Sb(O₂CCH=CH₂)₂ at T = 298.15 K was determined.     

Use of heteronuclear multiple bond coherence NMR spectroscopy to monitor nitrogen metabolism in a transformed root culture of Datura stramonium

In order to examine the factors that regulate the flux of primary metabolites into tropane alkaloids, Nuclear Magnetic Resonance spectroscopy (NMR) has been used to monitor nitrogen metabolism in transformed root cultures of Datura stramonium fed with (¹⁵NH₄)₂SO₄ and K¹⁵NO₃. This study employs the technique of Heteronuclear Multiple Bond Coherence (HMBC) NMR spectroscopy, which combines the advantages of both bidimensional resolution and ¹H NMR sensitivity. Moreover, the HMBC sequence allows the ¹⁵N bound to labile protons to be observed via ²J and/or ³J couplings, which is very appropriate for the observation of tropane alkaloids. In transformed roots of Datura stramonium, in addition to the amino acids normally observed in monodimensional ¹⁵N NMR, other amino acids are resolved by HMBC. Labelled peaks due to N-acetyl compounds and to uridine are identified. While these primary metabolites were also seen in cell cultures of Nicotiana plumbaginifolia, cross peaks corresponding to secondary metabolites, such as tropine, were also observed. This is the first time that such secondary products have been found by this technique.     

Furanose ring conformations in a 1′-alkynyl C-nucleoside and the dinucleotide

A large-scale synthesis was carried out for alkynyl C-nucleotide oligomers in order to clarify the structural details of the artificial DNA. A liquid-phase synthesis by means of phosphoramidite methodology enabled us to handle ～0.1 g of the DNA oligomers possessing a pseudouracil derivative (T*) as a non-natural nucleobase. ¹H NMR measurements in aqueous media were carried out for the oligomers, succeeded in the accurate assignments of the protons accompanying with determination of the coupling constants (J values). These J values revealed that average conformation of the alkynylribose rings in the DNA was substantially biased toward the S-type conformers (²T₁/E₁/^OT₁). X-ray crystal analysis of the non-natural nucleoside also supported the S-type conformation (²E/²T₁) as seen in natural B-DNA.     

Proton induced X-ray emission (PIXE) technique for determining multi-element composition of transformed hairy root cultures of Boerhaavia diffusa L.: an important medicinal herb

Therapeutically important inorganic elements in Agrobacterium rhizogenes-mediated genetically transformed hairy root cultures (HRCs) of a pharmaceutically significant herb Boerhaavia diffusa were quantified using proton induced X-ray emission technique. This was compared with that of roots from the naturally grown donor plant. Two macro-elements (Ca & K) and eight different trace elements namely V, Cr, Mn, Fe, Co, Cu Zn, and Ni were detected and their content was determined. In HRCs of a transformed rhizoclone, calcium and potassium had values which were significantly higher than that of in vivo roots. The concentrations of several trace elements, which are known to have a positive implication in human healthcare, were found to be either comparable (Fe) to that in the natural root samples or higher (Mn, Zn, Cr, Cu, Co) in the transformed rhizoclone. The genetically transformed HRCs can thus serve as a fast-proliferating renewable resource of medicinally useful minerals targeting specific diseases.     

Investigation of Tropane Alkaloids in Genetically Transformed <Emphasis Type="Italic">Atropa belladonna</Emphasis> L. Cultures

The purpose of the present study was to determine the tropane alkaloid content of genetically transformed hairy root cultures of Atropa belladonna L. Determination of alkaloids was performed by HPLC method. Samples were extracted with chloroform – methanol - cc. ammonia 15:5:1 (v/v/v). Crude extracts were purified on Extrelut columns. HPLC separation was performed on Luna C8 reversed phase column. An isocratic mixture of acetonitrile – 30 mM phosphate buffer - methanol 12.2:79.7:8.1(v/v/v) was used as eluent. Peaks were identified by addition of standards and diode-array detection. Hyoscyamine, scopolamine and apoatropine were determined by external standard method at 210 nm. We measured the alkaloid content of genetically transformed in vitro cultures (hairy roots and reorganised plants) cultivated on Gamborg B5 basic media. The highest hyoscyamine and scopolamine content was found in hairy root clone #K₅ (0,223 m/m%) and in hairy root clone #K₄ (0,018 m/m%) respectively. Alkaloid contents were higher in the hairy roots than in the reorganised plants.     

Abietane Diterpenoids from the Hairy Roots of Salvia corrugata

Salvia corrugata Vahl. is an interesting source of abietane and abeo-abietane compounds that showed antibacterial, antitumor, and cytotoxic activities. The aim of the study was to obtain transformed roots of S. corrugata and to evaluate the production of terpenoids in comparison with in vivo root production. Hairy roots were initiated from leaf explants by infection with ATCC 15834 Agrobacterium rhizogenes onto hormone-free Murashige and Skoog (MS) solid medium. Transformation was confirmed by polymerase chain reaction analysis of rolC and virC1 genes. The biomass production was obtained in hormone-free liquid MS medium using Temporary Immersion System bioreactor RITA^®. The chromatographic separation of the methanolic extract of the untransformed roots afforded horminone, ferruginol, 7-O-acetylhorminone and 7-O-methylhorminone. Agastol and ferruginol were isolated and quantified from the hairy roots. The amount of these metabolites indicated that the hairy roots of S. corrugata can be considered a source of these compounds.     

In vivo transformations of dihydroartemisinic acid in Artemisia annua plants

[15-¹³C²H₃]-dihydroartemisinic acid (2a) and [15-C²H₃]-dihydroartemisinic acid (2b) have been fed via the root to intact Artemisia annua plants and their transformations studied in vivo by one-dimensional ²H NMR spectroscopy and two-dimensional ¹³C-²H correlation NMR spectroscopy (¹³C-²H COSY). Labelled dihydroartemisinic acid was transformed into 16 12-carboxy-amorphane and cadinane sesquiterpenes within a few days in the aerial parts of A. annua, although transformations in the root were much slower and more limited. Fifteen of these 16 metabolites have been reported previously as natural products from A. annua. Evidence is presented that the first step in the transformation of dihydroartemisinic acid in vivo is the formation of allylic hydroperoxides by the reaction of molecular oxygen with the Δ^4,5-double bond in this compound. The origin of all 16 secondary metabolites might then be explained by the known further reactions of such hydroperoxides. The qualitative pattern for the transformations of dihydroartemisinic acid in vivo was essentially unaltered when a comparison was made between plants, which had been kept alive and plants which were allowed to die after feeding of the labelled precursor. This, coupled with the observation that the pattern of transformations of 2 in vivo demonstrated very close parallels with the spontaneous autoxidation chemistry for 2, which we have recently demonstrated in vitro, has lead us to conclude that the main ‘metabolic route’ for dihydroartemisinic acid in A. annua involves its spontaneous autoxidation and the subsequent spontaneous reactions of allylic hydroperoxides which are derived from 2. There may be no need to invoke the participation of enzymes in any of the later biogenetic steps leading to all 16 of the labelled 11,13-dihydro-amorphane sesquiterpenes which are found in A. annua as natural products.     

Vibrational analysis of trans,Trans,trans-2,3,4,5-tetrachlorohexa-1,3,5-trienes

Infrared and Raman spectra of t,T,t-2,3,4,5-tetrachlorohexa-1,3,5-triene were measured in liquid and crystalline phases. It is suggested that the non-planar structure of this compound may be due to the 1–3 effect. In the liquid state a mixture of rotational isomers, of C₂ and C_i symmetry, was confirmed by the freezing-out of some characteristic bands in the 1600 cm⁻¹ region in the IR and Raman spectra of the t,T,t-compound. Normal coordinate analysis was carried out using force constant values from the force field previously determined for chloroderivatives of buta-1,3-diene. A complete assignment of observed frequencies is proposed.     

X-ray diffraction and thermodynamic characteristics for tellurite of the composition Li<Subscript>2</Subscript>CeTeO<Subscript>5</Subscript>

Tellurite of the composition Li₂CeTeO₅ is synthesized by solid-phase method from cerium(IV) and tellurium(IV) oxides and lithium carbonate. The type of syngony, the unit cell parameters, and the compound’s X-ray and pycnometry densities are determined via X-ray diffraction analysis. The isobaric heat capacity of lithium–cerium tellurite is studied by means of dynamic calorimetry in the temperature range of 298.15–673 K; the results serve as the basis for deriving C _p ^° ~ f(T) dependency equations and determining the compound’s thermodynamic functions. λ-shaped anomalous effects, due probably to Type II phase transitions, are found on the C _p ^° ~ f(T) dependence.     

Magnetic order and heavy fermion behavior in CePd1+xAl6−x: Synthesis, structure, and physical properties

The physical properties including magnetic susceptibility, specific heat, and electrical resistivity of single crystals are reported for the compound CePd_1+xAl_6−x (x=0.5) which crystallizes in the tetragonal SrAu₂Ga₅-type structure (space group P4/mmm). The compound was grown from an excess of molten Al flux from the respective elements and the crystal structure was established from single-crystal X-ray diffraction. Anomalies in the low temperature specific heat C_p(T) and electrical resistivity ρ(T) show that the compound undergoes ferromagnetic order at T_C=2.8 K. In the ordered state, CePd_1.5Al_5.5 displays heavy fermion behavior with a Sommerfeld coefficient of ca. 500 mJ/mol-K².     

NMR study of molecular reorientations in NH4H2AsO4

Zeeman (T_1Z) and dipolar (T_1D) spin-lattice relaxation times of protons in NH₄H₂AsO₄ were measured as a function of temperature. The existence of a slow motion (τ ≈ 10^?3 see) is established, which is most probably a low frequency hindered reorientation of H₂AsO₄ groups. This motion is slowed down below the Curie point T_c. A sharp increase of the dipolar relaxation rate above T = 314°K indicates the possibility of a new high temperature phase transition in this compound.     

Dielectric properties of films of Ag-ED20 epoxy nanocomposite synthesized in situ. Temperature dependence of direct current conductivity

The influence of silver myristate used as a precursor of silver nanoparticles on the direct current conductivity σ _dc of epoxy polymer within the concentration range of ≤0.8 wt % was investigated. The value of direct current conductivity was determined on the basis of analysis of the frequency dependence of complex permittivity within the frequency range of 10^?2–10⁵ Hz. The temperature dependence of σ _dc is composed of two regions. The dependence corresponds to the Vogel-Fulcher-Tammann empirical law σ _dc = σ _dc0exp{?DT ₀/(T-T ₀)} (where T ₀ is the Vogel temperature and D is the strength parameter) at temperatures higher than the glass transition temperature T _g. At the same time, T ₀ does not depend on the concentration of nanoparticles. The Arrhenius temperature dependence characterized by activation energy about 1.2 eV is observed at temperatures lower than T _g. The observed shape of the temperature dependence is related to the change in the mechanism of conductivity after “freezing” of ionic mobility at temperatures lower than T _g. The value of σ _dc is increased as the concentration of nanoparticles is raised within the temperature range of T > T _g. The obtained dependence of σ _dc on silver myristate concentration is similar to the root one, indicating the absence of percolation within the studied range of concentrations.     

Reversible intersystem crossing,fluorescence lifetime lengthening and collisionally induced phosphorescence in biacetyl

The emissions of biacetyl excited at 4200 Å were studied at pressures down to 10^?3 torr. Apart from the well-known nanosecond fluorescence, a new emission of the same spectral composition was found with a non-exponential decay in the microsecond range. Furthermore the phosphorescence, as defined by its spectral composition, was found to be collisionally induced.The results imply that after excitation, the molecule rapidly transfers (rate constant k_S→T) to the triplet state, giving rise to the nanosecond decay time; and can then transfer back to the singlet state (rate constant k_T→S), giving rise to the microsecond emission. At the same time internal conversion can occur (k_S→S0). From an analysis of the data we find for k_S→S0 = 2.4 × 10⁷ sec^?1, k_S→T = 7.6 × 10⁷ sec^?1, k_T→S = 1.9 × 10⁵ sec^?1. The kinetic treatment can be transformed to a quantum mechanical one, yielding values for the triplet level density (?_T), the coupling element V_ST and the number of triplet states (N) coupled to the singlet excited. At 4200 Å we find ?_T = 6.3 × 10⁵cm, V_ST = 1.0 × 10^?5 cm^?1, N = 400.Phosphorescence occurs only when the molecule is deactivated by collisions to a vibronic triplet state below the vibrationless excited singlet state. The efficiency of biacetyl collisions is 0.54.     

Spin-lattice relaxations study of water mobility in natural natrolite

The mobility of water molecules in natural natrolite (Na₂Al₂Si₃O₁₀?2H₂O) is investigated by the ¹H NMR method. The spin-lattice relaxation times in the laboratory and rotating frames (T₁ and T_1ρ) are measured as a function of the temperature for a polycrystalline sample. From experimental T₁ data it follows that at T > 286 K the diffusion of water molecules along channels parallel to the c axis is observed. From experimental T_1ρ data it follows that at T > 250 K the diffusion of water molecules in transversal channels of natrolite is also observed. At a low temperature (T < 250 K) the dipolar interaction with paramagnetic impurities (presumably Fe³⁺ ions) becomes significant as a relaxation mechanism of ¹H nuclei.     

ESR study of the photo-induced decomposition of polypropylene in the presence of N,N,N′,N′-tetramethyl-p-phenylenediamine

The mechanism of uv (λ > 325 nm) photodegradation of polypropylene (PP) containing N,N,N′,N′-tetramethyl-p-phenylenediamine (T₄MPD) has been investigated by means of ESR spectroscopy. The observed spectra after uv irradiation of both isotactic-PP (IPP) and stereoblock-PP (SPP) samples in vacuum at 77 K consisted principally of a broad singlet which was assigned to a T₄MPD cation radical (T₄MPD^+·). On the other hand, the spectrum observed after irradiation of an atactic polypropylene (APP) sample at 77 K in vacuum was resolved into several components which decayed almost up to ca. 263 K to give rise to the broad singlet of T₄MPD^+·. One component was a sharp quartet which was assigned to a methyl radical, ·CH₃·. The other component, a singlet, was attributed to a trapped electron, e_t^?.By comparison of the ESR spectrum of deuterated T₄MPD with that of the normal compound it was found that 60 ～ 70% of the methyl radicals arose from the added T₄MPD due to β-scission, which also formed the N,N,N′-trimethyl-p-phenylenediamine radical, T₃MPD·. The T₃MPD· radical presumably captures an electron at lower temperatures to become a carbanion, T₃MPD^?, which releases the electron to reproduce the T₃MPD· radical at elevated temperatures. This production of the radical T₃MPD· due to the liberation of an electron provides an explanation for the observed increase in intensity of the decay curve in the temperature range from ? 168 K to 185 K. The remaining fraction, 30 ～ 40%, of the total methyl radicals was produced from the PP matrix by an energy transfer from the excited T₄MPD¹ to the PP matrix. The broad singlet which appeared in the temperature range near 195 K was attributed to an acyl radical ～CH₂CH(CH₃)CH₂?O from the observed g-value. By photoillumination of this sample this broad singlet was converted reversibly into the quartet which was assigned to the radical ～CH₂CH(CH₂·)CH₂CHO.