Synthesis of some new <Emphasis Type="Italic">N</Emphasis>-substituted-<Emphasis Type="Italic">N</Emphasis>-(2,3-Dihydro-[1,4]benzodioxin-6-yl)-4-acetamidobenzenesulfonamides as valuable antibacterial agents

The aim of the research was to investigate the anti-bacterial potential of some N-substituted sulfonamides bearing benzodioxane moiety. The synthesis was started by reaction of N-2,3-dihydrobenzo[1,4]dioxin-6-amine with 4-acetamidobenzene-1-sulfonyl chloride in the presence of 10% aqueous Na₂CO₃ solution to yield N-(2,3-dihydrobenzo[1,4]-dioxin-6-yl)-4-acetamidobenzenesulfonamide, which was further reacted with alkyl/aralkyl halides in DMF and lithium hydride as a base to afford N-substituted-N-(2,3dihydro-[1,4]-benzodioxin-6-yl)-4-acetamidobenzenesulfonamides. All the synthesized compounds were characterized by spectral data (IR, ¹H NMR, EI-MS, and HR-MS). The compounds were tested for antibacterial activity and most of them exhibited potent therapeutic potential against various Gram-negative and Gram-positive strains.     

New Charge-Deficient Agmatine Analogues

N,N′-Di-Boc-N″-triflylguanidine was demonstrated to be an efficient guanidinylation reagent for O-substituted hydroxylamines. N-(3-Aminooxypropyl)- and N-(3-aminopropoxy)guanidines, previously unknown isosteric and charge-deficient agmatine analogues, have been synthesized. The possibilities of using these compounds in studying polyamine metabolism are discussed.     

Synthesis of <Emphasis Type="Italic">N</Emphasis>-acetyllactosamine-containing oligosaccharides,galectin ligands

The following spacered oligosaccharides were synthesized: GlcNAcβ1-3Galβ1-4GlcNAcβ-sp, GlcNAcβ1-6Galβ1-4GlcNAcβ-sp, GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAcβ-sp, Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-sp, Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAcβ-sp, Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAcβ-sp, GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAcβ-sp, and Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAcβ-sp (sp = O(CH₂)₂NH₂). They represent N-acetyllactosamines substituted with N-acetylglycosamine or N-acetyllalctosamine residue at O3, O6, or at both positions of galactose. Glycosylation was achieved by coupling with N-trichloroethoxycarbonyl-protected glucosamine bromide in the presence of silver triflate.     

Attaching an alkali metal atom to an alkaline earth metal oxide (BeO,MgO, or CaO) yields a triatomic metal oxide with reduced ionization potential and redirected polarity

The existence of a series of neutral triatomic metal oxides MON and their corresponding cations MON ⁺ (M = Be, Mg, Ca; N = Li, Na, K) was postulated and verified theoretically using ab initio methods at the CCSD(T)/6-311+G(3df)//MP2/6-311+G(3df) level of theory. The calculations revealed that the vertical ionization potentials (IPs) of the MON radicals (calculated using the outer-valence Green’s function technique (OVGF) with the 6-311+G(3df) basis set) were ca. 2–3 eV smaller than the IPs of the corresponding MO and NO systems or that of the isolated M atom. Population analysis of the neutral triatomic MON molecules and their corresponding MO counterparts indicated that the attachment of an alkali metal atom to any oxide MO (BeO, MgO, CaO) reverses its polarity, which manifests itself as the redirection of the dipole moment vector.     

Structure of carbohydrate chains of fucolectin from the bark of golden rain shrub <Emphasis Type="Italic">Laburnum anagyroides</Emphasis>

A reductive LiBH₄-Bu^tOH cleavage of N-glycosylamide carbohydrate-peptide bond allowed splitting off of oligosaccharide chains of the fucolectin, the bark agglutinin from the shrub golden rain Laburnum anagyroides (LABA). Four N-glycans were isolated by HPLC, and their structures were elucidated by monosaccharide analysis and ¹H NMR (500 MHz) spectroscopy: Man₂Fuc₁Xyl₁GlcNAc₂ (M2FX), Man₃Xyl₁GlcNAc₂ (M3X), Man₃Fuc₁Xyl₁GlcNAc₂ (M3FX), and Man₃Xyl₁Fuc₁GlcNAc₃ (NM3FX). All the N-glycans contain D-xylose and three of them, L-fucose; they were found to be in a 1:8:3:1 ratio.     

Precise transfers of genes for high grain iron and zinc from wheat-<Emphasis Type="Italic">Aegilops</Emphasis> substitution lines into wheat through pollen irradiation

Nearly 2 billion people worldwide are suffering from iron (Fe) deficiency anemia and zinc (Zn) deficiency. The available elite bread wheat cultivars have inherently low grain micronutrient content. Biofortification for grain Fe and Zn content is one of the most feasible and cost-effective approach for combating widespread deficiency of the micronutrients. QTL controlling high grain Fe and Zn have been mapped on groups 2 and 7 chromosomes of Triticeae. The present study was initiated for precise transfers of genes for high grain Fe and Zn on group 2 and 7 chromosomes of wheat-Aegilops substitution lines to wheat cultivars using pollen radiation hybridization. The pollen radiation hybrids (PRH₁) derived from 1.75 krad irradiated spikes showed the presence of univalents and multivalents in meiotic metaphase-I indicating the effectiveness of radiation dose. In the advanced generation PRH₅, the plants selected with stable chromosome number and high grain Fe and Zn content were analyzed with wheat groups 2 and 7 chromosome specific intron targeted amplified polymorphism (ITAP) markers of the metal homeostasis genes to monitor the transfers of alien genes from the substituted Aegilops chromosomes. The group 2 chromosome derivatives showed the presence of NAS2, FRO2, VIT1, and ZIP2 Aegilops genes whereas the group 7 derivatives had YSL15, NAM, NRAMP5, IRO3, and IRT2 Aegilops genes. The pollen radiation hybrids of both the groups 2 and 7 chromosomes showed more than 30% increase in grain Fe and Zn content with improved yield than the elite wheat cultivar PBW343 LrP indicating small and compensating transfers of metal homeostasis genes of Aegilops into wheat.     

Assessment of photosynthetic potential of indoor plants under cold stress

Photosynthetic parameters including net photosynthetic rate (P_N), transpiration rate (E), water-use efficiency (WUE), and stomatal conductance (g_s) were studied in indoor C₃ plants Philodendron domesticum (Pd), Dracaena fragans (Df), Peperomia obtussifolia (Po), Chlorophytum comosum (Cc), and in a CAM plant, Sansevieria trifasciata (St), exposed to various low temperatures (0, 5, 10, 15, 20, and 25°C). All studied plants survived up to 0°C, but only St and Cc endured, while other plants wilted, when the temperature increased back to room temperature (25°C). The P_N declined rapidly with the decrease of temperature in all studied plants. St showed the maximum P_N of 11.9 μmol m^?2 s^?1 at 25°C followed by Cc, Po, Pd, and Df. E also followed a trend almost similar to that of P_N. St showed minimum E (0.1 mmol m^?2 s^?1) as compared to other studied C₃ plants at 25°C. The E decreased up to ≈4-fold at 5 and 0°C. Furthermore, a considerable decline in WUE was observed under cold stress in all C₃ plants, while St showed maximum WUE. Similarly, the g_s also declined gradually with the decrease in the temperature in all plants. Among C₃ plants, Pd and Po showed the maximum g_s of 0.07 mol m^?2 s^?1 at 25°C followed by Df and Cc. However, St showed the minimum g_s that further decreased up to ～4-fold at 0°C. In addition, the content of photosynthetic pigments [chlorophyll a, b, (a+b), and carotenoids] was varying in all studied plants at 0°C. Our findings clearly indicated the best photosynthetic potential of St compared to other studied plants. This species might be recommended for improving air quality in high-altitude closed environments.     

<Emphasis Type="Italic">N</Emphasis>-Terminal seven-amino-acid extension simultaneously improves the pH stability,optimal temperature,thermostability and catalytic efficiency of chitosanase CsnA

Objective

To determine the effects of the extra N-terminal seven-amino-acid sequence on the function of chitosanase CsnA.

Results

Sequence and structure analysis indicated that the mature CsnA contains a seven-amino-acid extension in a disordered form at the N-terminus. To determine the function of this sequence, both mature CsnA and its N-terminus-truncated mutant, CsnAΔN, were expressed in Escherichia coli and characterized. Compared with CsnAΔN, CsnA exhibited a 15 °C higher temperature optimum, enhanced pH stability, thermostability and catalytic efficiency. The underlying mechanisms responsible for these changes were analyzed by circular dichroism (CD) spectroscopy. CD analysis revealed that the deletion of the N-terminal sequence resulted in a decrease in the T_m of 4.3 °C and this sequence altered the secondary structure of the enzyme.

Conclusions

The N-terminal sequence is essential for the stability and activity of chitosanase CsnA.

     

Species differences and mechanism of action of A<Subscript>3</Subscript> adenosine receptor allosteric modulators

Activity of the A₃ adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A₃AR species homologs used in preclinical drug development. In guanosine 5′-[γ-[³⁵S]thio]triphosphate ([³⁵S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A₃ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A₃ARs but provided only weak activity at mouse A₃ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A₃AR agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N ⁶-(4-amino-3-[¹²⁵I]iodobenzyl)adenosine-5′-N-methylcarboxamide ([¹²⁵I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A₃AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [¹²⁵I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A₃AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.     

Altered torquoselectivity of fluorine in the iron-tricarbonyl-mediated thermal ring opening of 3-fluorocyclobutene: a density-functional exploration

There are eight possible pathways for the iron-tricarbonyl-assisted thermal electrocyclic ring opening of fluorocyclobutene due to variations in the orientation of the binding of Fe(CO)₃ relative to the fluorine substituent (R₁ or R₂), stereoselectivity (conrotatory or disrotatory, i.e., C or D), and the torquoselectivity of fluorine (inward-facing or outward-facing, i.e., in or out). A density functional study revealed that the energetically favored pathway is R₁ Din. Not only is the D mode favored energetically, but the in configuration was observed to be the lowest-lying mode of R₁, despite the general tendency of fluorine substituents to prefer an out configuration. Data on the activation hardness and aromaticity indices such as BAC and HOMA lead to the same conclusion. However, the R₂ mode surprisingly shows no particular preference for either the Cout or the Dout pathway (i.e., the R₂ mode shows less stereoselectivity than R₁). This behavior occurs due to the influences of both the fluorine substituent and metal coordination. Also, the geminal bond orbitals σ(C–F) and σ*(C–F) appear to participate in ring opening, given the excellent correlation of ?BE with the activation barrier of the transition state.     

Comparative theoretical studies of differently bridged nitramino-substituted ditetrazole 2-<Emphasis Type="Italic">N</Emphasis>-oxides with high detonation performance and an oxygen balance of around zero

In this work, six (A–F) nitramino (–NHNO₂)-substituted ditetrazole 2-N-oxides with different bridging groups (–CH₂–, –CH₂–CH₂–, –NH–, –N=N–, and –NH–NH–) were designed. The six compounds were based on the parent compound tetrazole 2-N-oxide, which possesses a high oxygen balance and high density. The structure, heat of formation, density, detonation properties (detonation velocity D and detonation pressure P), and the sensitivity of each compound was investigated systematically via density functional theory, by studying the electrostatic potential, and using molecular mechanics. The results showed that compounds A–F all have outstanding energetic properties (D: 9.1–10.0 km/s; P: 38.0–46.7 GPa) and acceptable sensitivities (h ₅₀: 28–37 cm). The bridging group present was found to greatly affect the detonation performance of each ditetrazole 2-N-oxide, and the compound with the –NH–NH– bridging group yielded the best results. Indeed, this compound (F) was calculated to have comparable sensitivity to the famous and widely used high explosive 1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX), but with values of D and P that were about 8.7% and 19.4% higher than those for HMX, respectively. The present study shows that tetrazole 2-N-oxide is a useful parent compound which could potentially be used in the design of new and improved high-energy compounds to replace existing energetic compounds such as HMX.     

Structure–activity relationships of <Emphasis Type="Italic">N</Emphasis>- and <Emphasis Type="Italic">S</Emphasis>-analogs of the seed germination inhibitor (3,4,5-trimethylfuran-2(5<Emphasis Type="Italic">H</Emphasis>)-one) for mode of action elucidation

Smoke-derived butenolides are important germination signaling molecules which may be useful in promoting or controlling germination in agriculture and horticulture. The butenolide 3,4,5-trimethylfuran-2(5H)-one (trimethylbutenolide; TMB) was previously isolated from plant-derived smoke and was found to significantly reduce the germination stimulatory activity of the highly active germination promoter, 3-methyl-2H-furo[2,3-c]pyran-2-one (karrikinolide; KAR₁), another smoke-derived butenolide, when applied together. In this study, the germination inhibitory activity of eight N-analogs and one S-analog of TMB, modified in position 1 were evaluated. All synthesized analogs significantly reduced the germination of ‘Grand Rapids’ lettuce (Lactuca sativa) seeds compared to the respective controls, when applied at 1000 µM alone, or in combination with 0.01 µM KAR₁. The S-analog was the most active of the test analogs in this study, with inhibitory activity ranging from 10 to 1000 µM. This was the only compound with activity similar to that of TMB. Thus, these results indicate that N-alkylation of the lactam ring reduces its germination inhibitory activity, while the activity of the S-analog is comparable to TMB. This provides valuable information toward understanding the mode of action of these compounds in regulating germination and for the design of related synthetic compounds with potential use in agriculture or horticulture.     

New amphiphilic chlorins of the chlorophyll <Emphasis Type="Italic">a</Emphasis> series

We treated by nucleophiles derivatives of chlorophyll a with additional six-membered cycles: δ-lactone at pyrrole D and anhydride at pyrrole C bearing a γ-meso-bridge. In the first case, this led to the formation of substituted amides at the propionic acid residue and the introduction of hydroxy group at position 18 of the macrocycle, and, in the second case, to N-substituted cycloimides of chlorin p6.     

Fine-scale differences in genetic and census population size ratios between two stream fishes

Comparing the ratio of effective number of breeders (N _b ) to adult population size (N) among closely related coexisting species can provide insights into the role of life history on N _b /N ratios and inform conservation programs towards limiting the loss of evolutionary potential in natural populations. We estimated N _b and N in two coexisting salmonid fishes (Brook trout and Atlantic salmon) for 3–4 consecutive years in two small, adjacent streams in Newfoundland, Canada, using mark-recapture (N), linkage disequilibrium (N _b(LD)), and sibship frequency approaches (N _b(Sib) ). We found that N _b /N ratios were about 20-fold greater in Atlantic salmon than in brook trout (mean 0.20, range 0.06–0.56 vs. mean 0.02, range 0.01–0.05, respectively). This difference was consistent across N _b estimators. In addition, we found that removing migrants reduced N _b : the strength of the effect was weak for N _b(LD) and much stronger for N _b(Sib). Our results highlight the importance of subtle ecological differences and gene flow in shaping N _b /N. They also provide some evidence that the linkage between demographic and evolutionary processes varies between closely related taxa and suggest that a more complete understanding of the N _b /N range across various species is an important component of conservation genetics and management.     

Microwave electrode discharge in nitrogen: Structure and characteristics of the electrode region

The parameters of the electrode region of an electrode microwave discharge in nitrogen are studied by emission spectroscopy. The radial and axial distributions of the intensities of the bands of the second (N₂(C ³Π _u → B ³Π _g )) and first (N₂(B ³Π _g → A ³Σ _u ⁺ )) positive systems of molecular nitrogen and the first negative system of nitrogen ions (N ₂ ⁺ (B ²Σ _u ⁺ → X ²Σ _g ⁺ )), the radial profiles of the electric field E and the electron density N _e , and the absolute populations of the vibrational levels v _C = 0–4 of the C ³Π _u excited state of N₂ and the vibrational level v _Bi = 0 of the B ²Σ _u ⁺ excited state of a molecular nitrogen ion are determined. The population temperature of the first vibrational level T _V of the ground electronic state X ¹Σ _g ⁺ of N₂ and the excitation temperature T _C of the C ³Π _u state in the electrode region of the discharge are measured. The radius of the spherical region and the spatially integrated plasma emission spectra are studied as functions of the incident microwave power and gas pressure. A method for determining the electron density and the microwave field strength from the plasma emission characteristics is described in detail.     

Study of the oxidation of alkanes in their mixtures with oxygen and air under the action of a pulsed volume nanosecond discharge

The slow oxidation of alkanes (from methane to hexane) in their stoichiometric mixtures with oxygen or air under the action of nanosecond pulsed discharges was investigated. The discharges were excited in a tube of diameter 5 cm and length of 20 cm by 25-ns voltage pulses with an amplitude of 10 kV and a repetition rate of 40 Hz. The initial pressure in the mixture was varied in the range 0.76–10.1 torr. The current, the electric field strength, and the power deposited in a discharge were measured with a nanosecond time resolution. In time-resolved and time-integrated measurements, the intensities of the following bands were determined: CO ₂ ⁺ (B²Σ → X²Π, δv=0), CH(A²Δ, v′=0 → X²Π, v″=0), OH(A²Σ, v′=0 → X²Π, v″=0), CO(B¹Σ, v′=0 → A¹Π, v″=2), NO(A²Σ → X²Π, δv=3), N₂(C³Π, v′=1 → B³Π, v″=7), N₂(B³Π, v′=6 → A³Σ, v″=3), and N ₂ ⁺ (B²Σ, v′=0 → X²Σ, v″=2). The methane concentration was measured from the absorption of He-Ne laser radiation. Based on the results of optical measurements, the times of the complete oxidation of hydrocarbons were determined.     

Cytotoxicity and Glycan-Binding Profile of a <Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare (<Emphasis Type="Italic">Aplysia kurodai</Emphasis>)

A divalent cation-independent 16 kDa d-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized d-galactose and d-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl d-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k _ass and k _diss values are 2.4 × 10³ M^?1 s^?1 and 3.8 × 10^?3 s^?1, respectively. AKL-2 appeared cytotoxicity against both Burkitt’s lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.     

Engineering a highly thermostable and stress tolerant superoxide dismutase by N-terminal modification and metal incorporation

Thermophilic or hyperthermophilic SODs (superoxide dismutase) usually offer substantial biotechnological advantages over mesophilic SODs. Previously a 244-amino acid N-terminal domain (NTD) from a heatresistant SOD of Geobacillus thermodenitrificans NG80-2 was discovered and demonstrated to be able to confer thermostability to homologous mesophilic SODs, which revealed a new type of heat resistance mechanism. To further improve the heat resistance and stress tolerance of thermophilic cambialistic superoxide dismutase (Fe/Mn- SOD _Ap ) from Aeropyrum pernix K1 through metal incorporation and fusion with the newly found peptide NTD for broadening its industrial application, the wildtype SOD _Ap and NTD-fused ntdSOD _Ap were expressed in E. coli BL21 and incorporated with metal cofactors by two ways. Recombinant fusion SOD obtained by in vitro reconstitution (Mn-rec ntdSOD _Ap ) exhibited improved optimum temperature at 70°C and dramatically enhanced thermostability especially at 110°C with enhanced pH stability from 4 to 10 and higher tolerance for denaturants and organic media than Mn-rec SOD _Ap . To the best of our knowledge, Mn-rec ntdSOD _Ap could be the most heat resistant SOD. In addition, metal incorporation of SOD _Ap and ntdSOD _Ap via in vivo modification have been developed and proved to be more practical for industrial use. These results indicate that fusion with NTD along with metal incorporation can generate superimposed effect and be applied to enhance the stability of cambialistic thermophilic SODs, thus providing a universal and convenient bioengineering method for generating extremely stable SODs.     

Alteration of a recombinant protein <Emphasis Type="Italic">N</Emphasis>-glycan structure in silkworms by partial suppression of <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase gene expression

Objective

To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae.

Results

Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man₃GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins.

Conclusions

Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.