Highly enantioselective reduction of 4-(trimethylsilyl)-3-butyn-2-one to enantiopure (R)-4-(trimethylsilyl)-3-butyn-2-ol using a novel strain Acetobacter sp. CCTCC M209061

The biocatalytic reduction of 4-(trimethylsilyl)-3-butyn-2-one to enantiopure (R)-4-(trimethylsilyl)-3-butyn-2-ol was successfully conducted with high enantioselectivity using immobilized whole cells of a novel strain Acetobacter sp. CCTCC M209061, newly isolated from kefir. Compared with other microorganisms that were investigated, Acetobacter sp. CCTCC M209061 was shown to be more effective for the bioreduction reaction, and afforded much higher yield and product enantiomeric excess (e.e.). The optimal buffer pH, co-substrate concentration, reaction temperature, substrate concentration and shaking rate were 5.0, 130.6 mM, 30 °C, 6.0 mM and 180 r/min, respectively. Under the optimized conditions, the maximum yield and the product e.e. were 71% and >99%, respectively, which are much higher than those reported previously. Additionally, the established biocatalytic system proved to be efficient for the bioreduction of acetyltrimethylsilane to (R)-1-trimethylsilylethanol with excellent yield and product e.e. The immobilized cells manifested a good operational stability under the above reaction conditions since they retained 70% of their catalytic activity after ten cycles of use.     

Synthesis and characterization of copper and manganese complexes of monodentate functionalized isocyanide: trimethylsilylmethylisocyanide and p-tolylsulfonylmethylisocyanide

Reaction of excess CNCH₂SiMe₃(L) with CuX₂·nH₂O (X=NO₃⁻, n=3; ClO₄⁻, n=6) in THF gives the Cu^I complexes [CuL₄]NO₃ (1) and [CuL₄]ClO₄ (2). When CuCN is used as starting material, complex 3, Cu(L₃)CN, C₄H₁₀O·3H₂O, is obtained. Immediate reduction occurs with AgNO₃ precipitating metallic Ag. Reactions with MnCl₂·6H₂O and Mn(NO₃)₂·6H₂O in THF produce two new compounds which analyze as MnL₄Cl₂·4H₂O (6) and MnL₂(NO₃)₂·H₂O (7). When excess p-tolylsulfonylmethylisocyanide (L′) is reacted with Cu(NO₃)₂, the mixed-valence Cu^I---Cu^II complex Cu₂L′₆(NO₃)₃ (5) is precipitated, while using CuCN gives the Cu^I dimer Cu₂L′₄(CN)₂ (4). In analogous conditions the manganese complex MnL′₂(NO₃)₂·C₃H₆O·3H₂O (8) is precipitated. All these complexes have been isolated, characterized by IR, NMR for diamagnetic species, magnetic susceptibilities, EPR measurements and electrochemical analyses. Influence of the two substituents is discussed.     

Stereoselective synthesis of (R)-1-chloro-3(3,4-difluorophenoxy)-2-propanol using lipases from Pseudomonas aeruginosa in ionic liquid-containing system

Direct transesterification of (R,S)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) (a key intermediate in the synthesis of the chiral drug (S)-lubeluzole) with vinyl butyrate by lipases from Pseudomonas aeruginosa (P. aeruginosa) MTCC 5113 was performed in hexane with ionic liquids (ILs) 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIm][PF₆] and 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIm][BF₄] as co-solvents. The maximum conversion (>49%) and enantiomeric excess (ee > 99.9%) was achieved in 6 h of incubation at 30 °C with [BMIm][PF₆] as co-solvent in a two-phase system. The enzyme was able to perform with the same specificity even at 60 °C in the presence of ILs. It was possible to use lipases repeatedly for more than 10 times while still maintaining absolute enantioselectivity and reactivity. Stability studies on lipases from P. aeruginosa in ILs revealed the fact that the enzyme constancy and the reactivity in catalyzing transesterification of rac-CDPP into (S)-1-chloro-3-(3,4-difluorophenoxy)-2-butanoate was of the order of [BMIm][PF₆] > [BMIm][BF₄] in two-phase system.     

Thyroid hormone: Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T₃) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T₃ on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10⁻⁸ M T₃ to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T₃ completely inhibits the maximal increase in short-circuit current in response to 1·10⁻⁷ M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T₃ and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10⁻⁹ M T₃. T₃ has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T₃ on nuclear binding of [³H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K′_d1 = (0.82 ± 0.36)·10⁻¹⁰ M and K′_d2 = (3.2±0.60)·10⁻⁸ M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10⁻⁸ M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10⁻⁸ M T₃ showed a K′_d1 = (0.15 ± 0.10)·10⁻¹⁰ M and K′_d2 = (3.5 ± 0.10)·10⁻⁸ M. We conclude that T₃ i action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

Application of response-surface methodology to evaluate the optimum medium components for the enhanced production of lichenysin by Bacillus licheniformis R2

Biosurfactants have gained attention because they exhibit some advantages such as biodegradability, low toxicity, ecological acceptability and ability to be produced from renewable and cheaper substrates. They are widely used for environmental applications for bioremediation and also in biomedical field. However, the high cost of production is the limiting factor for widespread industrial applications. Thus, optimization of the growth medium for biosurfactant-lichenysin production by Bacillus licheniformis R2 was carried out using response-surface methodology. A preliminary screening phase based on a two-level fractional factorial design led to the identification of NH₄NO₃, glucose, Na₂HPO₄ and MnSO₄·4H₂O concentrations as the most significant variables affecting the fermentation process. The 2⁴ full-factorial central composite design was then applied to further optimize the biosurfactant production. The optimal levels of the aforementioned variables were (g/l): NH₄NO₃, 1.0; glucose, 34.0; KH₂PO₄, 6.0; Na₂HPO₄, 2.7; MgSO₄·7H₂O, 0.1; CaCl₂, 1.2 × 10⁻³; FeSO₄·7H₂O, 1.65 × 10⁻³; MnSO₄·4H₂O, 1.5 × 10⁻³ and Na–EDTA, 2.2 × 10⁻³. With the optimization procedure, the relative lichenysin yield expressed as the critical micelle dilution (CMD) was fourfold higher than that obtained in the non-optimized reference medium.     

Nitrogen utilization by the raphidophyte Heterosigma akashiwo: Growth and uptake kinetics in laboratory cultures

The nitrogen uptake and growth capabilities of the potentially harmful, raphidophycean flagellate Heterosigma akashiwo (Hada) Sournia were examined in unialgal batch cultures (strain CCMP 1912). Growth rates as a function of three nitrogen substrates (ammonium, nitrate and urea) were determined at saturating and sub-saturating photosynthetic photon flux densities (PPFDs). At saturating PPFD (110 μE m⁻² s⁻¹), the growth rate of H. akashiwo was slightly greater for cells grown on NH₄⁺ (0.89 d⁻¹) compared to cells grown on NO₃⁻ or urea, which had identical growth rates (0.82 d⁻¹). At sub-saturating PPFD (40 μE m⁻² s⁻¹), both urea- and NH₄⁺-grown cells grew faster than NO₃⁻-grown cells (0.61, 0.57 and 0.46 d⁻¹, respectively). The N uptake kinetic parameters were investigated using exponentially growing batch cultures of H. akashiwo and the ¹⁵N-tracer technique. Maximum specific uptake rates (V_max) for unialgal cultures grown at 15 °C and saturating PPFD (110 μE m⁻² s⁻¹) were 28.0, 18.0 and 2.89 × 10⁻³ h⁻¹ for NH₄⁺, NO₃⁻ and urea, respectively. The traditional measure of nutrient affinity—the half saturation constants (K_s) were similar for NH₄⁺ and NO₃⁻ (1.44 and 1.47 μg-at N L⁻¹), but substantially lower for urea (0.42 μg-at N L⁻¹). Whereas the α parameter (α = V_max/K_s), which is considered a more robust indicator for substrate affinity when substrate concentrations are low (<K_s), were 19.4, 12.2 and 6.88 × 10⁻³ h⁻¹/(μg-at N L⁻¹) for NH₄⁺, NO₃⁻ and urea, respectively. These laboratory results demonstrate that at both saturating and sub-saturating N concentrations, N uptake preference follows the order: NH₄⁺ > NO₃⁻ > urea, and suggests that natural blooms of H. akashiwo may be initiated or maintained by any of the three nitrogen substrates examined.     

1-Methoxy-, 1-deoxy-11-hydroxy- and 11-Hydroxy-1-methoxy-Δ-tetrahydrocannabinols: new selective ligands for the CB2 receptor

Three series of new cannabinoids were prepared and their affinities for the CB₁ and CB₂ cannabinoid recptors were determined. These are the 1-methoxy-3-(1′,1′-dimethylalkyl)-, 1-deoxy-11-hydroxy-3-(1′,1′-dimethylalkyl)- and 11-hydroxy-1-methoxy-3-(1′,1′-dimethylalkyl)-Δ⁸-tetrahydrocannabinols, which contain alkyl chains from dimethylethyl to dimethylheptyl appended to C-3 of the cannabinoid. All of these compounds have greater affinity for the CB₂ receptor than for the CB₁ receptor, however only 1-methoxy-3-(1′,1′-dimethylhexyl)-Δ⁸-THC (JWH-229, 6e) has effectively no affinity for the CB₁ receptor (K_i=3134±110 nM) and high affinity for CB₂ (K_i=18±2 nM).     

Medium optimization for the production of a novel bioflocculant from Halomonas sp. V3a′ using response surface methodology

The novel exopolysaccharide bioflocculant HBF-3 is produced by Halomonas sp. V3a′, which is a mutant strain of the deep-sea bacterium Halomonas sp. V3a. Response surface methodology (RSM) was employed to optimize the production medium for increasing HBF-3 production. Using a Plackett–Burman experimental design to aid in the first step of optimization, edible glucose, MgSO₄·7H₂O, and NH₄Cl were found to be significant factors affecting HBF-3 production. To determine the optimal concentration of each significant variable, a central composite design was employed. Based on response surface and canonical analysis, the optimum concentrations of the critical components were obtained as follows: edible glucose, 16.14 g/l; MgSO₄·7H₂O, 2.73 g/l; and NH₄Cl, 1.97 g/l. HBF-3 production obtained by using the optimized medium was 4.52 g/l, which was in close agreement with the predicted value of 4.55 g/l. By scaling up fermentation from flask to fermenter, HBF-3 production was further increased to 5.58 g/l.     

Synthesis and biological activity of 17α-alkynylamide derivatives of estradiol

Seven estradiol (E₂) derivatives with an alkynylamide side chain at the 17α position were synthesized starting from ethynylestradiol (EE₂). The main chemical step was the coupling reaction of the acetylide ion of EE₂ with carbon dioxide, glutaric anhydride or bromoalkyl ortho ester. The synthesis of these compounds is fast (3–6 steps according to the compound) and is easily achieved with good yield. Five compounds with different side chain lenghts were evaluated for uterotrophic and antiuterotrophic activity in the CD-1 mouse. None of the tested compounds shows estrogenic activity in this sensitive in vitro system. At low doses (1 and 3 μg), a 14–57% inhibition of E₂-induced uterine growth was observed while no additional inhibition was observed at the 10, 20 and 30 μg doses. In human breast carcinoma cells in culture, all compounds show estrogenic activity at high concentrations while only compound 39 (N-buty,N-methyl-8-[3′,17′β-dihydroxy estra-1′,3′,5′(10′)-trien-17′α-yl]-7-octynamide) possesses antiproliferative or antiestrogenic effects. No significant correlation could be demonstrated between alkynylamide side chain length and estrogenic or antiestrogenic activity. Among the compounds tested, the derivative of EE₂ possessing a five-methylene (CH₂) side chain (compound 39) possesses the best antiestrogenic activity (44 ± 7% in the CD-1 mouse uterus assay at the 3μg dose and 57 ± 4% at 0.1 nM in human ZR-75-1 cancer cells in culture).     

The crystal structures of 4,4′-bipyridinium μ-(4,4′-bipyridine)bis[diaquatetranitratoneodymate(III)]-tris(4,4′-bipyridine) and a second monoclinic form of triaquatrinitratoholmium(III) – bis (4,4′-bipyridine)

The structures of (4-bipyH)₂[(μ-4-bipy)Nd₂(NO₃)₈(H₂O)₄]·3(4-bipy) (4-bipy = 4,4′-bipyridine; P2₁/c, a = 18.723(10), b = 10.720(6), c = 18.027(10) Å, β = 94.43(5)°, Z = 2; R = 0.066 for 4931 (diffractometer data) and of a second monoclinic form of [Ho(NO₃)₃(H₂O)₃]·2(4-bipy) (P2₁/c, a = 15.830(10), b = 21.44(3), c = 15.70(3) Å, β = 100.4(2)°, Z = 8; R = 0.091 for 2335 film data) are reported. In the first compound pairs of Nd atoms are bridged across a crystal inversion centre by a 4-bipy ligand, and 10-coordination is completed by one monodentate NO₃, three bidentate NO₃, and two H₂O ligands, with bond lengths Nd---N 2.70, Nd---OH₂(av.) 2.44, Nd---O(NO₃, av.) 2.56 Å. The second compound has a variant of the previously-reported monoclinic [Y(NO₃)₃(H₂O)₃]·2(4-bipy) structure, with doubling of the unit cell on a but with essentially no change in the geometry and orientation of the nine-coordinate complex. In both compounds the non-coordinated, non-protonated 4-bipy N atoms form hydrogen bonds with ligand H₂O.     

Synthesis and magnetic properties of bis(μ-hydroxo)bis[(2,2 ′-bipyridyl)copper(II)] squarate. Crystal structure of bis(μ-hydroxo)bis[(2,2′-bipyridyl)copper(II)] squarate tetrahydrate

The compound [Cu₂(bipy)₂(OH)₂](C₄O₄)·5.5H₂O, where bipy and C₄O₄²⁻ correspond to 2,2′-bipyridyl and squarate (dianion of 3,4-dihydroxy-3-cyclo- butene-1,3-dione) respectively, has been synthesized. Its magnetic properties have been investigated in the 2–300 K temperature range. The ground state is a spin-triplet state, with a singlet-triplet separation of 145 cm⁻¹. The EPR powder spectrum confirms the nature of the ground state.Well-formed single crystals of the tetrahydrate, [Cu₂(bipy)₂(OH)₂](C₄O₄)·4H₂O, were grown from aqueous solutions and characterized by X-ray diffraction. The system is triclinic, space group P , with a = 9.022(2), b = 9.040(2), c = 8.409(2) Å, α = 103.51(2), β = 103.42(3), γ = 103.37(2)°, V = 642.9(3) ų, Z = 1, D_x = 1.699 g cm⁻³, μ(Mo Kα) = 17.208 cm⁻¹, F(000) = 336 and T= 295 K. A total of 2251 data were collected over the range 1θ25°; of these, 1993 (independent and with I3σ(I)) were used in the structural analysis. The final R and R_w residuals were 0.034 and 0.038 respectively. The structure contains squarato-O¹, O³-bridged bis(μ-hydroxo)bis[(2,2′-bipyridyl)copper(II)] units forming zigzag one-dimensional chains. Each copper atom is in a square-pyramidal environment with the two nitrogen atoms of 2,2′-bipyridyl and the two oxygen atoms of the hydroxo groups building the basal plane and another oxygen atom of the squarate lying in the apical position.The magnetic properties are discussed in the light of spectral and structural data and compared with the reported ones for other bis(μ-hydroxo)bis[(2,2′-bipyridyl)copper(II)] complexes.     

Cyclodextrin production by Bacillus firmus strain 37 immobilized on inorganic matrices and alginate gel

Production of β-cyclodextrin (β-CD) by Bacillus firmus strain 37 cells, immobilized by adsorption on silica–titania (SiO₂/TiO₂) and silica–manganese dioxide (SiO₂/MnO₂) matrices, was optimized for temperature, substrate concentration and initial biomass. The immobilization process was most efficient at 60 °C with 10% maltodextrin and 1.0 g of cells, resulting, after a 5-day assay, in a β-CD production of 11.7 ± 0.1 mM for cells immobilized on SiO₂/TiO₂ and 11.2 ± 0.1 mM in SiO₂/MnO₂. Entrapment in alginate gel resulted in a maximum β-CD production of 4.1 ± 0.1 mM, which was maintained constantly until the end of a 10-day assay. During this same period, free cells produced 8.3 ± 0.2 mM, and cells immobilized on SiO₂/TiO₂ and SiO₂/MnO₂, 16.7 ± 0.4 and 17.3 ± 0.5 mM, respectively. β-CD production by cells immobilized in calcium alginate in four repetitive cycles of 5 days each, showed an increase up to the third cycle, reaching 4.8 ± 0.2 mM, while production by free cells started falling from the second cycle. In this same assay, cells immobilized on SiO₂/TiO₂ and SiO₂/MnO₂, showed the best β-CD production results at the end of the first cycle, with a gradual fall occurring due to the desorption of cells thereafter.     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

Hydroxylation of naringin by Trichoderma harzianum to dramatically improve its antioxidative activity

Transforming naringin using the mycelium of Trichoderma harzianum CGMCC 1523 produces two metabolites, 3′,4′,5,7-tetrahydroxy flavanone-7-rhamnoglucoside (3′-OHN) and 3′,4′,5′,5,7-pentahydroxy flavanone-7-rhamnoglucoside (3′,5′-DOHN), both of which were characterized by ESI–MS, ¹H NMR and ¹³C NMR analyses. The time course of the biotransformation by T. harzianum showed that 3′-OHN and 3′,5′-DOHN appeared simultaneously at 6 h, and the conversion yield (32.6%) of 3′,5′-DOHN was higher (10.6%) than that of 3′-OHN at 56 h. The optimal biotransformation temperature was 30 °C, the optimal pH was 5.0, and the optimal concentration of naringin was 400 mg/l. The bigger volume of biotransformation mixture and lower shaking speed did not favor hydroxylation reactions. The radical scavenging activity of naringin at 2000 μM was 11.1%, whereas activity of 3′-OHN at 100 μM could reach 38.4%, which is 68.6 times more than naringin. Antioxidative activity of 3′,5′-DOHN was increased 13.5% at 100 μM compared to 3′-OHN.     

A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A

The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3′,5′-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A (K_I = 43.4 ± 7.2 μM and k_cart = 0.007 ± 0.0006 min⁻¹). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (K_d = 72 μM) > Sp-cGMPS (124), Sp-cAMPS (182) > GMP (1517), Rp-cAMPS (3762), AMP (4370 μM). NAD⁺, neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes.     

3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-Δ⁸-THC analogues, several of which have high affinity for the CB₂ receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ⁸-THC (5), 1-deoxy-Δ⁸-THC (6), 1-deoxy-3-butyl-Δ⁸-THC (7), 1-deoxy-3-hexyl-Δ⁸-THC (8) and a series of 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=0–4, 6, 7, where n=the number of carbon atoms in the side chain−2). Three derivatives (17–19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB₁ and CB₂ receptors were determined employing previously described procedures. Five of the 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=1–5) have high affinity (K_i=<20 nM) for the CB₂ receptor. Four of them (2, n=1–4) also have little affinity for the CB₁ receptor (K_i=>295 nM). 3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ⁸-THC (2, n=2) has very high affinity for the CB₂ receptor (K_i=3.4±1.0 nM) and little affinity for the CB₁ receptor (K_i=677±132 nM).

Scheme 3. (a) (C₆H₅)₃PCH₃⁺ Br⁻, n-BuLi/THF, 65°C; (b) LiAlH₄/THF, 25°C; (c) KBH(sec-Bu)₃/THF, −78 to 25°C then H₂O₂/NaOH.     

DNA adduct formation in human hepatoma cells treated with 3-nitrobenzanthrone: analysis by the P-postlabeling method

3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a powerful mutagen and a suspected human carcinogen existing in diesel exhaust and airborne particulates. Recently, one of the major presumed metabolites of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in human urine samples. Here we analyzed DNA adducts formed in 3-NBA-exposed human hepatoma HepG2 cells by a ³²P-postlabeling/thin layer chromatography (TLC) method and a ³²P-postlabeling/polyacrylamide gel electrophoresis (PAGE) method. With HepG2 cells exposed to 3-NBA (0.36–36.4 μM) for 3 h, we obtained three spots or bands corresponding to adducted nucleotides. Two were assigned as 2-(2′-deoxyadenosin-N⁶-yl)-3-aminobenzanthrone-3′-phosphate (dA3′p-N⁶-C2-ABA) and 2-(2′-deoxyguanosin-N²-yl)-3-aminobenzanthrone-3′-phosphate (dG3′p-N²-C2-ABA), with identical mobilities to those of synthetic standards on PAGE analysis. The chemical structure of the substance corresponding to the other spot or band could not be identified. Quantitative analyses revealed that the major adduct was dA3′p-N⁶-C2-ABA and its relative adduct labeling (RAL) value at 36.4 μM of 3-NBA was 200.8 ± 86.1/10⁸ nucleotide.     

Characterization of galactomannans isolated from legume endosperms of Caesalpinioideae and Faboideae subfamilies by multidetection aqueous SEC

Galactomannans isolated from legume seed endosperms, including those of commercial interest, have been characterized by multidetection aqueous SEC. Galactomannans derived from seeds of the Faboideae subfamily had substantially higher M_w than those from Caesalpinioideae seeds (M_w,Fab = 2.4–3.1 × 10⁶ g/mol, M_w,Caes. = 0.86–2.1 × 10⁶ g/mol) and within the latter botanical subfamily, an apparent correlation between M_w and the degree of galactose substitution DG was found. The molar mass distributions were unimodal and differed primarily by a scale factor, with distributional widths narrower than a true Flory ‘most-probable distribution’; good fits to Schulz–Zimm model were obtained. Across subfamilies no differences were found in the exponents of [η]–M and R_v–M relationships (0.61 ± 0.02, 0.54 ± 0.01, respectively), the Flory chain stiffness ratio (C_∞ = 20 ± 1 (BSF analysis)), or the persistence length (L_p = 5.5 ± 0.2 nm) obtained from SEC fraction data. However, it was found that prefactors in the [η]–M and R_v–M relationships as well as the unperturbed parameter K_Θ decrease in proportion to DG and therefore chain density. Generalized relationships incorporating galactose-dependent prefactors were therefore developed to model SEC fraction data of native galactomannans ([η]_GM = (1800 ± 200) × M_o^−1.61 × M^0.61±0.02, R_v,GM = 0.63 ± 0.05 × M_o^−0.54 × M^0.54±0.01) as well as lower-M fractions obtained by ultrasonication ([η]_GM = (730 ± 100) × M_o^−1.71 × M_w^0.71±0.02, R_v,GM = 0.49 ± 0.05 × M_o^−0.57 × M_w^0.57±0.01, M ≈ 1 × 10⁵-native). As a consequence of this dependence and the observed patterns in molar mass variation, [η] varies within a narrow range for galactomannans as a whole despite substantial M_w differences.     

Selectivity and stability of alkaline protease AL-89 in hydrophilic solvents

The alkaline protease from Bacillus pseudofirmus strain AL-89 used vinyl fatty acid esters of increasing chain length from C10 to C18 equally well as substrates for esterification of sucrose in a reaction mixture of DMF and DMSO (1:1, v/v). The synthesized esters were purified and characterized by NMR and nano-electron spray MS. As evaluated by the initial reaction rates, the primary site of substitution of sucrose was at the C-2 position with the C-3 and C-3′ as secondary substitution sites. The enzyme catalysed the formation of 3-O-acyl sucrose from 2-O-acyl sucrose. The investigation did not reveal if the 3′-O-acyl sucrose was formed the same way. The synthesis of the 2-O-esters showed the characteristics of kinetically controlled reactions, whereas the formation of the 3-O- and 3′-O-esters showed the characteristics of equilibrium controlled reactions. The enzyme catalysed process was effected by initial water content, substrate molar ratio and reaction temperature. Under the reaction conditions of 0% initial water content, a molar ratio of sucrose to vinyl stearate of 1:1.5 and 70 °C an initial formation rate of 13.5, 2.9 and 2.1 μmol min⁻¹ was achieved for 2-O-, 3-O- and 3′-O-stearoyl sucrose respectively with a specific initial synthesis rate of 2-O-stearoyl sucrose of 0.27 μmol min⁻¹ mg⁻¹ biocatalyst. In the absence of substrates the enzyme proved to be more stable in DMF than in water and DMSO at 50 °C. Mixing DMF with DMSO 1:1 (v/v) increased the stability and the half-life was found equal to that in water. In the presence of substrates a residual activity of 40% was observed after 24 h of incubation in the 1:1 (v/v) mixture of DMF and DMSO at 70 °C.