Crystallography and Structure–Property Relationships in 2,2′,2″,2′′′,4,4′,4″,4′′′,6,6′,6″,6′′′‐Dodecanitro‐1,1′ : 3′1″ : 3″,1′′′‐ Quaterphenyl (DODECA)

X‐ray crystallographic study of 2,2′,2″,2′′′,4,4′,4″,4′′′,6,6′,6″,6′′′‐dodecanitro‐1,1′ : 3′1″ : 3″,1′′′‐quaterphenyl (DODECA) has been carried out. Nonbonding interatomic distances of oxygen atoms inside of all the nitro groups are shorter than those corresponding to the intermolecular contact radii for oxygen. By means of the DFT B3LYP/6‐31(d, p) method a difference of 136 kJ mol⁻¹ between the X‐ray and DFT structures of DODECA was found. The bearer of the highest initiation reactivity in its molecule in solid phase should be the nitro group at 4′′′‐position, in contrast to those at 2′‐ or 2″‐positions in its isolated molecule. The most reactive nitro group in the DODECA molecule can be well specified by the relationship between net charges on nitro groups and charges on their nitrogen atoms, both of them for the X‐ray structure. The ¹⁵N chemical shift, corresponding to this nitro group for the initiation by impact and shock, correlates very well with these shifts of the reaction centers of the other six “genuine” polynitro arenes.     

Selective Oxidation of Acetophenones Bearing Various Functional Groups to Benzoic Acid Derivatives with Molecular Oxygen

Acetophenones substituted by alkyl, alkoxy, acetoxy, and halogen groups were selectively oxidized with molecular oxygen to the corresponding benzoic acids by using the N,N′,N′′‐trihydroxyisocyanuric acid (THICA)/cobalt(II) acetate [Co(OAc)₂] and THICA/Co(OAc)₂/manganese(II) acetate [Mn(OAc)₂]. For example, 4‐methylacetophenone was selectively oxidized with molecular oxygen to 4‐acetylbenzoic acid (85%) by THICA/Co(OAc)₂ and to 4‐methylbenzoic acid (93%) by Mn(OAc)₂, while terephthalic acid was obtained in 93% with the THICA/Co(OAc)₂/Mn(OAc)₂ catalytic system. It is interesting that the acetyl group on the aromatic ring is efficiently converted by a very small amount of Mn(OAc)₂ to the corresponding carboxylic acid, and that the present method provides a versatile route to acetylbenzoic acids which are difficult to prepare by conventional methods.     

The use of 3,3′,4′,5‐tetrachlorosalicylanilide as a chemical uncoupler to reduce activated sludge yield

To determine whether chemical additions can be used to reduce sludge production in biological wastewater treatment, 3,3′,4′,5‐tetrachlorosalicylanilide (TCS) was added to activated sludge cultures as a metabolic uncoupler. Batch tests confirmed that TCS is an effective chemical uncoupler in reducing the sludge yield at concentrations greater than 1.0 mg dm^?3; a TCS concentration of 1.0 mg dm^?3 reduced sludge yield by approximately 50%. Substrate removal capability and effluent nitrogen concentration were not affected adversely by the presence of TCS when dosed every other day in a range of 2.0–3.6 mg dm^?3 during the 40‐day operation of activated sludge batch cultures. Such sludge growth reduction was associated with the enhancement of microbial activities in terms of the specific oxygen uptake rate and dehydrogenase activity. Sludge settleability of the treated and control samples was qualitatively comparable and not significantly different. Filamentous bacteria continued to grow in sludge flocs only in the control reactor at the end of the 40‐day trial. These results suggest that TCS treatment of activated sludge systems may reduce excess sludge yield. Copyright © 2003 Society of Chemical Industry     

Crystal Structure of a Soluble Form of Human CD73 with Ecto‐5′‐Nucleotidase Activity

CD73 is a dimeric ecto‐5′‐nucleotidase that is expressed on the exterior side of the plasma membrane. CD73 has important regulatory functions in the extracellular metabolism of certain nucleoside monophosphates, in particular adenosine monophosphate, and has been linked to a number of pathological conditions such as cancer and myocardial ischaemia. Here, we present the crystal structure of a soluble form of human soluble CD73 (sCD73) at 2.2 Å resolution, a truncated form of CD73 that retains ecto‐5′‐nucleotidase activity. With this structure we obtained insight into the dimerisation of CD73, active site architecture, and a sense of secondary modifications of the protein. The crystal structure reveals a conserved loop that is directly involved in the dimer‐dimer interaction showing that the two subunits of the dimer are not linked by disulfide bridges. Using biophotonic microarray imaging we were able to confirm glycosylation of the enzyme and show that the enzyme is decorated with a variety of oligosaccharide structures. The crystal structure of sCD73 will aid the design of inhibitors or activator molecules for the treatment of several diseases and prove useful in explaining the possible roles of single nucleotide polymorphisms in physiology and disease.     

β-Enaminoesters as building blocks in heterocyclic synthesis. A novel synthesis of fused azines by using blaise reaction as a key step

Ethyl bromoacetate 4 reacts with nitriles in the presence of activated zinc dust to afford the β-enaminoesters 7 and 19 . Compounds 7 and 19 show high reactivity towards a variety of reagents to give polyfunctional heterocyclic compounds. For example, the enaminoesters 7 and 19 react with benzaldehyde to give the 1,4 dihydropyridine derivatives 9 and 20 respectively. Isothiocyanates 24 (R = Ph, OEt) added to 19 to furnish pyrimidine-thiones 26 and 27 respectively. Structures and conceivable mechanisms are discussed     

Synthesis and adsorption properties,toward some heavy metal ions,of a new polystyrene‐based terpyridine polymer

A novel polymeric ligand having 2,2′:6′,2″‐terpyridine as pendant group was prepared through a Williamson type etherification approach for the reaction between 4′‐hydroxy‐2,2′: 6′,2″‐terpyridine and the commercially available 4‐chloromethyl polystyrene. The chelating properties of the new polymer toward the divalent metal ions (Cu²⁺, Zn²⁺, Ni²⁺, and Pb²⁺) in aqueous solutions was studied by a batch equilibration technique as a function of contact time, pH, mass of resin, and concentration of metal ions. The amount of metal‐ion uptake of the polymer was determined by using atomic absorption spectrometry. Results of the study revealed that the resin exhibited higher capacities and a more pronounced adsorption toward Pb²⁺ and that the metal‐ion uptake follows the order: Pb²⁺ > Cu²⁺ > Zn²⁺ > Ni²⁺. The adsorption and binding capacity of the resin toward the various metal ions investigated are discussed. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Alterations in fatty acid composition and fluidity of cell membranes affect the accumulation of PCB congener 2,2′,5,5′‐tetrachlorobiphenyl by Ralstonia eutropha H850

The effect of alterations in fatty acid composition and fluidity of cell membranes on the accumulation of PCB congener 2,2′,5,5′‐tetrachlorobiphenyl (TeCB) by Ralstonia eutropha (formerly Alcaligenes eutrophus) H850 was studied. Cells of R eutropha H850 grown on either biphenyl or fructose were used. Significant increases in saturated fatty acid composition and decreases in membrane fluidity of bacteria grown on biphenyl at 28 °C were observed compared with those grown on fructose at 17 or 28 °C. The ratio of saturated fatty acids to unsaturated fatty acids and membrane fluidity of R eutropha H850 grown on biphenyl at 28 °C resembled those of cells grown on fructose at 37 °C. No inhibition effect of the uncoupler 2,4‐dinitrophenol (2,4‐DNP) on TeCB accumulation was observed, suggesting an energy‐independent mechanism for TeCB accumulation in cells of R eutropha H850. The amount of TeCB accumulated was considerably higher in R eutropha H850 grown on fructose at 17 and 28 °C than when grown on biphenyl at 28 °C. Similar amounts of TeCB accumulated in bacteria grown on biphenyl at 28 °C as compared with those grown on fructose at 37 °C. These results suggest the alterations in fatty acid composition and membrane fluidity of R eutropha H850 may affect the accumulation of TeCB. © 2002 Society of Chemical Industry     

Chemical Synthesis of Rare Natural Bile Acids: 11α‐Hydroxy Derivatives of Lithocholic and Chenodeoxycholic Acids

A method for the preparation of 11α‐hydroxy derivatives of lithocholic and chenodeoxycholic acids, recently discovered to be natural bile acids, is described. The principal reactions involved were (1) elimination of the 12α‐mesyloxy group of the methyl esters of 3α‐acetate‐12α‐mesylate and 3α,7α‐diacetate‐12α‐mesylate derivatives of deoxycholic acid and cholic acid with potassium acetate/hexamethylphosphoramide; (2) simultaneous reduction/hydrolysis of the resulting △¹¹‐3α‐acetoxy and △¹¹‐3α,7α‐diacetoxy methyl esters with lithium aluminum hydride; (3) stereoselective 11α‐hydroxylation of the △¹¹‐3α,24‐diol and △¹¹‐3α,7α,24‐triol intermediates with B₂H₆/tetrahydrofuran (THF); and (4) selective oxidation at C‐24 of the resulting 3α,11α,24‐triol and 3α,7α,11α,24‐tetrol to the corresponding C‐24 carboxylic acids with NaClO₂ catalyzed by 2,2,6,6‐tetramethylpiperidine 1‐oxyl free radical (TEMPO) and NaClO. In summary, 3α,11α‐dihydroxy‐5β‐cholan‐24‐oic acid and 3α,7α,11α‐trihydroxy‐5β‐cholan‐24‐oic acid have been synthesized and their nuclear magnetic resonance (NMR) spectra characterized. These compounds are now available as reference standards to be used in biliary bile acid analysis.     

Characterization of Enzymes Catalyzing Transformations of Cysteine S‐Conjugated Intermediates in the Lincosamide Biosynthetic Pathway

Lincosamides such as lincomycin A, celesticetin, and Bu‐2545, constitute an important group of antibiotics. These natural products are characterized by a thiooctose linked to a l ‐proline residue, but they differ with regards to modifications of the thioacetal moiety, the pyrrolidine ring, and the octose core. Here we report that the pyridoxal 5′‐phosphate‐dependent enzyme CcbF (celesticetin biosynthetic pathway) is a decarboxylating deaminase that converts a cysteine S‐conjugated intermediate into an aldehyde. In contrast, the homologous enzyme LmbF (lincomycin biosynthetic pathway) catalyzes C?S bond cleavage of the same intermediate to afford a thioglycoside. We show that Ccb4 and LmbG (downstream methyltransferases) convert the aldehyde and thiol intermediates into a variety of methylated lincosamide compounds including Bu‐2545. The substrates used in these studies are the β‐anomers of the natural substrates. The findings not only provide insight into how the biosynthetic pathway of lincosamide antibiotics can bifurcate to generate different lincosamides, but also reveal the promiscuity of the enzymes involved.     

Glucose 1‐Phosphate Thymidylyltransferase in the Synthesis of Uridine 5′‐Diphosphate Galactose and its Application in the Synthesis of N‐Acetyllactosamine

In this study, we describe the direct synthesis of uridine 5′‐diphosphate galactose (UDP‐Gal) by a wild‐type bacterial thymidylyltransferase (RmlA), which is used to synthesize thymidine 5′‐diphosphate glucose (TDP‐glucose) in nature. By using magnesium (Mg²⁺) as a cofactor and a reaction temperature of 55 °C, a one hundred milligram‐scale synthesis of UDP‐Gal was achieved by RmlA. In addition, RmlA was site‐specifically and covalently immobilized on magnetic nanoparticles (MNPa) The resulting RmlA‐MNP complex retained almost 95% of its activity after reuse in ten consecutive enzyme assays. Furthermore, β‐1,4‐galactosyltransferase (GalT) from Neisseria meningitides was successfully overexpressed and purified by using an intein‐mediated protein expression system. GalT was relatively stable at 25 °C, and its activity was enhanced in the presence of DTT and BSA. Thus, it was feasible to synthesize N‐acetyllactosamine (LacNAc) using RmlA and GalT in a sequential addition of enzyme and adjustment of thereaction temperature. These results demonstrate the potential applications of bacterial RmlA in carbohydrate synthesis.     

Three in One: Temperature,Solvent and Catalytic Stability by Engineering the Cofactor‐Binding Element of Amine Transaminase

Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate‐induced inactivation mechanism involving dissociation of the enzyme–cofactor intermediate. Here, we report on engineering the cofactor‐ring‐binding element, which also shapes the active‐site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme–cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5′‐phosphate supply, thus making large‐scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural “hotspot” is a common feature of other transaminases it could serve as a general engineering target.     

Impregnation synthesis of a novel macroporous silica-based crown ether polymeric material modified by 1-dodecanol and its adsorption for strontium and some coexistent metals

4,4′,(5′)-Di-(tert-butylcyclohexano)-18-crown-6(DtBuCH18C6) is a chelating agent having high selectivity mostly for Sr(II). To significantly reduce its leakage by molecular modification, a macroporous silica-based DtBuCH18C6 polymeric composite (DtDo/SiO₂–P) was synthesized. It was performed by impregnating and immobilizing DtBuCH18C6 and 1-dodecanol molecules into the pores of the SiO₂–P particles utilizing an advanced vacuum sucking technique. The adsorption of a few fission and non-fission products Sr(II), Ba(II), Cs(I), Ru(III), Mo(VI), Na(I), K(I), Pd(II), La(III), and Y(III) onto DtDo/SiO₂–P was investigated. It was done by examining the effects of contact time and the HNO₃ concentration in a range of 0.1–5.0 M at 298 K. At the optimum concentration of 2.0 M HNO₃, DtDo/SiO₂–P exhibited strong adsorption ability and high selectivity for Sr(II) great over all of the tested elements, which showed very weak or almost no adsorption except Ba(II). Meanwhile, It was found that the quantity of total organic carbon (TOC) leaked from DtDo/SiO₂–P in 2.0 M HNO₃, 187.5 ppm, was lower than 658.4 ppm that leaked from DtBuCH18C6/SiO₂–P, which was not modified. This was ascribed to the effective association of DtBuCH18C6 and 1-dodecanol through intermolecular interaction. The reduction of DtBuCH18C6 leakage by molecular modification with 1-dodecanol was achieved. It was of great benefit to application of DtDo/SiO₂–P in chromatographic partitioning of Sr(II), one of the main heat generators, from high level liquid waste (HLLW) in reprocessing of nuclear spent fuel in the MAREC (Minor Actinides Recovery from HLLW by Extraction Chromatography) process developed recently.     

Enzymatic Synthesis of Chiral Phenylalanine Derivatives by a Dynamic Kinetic Resolution of Corresponding Amide and Nitrile Substrates with a Multi‐Enzyme System

Mutant α‐amino‐ε‐caprolactam (ACL) racemase (L19V/L78T) from Achromobacter obae with improved substrate specificity toward phenylalaninamide was obtained by directed evolution. The mutant ACL racemase and thermostable mutant D ‐amino acid amidase (DaaA) from Ochrobactrum anthropi SV3 co‐expressed in Escherichia coli (pACLmut/pDBFB40) were utilized for synthesis of (R)‐phenylalanine and non‐natural (R)‐phenylalanine derivatives (4‐OH, 4‐F, 3‐F, and 2‐F‐Phe) by dynamic kinetic resolution (DKR). Recombinant E. coli with DaaA and mutant ACL racemase genes catalyzed the synthesis of (R)‐phenylalanine with 84% yield and 99% ee from (RS)‐phenylalaninamide (400 mM) in 22 h. (R)‐Tyrosine and 4‐fluoro‐(R)‐phenylalanine were also efficiently synthesized from the corresponding amide compounds. We also co‐expresed two genes encoding mutant ACL racemase and L ‐amino acid amidase from Brevundimonas diminuta in E. coli and performed the efficient production of various (S)‐phenylalanine derivatives. Moreover, 2‐aminophenylpropionitrile was converted to (R)‐phenylalanine by DKR using a combination of the non‐stereoselective nitrile hydratase from recombinamt E. coli and mutant ACL racemase and DaaA from E. coli encoding mutant ACL racemase and DaaA genes.     

Released ATP Mediates Spermatozoa Chemotaxis Promoted by Uterus-Derived Factor (UDF) in Ascaris suum

Fertilization requires sperm migration toward oocytes and subsequent fusion. Sperm chemotaxis, a process in which motile sperm are attracted by factors released from oocytes or associated structures, plays a key role in sperm migration to oocytes. Here, we studied sperm chemotaxis in the nematode Ascaris suum. Our data show that uterus-derived factor (UDF), the protein fraction of uterine extracts, can attract spermatozoa. UDF is heat resistant, but its activity is attenuated by certain proteinases. UDF binds to the surface of spermatozoa but not spermatids, and this process is mediated by membranous organelles that fuse with the plasma membrane. UDF induces spermatozoa to release ATP from intracellular storage sites to the extracellular milieu, and extracellular ATP modulates sperm chemotaxis. Moreover, UDF increases protein serine phosphorylation (pS) levels in sperm, which facilitates sperm chemotaxis. Taken together, we revealed that both extracellular ATP and intracellular pS signaling are involved in Ascaris sperm chemotaxis. Our data provide insights into the mechanism of sperm chemotaxis in Ascaris suum.     

The role of montmorillonite in its catalysis of RNA synthesis

Montmorillonite catalyzes the oligomerization of activated nucleotides to form RNA. However, the mechanism of the montmorillonite catalysis, used as a model for similar prebiotic chemistry, is still not well understood. We have observed that the extent of catalysis depends not only upon the magnitude of the negative charge on the montmorillonite lattice and the number of cations associated with it, but also on the pH at which the reaction is promoted. The isotherm and catalysis studies have been extended to provide binding information and catalytic outcomes over a wide pH range. The optimal binding occurs in the region of maximal oligomer formation. X-ray diffraction studies revealed changes in layer separations of montmorillonite as reaction occurs. The application of the Scherrer equation to the X-ray diffraction data showed differences in domain size. Modeling of the size of the activated nucleotide monomers and the charge on the montmorillonite surface provided an interpretation of how these factors influence adsorption. This research provides a basis for further understanding of the physical processes in the mechanism of this catalysis.     

Friedel–Crafts Reaction of Indoles with Isatin‐Derived β,γ‐Unsaturated α‐Keto Esters Using a BINOL‐Derived Bisoxazoline (BOX)/Copper(II) Complex as Catalyst

Several chiral BINOL‐derived bisoxazoline (BOX)/copper(II) complexes were synthesized and evaluated as catalysts for the Friedel–Crafts reaction of indoles with isatin‐derived β,γ‐unsaturated α‐keto esters. The resulting bis‐indole products bearing a quaternary stereocenter were obtained in excellent yields and enantioselectivities. Additionally, the desired products were practically transformed to α‐amino esters, α‐hydroxy esters and α‐keto amides. It is noteworthy that this catalytic procedure was conducted with a catalyst loading of 0.5 mol% without any discernible decrease in the reactivity or enantioselectivity.

     

Synthesis of 5,5′-Oxydimethylenebis (2-furfural) by Thermal Dehydration of 5-Hydroxymethyl-2-furfural in the Presence of Dimethylsulfoxide

A new method for the synthesis of 5,5′-oxydimethylenebis (2-furfural) by thermal dehydration of 5-hydroxymethyl-2-furfural in the presence of dimethyl-sulfoxide is described. Optimum conditions are studied by experimental design. Reaction media present a high concentration of HMF. Reaction is supposed to be rapid. Yields reach 55% with a 84% selectivity.