Syntheses and activities as trehalase inhibitors of N-arylglycosylamines derived from fluorinated anilines.

Twelve N-arylglycosylamines were prepared in a one-pot reaction by treatment of D-glucose, D-galactose, D-mannose or D-xylose with fluorinated anilines in the presence of small amount of hydrochloric acid. The inhibitory activity against porcine trehalase and the fungicidal activity of the title compounds toward Rhizoctonia solani have been tested.     

Reaction pathways for Zn(II)-catalyzed carboxylic acid esters hydrolysis

Reaction pathways have been investigated by quantum-mechanical procedures on gas phase models for the hydrolysis of methyl acetate catalyzed by a monohydroxo-Zn(II) complex formed by a phenanthroline-containing polyamine macrocycle. Based on consideration of energy barrier heights, the hydrolysis process is predicted to be bimolecular, consistently with kinetic data obtained for the hydrolysis of p-nitrophenyl acetate promoted by the modelled catalyst. Differences with respect to results of theoretical studies on the more extensively investigated hydrolysis processes catalyzed by the OH⁻ anion are discussed and appear to be mostly due to the presence of the metal centre close to the OH function in the system now investigated. The pervasive presence and path-controlling role of hydrogen bonds are also discussed.     

Reaction pathways of glucose oxidation by ozone under acidic conditions

The ozonation of d-glucose-1-¹³C, 2-¹³C, and 6-¹³C was carried out at pH 2.5 in a semi-batch reactor at room temperature. The products present in the liquid phase were analyzed by GC-MS, HPAEC-PAD, and ¹³C NMR spectroscopy. Common oxidation products of glucose have also been submitted to identical ozonation conditions. For the first time, a pentaric acid was identified and its formation quantitatively correlated to the loss of C-6 of glucose in the form of carbon dioxide. Potential mechanisms for the formation of this pentaric acid are discussed. The well-accepted pathway involving the anomeric position in glucose, gluconic acid, arabinose, and carbon dioxide is reinvestigated. The origin of small molecules such as tartaric, erythronic, and oxalic acids is clarified. Finally, new reaction pathways and tentative mechanisms consistent with the formation of ketoaldonic acids and smaller acids are proposed.     

A structural study of fluorinated cellulose: crystallization of fluorinated cellulose by conversion treatments used for cellulose

A deoxyfluorocellulose derivative, with regioselective substitution mostly at the C-6 carbon atoms, has been studied by an X-ray diffraction method. The results showed that the crystallinity of the fluorinated cellulose is low but can be enhanced by a specific conversion treatment. The hydrothermally treated sample displayed a spectrum similar to that of cellulose IV.     

Reaction of proteinases with alpha 2-macroglobulin: evidence for alternate reaction pathways in the inhibition of trypsin

Titration experiments were employed to measure the binding stoichiometry of alpha 2M for trypsin at high and low concentrations of reactants. These titration experiments were performed by measuring the SBTI-resistant trypsin activity and by direct binding measurements using 125I-labeled trypsin. The binding stoichiometry displayed a marked dependence upon protein concentration. At high alpha 2M concentrations (micromolar), 2 mol of trypsin are bound/mol of inhibitor. However, at low alpha 2M concentrations (e.g., 0.5 nM), only 1.3 mol of trypsin were bound/mol of inhibitor. Sequential additions of subsaturating amounts of trypsin to a single aliquot of alpha 2M also resulted in a reduction in the final binding ratio. A model has been formulated to account for these observations. A key element of this model is the observation that purified 1:1 alpha 2M-proteinase complexes are not capable of binding a full mole of additional proteinase [Strickland et al. (1988) Biochemistry 27, 1458-1466]. The model predicts that once the 1:1 alpha 2M-proteinase complex forms, this species undergoes a time-dependent conformational rearrangement to yield a complex with greatly reduced proteinase binding ability. According to this model, the ability of alpha 2M to bind 2 mol of proteinase depends upon the association rate of the second enzyme molecule with the binary (1:1) complex, the enzyme concentration, and the rate of the conformational alteration that occurs once the initial complex forms. Modeling experiments suggest that the magnitude of the rate constant for this conformational change is in the order of 1-2 s-1.     

Reaction pathways in the decomposition of hydrogen peroxide catalyzed by copper(II)

The kinetics of the decomposition of H(2)O(2) catalyzed by Cu(II) has been studied by the initial-rate method in aqueous phosphate media at near physiological pH. The activity of the catalyst is increased by [Fe(CN)(6)](3-) and decreased by VO(3)(-), CrO(4)(2-) and Zn(II). Three reaction pathways are involved in the Cu(II)-H(2)O(2) reaction, the kinetic orders of the catalyst being 1 (rate constant k1), 2 (rate constant k2) and 3 (rate constant k3). The three pathways present fractional apparent orders (>1) in H(2)O(2) and base catalysis. The apparent activation energies associated to rate constants k1, k2 and k3 are 102+/-4, 65+/-8 and 61+/-5 kJ mol(-1). Free-radical chain mechanisms are proposed for the three pathways.     

Pyrimidine methyl anilines: selective potentiators for the metabotropic glutamate 2 receptor

Pyrimidine methyl anilines as potent and selective mGlu2 potentiators are described. Findings from the structure-activity-relationship investigations are discussed.     

Biocatalytic synthesis of fluorinated polyesters

The biocatalytic synthesis of fluorinated polyesters from activated diesters and fluorinated diols has been investigated. The effects of time, continuous enzyme addition, enzyme concentration, and diol chain length were studied to determine the factors that would limit chain extension, such as enzyme inactivation, enzyme specificity, the equilibrium position for the reaction, hydrolytic side reactions, and polymer precipitation. An enzyme screen demonstrated that only Novozym 435, an immobilized lipase from Candida antarctica, was effective in producing the fluorinated polyester. Molecular weight and polydispersity analyses were performed by means of gel permeation chromatography. End group analysis was accomplished through the use of matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. Polymer molecular weight steadily increased and then leveled off after approximately 30 h, with a weight average molecular weight of approximately 1773. The majority of the polymer chains were terminated with either hydroxyl or vinyl groups. Polymers that were synthesized from bulk monomers had higher molecular weights, but high enzyme concentrations were required. Enzyme specificity toward shorter chain fluorinated diols appeared to be the governing factor in limiting chain growth. However, polymer molecular weight increased further (M(w) = 8094) when a fluorinated diol that contained an additional methylene spacer between the fluorine atoms and hydroxyl groups was used.     

NMR spectra of fluorinated carbohydrates

Recent advances in structural and conformational analysis of fluorinated carbohydrates by NMR spectroscopy are reviewed. Characteristic 1H, 13C, and 19F NMR chemical shifts and coupling constants for selected examples are given and the spectral data of a series of fluorinated carbohydrates were collected in continuation of the review of Csuk and Gl?nzer [Adv. Carbohydr. Chem. Biochem., 46 (1988) 73-177].     

2,4-Diaminopyrimidine inhibitors of c-Met kinase bearing benzoxazepine anilines

Elaboration of the SAR around a series of 2,4-diaminopyrimidines led to a number of c-Met inhibitors in which kinase selectivity was modulated by substituents appended on the C4-aminobenzamide ring and the nature of the C2-aminoaryl ring. Further lead optimization of the C2-aminoaryl group led to benzoxazepine analogs whose pharmaceutical properties were modulated by the nature of the substituent on the benzoxazepine nitrogen. Tumor stasis (with partial regressions) were observed when an orally bioavailable analog was evaluated in a GTL-16 tumor xenograft mouse model. Subsequent PK/PD studies suggested that a metabolite contributed to the overall in vivo response.     

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.     

Development of fluorinated CB2 receptor agonists for PET studies

A convergent strategy was followed to modify systematically carbazole based CB₂ receptor ligands. The length of the N-(fluoroalkyl) group (n in 7), the length of the alkanamide (m in 7) and the substitution pattern of the phenyl moiety (X and Y in 7) were varied systematically. The highest CB₂ affinity was found for the 2-fluoroethyl substituted carbazole derivative 20a (K_i = 5.8 nM) containing the propionamide and the 2-bromo-4-fluorophenyl moiety. According to docking studies 20a fits nicely into the binding pocket of the CB₂ receptor, but elongation of the fluoroethyl side chain leads to a different binding mode of the ligands. The high CB₂ affinity together with the high selectivity over the CB₂ subtype qualifies the fluoroethyl derivative 20a to be developed as a PET tracer.     

Transfection with fluorinated lipoplexes based on fluorinated analogues of DOTMA,DMRIE and DPPES

Fluorinated double-chain (poly)cationic lipids (one or both of these chains being ended by a highly fluorinated tail) which are close analogues of DOTMA, DMRIE or DPPES were designed as synthetic vectors for gene delivery. For N/P ratios (N=number of amine functions of the lipid; P=number of DNA phosphates) from 0.8 to 5, these fluorinated cationic lipids condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. No specific cell toxicity was evidenced for these new fluorinated lipoplexes. The efficiency of some of the fluorinated lipoplexes to transfect lung epithelial A549 cells was comparable to that of the first generation of fluorinated lipoplexes made from fluorinated analogues of DOGS (Transfectam) [Bioconjug. Chem. 12 (2001) 114]. These results, combined with the higher in vivo transfection potential found for fluorinated lipoplexes than for conventional lipoplexes or PEI polyplexes [J. Gene Med. 3 (2001) 109], confirm that fluorinated lipoplexes are very promising gene transfer systems.     

Transfection with fluorinated lipoplexes based on fluorinated analogues of DOTMA, DMRIE and DPPES

Fluorinated double-chain (poly)cationic lipids (one or both of these chains being ended by a highly fluorinated tail) which are close analogues of DOTMA, DMRIE or DPPES were designed as synthetic vectors for gene delivery. For N/P ratios (N=number of amine functions of the lipid; P=number of DNA phosphates) from 0.8 to 5, these fluorinated cationic lipids condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. No specific cell toxicity was evidenced for these new fluorinated lipoplexes. The efficiency of some of the fluorinated lipoplexes to transfect lung epithelial A549 cells was comparable to that of the first generation of fluorinated lipoplexes made from fluorinated analogues of DOGS (Transfectam) [Bioconjug. Chem. 12 (2001) 114]. These results, combined with the higher in vivo transfection potential found for fluorinated lipoplexes than for conventional lipoplexes or PEI polyplexes [J. Gene Med. 3 (2001) 109], confirm that fluorinated lipoplexes are very promising gene transfer systems.     

Synthesis of insecticidal fluorinated anthranilic diamides

A series of highly active fluorinated anthranilic diamide insecticides have been prepared and their biological activity assessed on two aphid species in the search for systemically active compounds that control Hemiptera. In addition, we have demonstrated a new synthesis of N-aryl 3-fluoropyrazoles.     

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway.

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.     

Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization

A straightforward protocol for the site-specific incorporation of a 19F label into any protein in vivo is described. This is done using a plasmid containing an orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) that incorporates L-4-trifluoromethylphenylalanine in response to the amber codon UAG. This method improves on other in vivo methods because the 19F label is incorporated into only one location on the protein of interest and that protein can easily be produced in large quantities at low cost. The protocol for producing 19F-labeled protein is similar to expressing protein in Escherichia coli and takes 4 d to obtain pure protein starting from the appropriate vectors.