Heterogeneously catalyzed asymmetric hydrogenation of C = C bonds directed by surface-tethered chiral modifiers

Asymmetric hydrogenation of C=C bonds is of the highest importance in organic synthesis, and such reactions are currently carried out with organometallic homogeneous catalysts. Achieving heterogeneous metal-catalyzed hydrogenation, a highly desirable goal, necessitates forcing the crucial enantiodifferentiating step to take place at the metal surface. By synthesis and application of six chiral sulfide ligands that anchor robustly to Pd nanoparticles and resist displacement, we have for the first time accomplished heterogeneous enantioselective catalytic hydrogenation of isophorone. High resolution XPS data established that ligand adsorption from solution occurred exclusively on the Pd nanoparticles and not on the carbon support. All ligands contained a pyrrolidine nitrogen to enable their interaction with the isophorone substrate while the sulfide functionality provided the required interaction with the Pd surface. Enantioselective turnover numbers of up to similar to 100 product molecules per ligand molecule were found with a very large variation in asymmetric induction between ligands: observed enantiomeric excesses increased with increasing size of the alkyl group in the sulfide. This likely reflects varying degrees of ligand dispersion on the surface: bulky substituent groups hinder close approach of ligand molecules to each other, inhibiting close-packed island formation, favoring dispersion as separate molecules, and leading to effective asymmetric induction. Conversely, small substituents favor island formation leading to very low asymmetric induction. Enantioselective reaction most likely involves initial formation of an enamine or iminium species, confirmed by use of an analogous tertiary amine, which leads to racemic product. Ligand rigidity and resistance to self-assembled monolayer formation are important attributes that should be designed into improved chiral modifiers.

Peroxynitrite-modified collagen-II induces p38/ERK and NF-kappa B-dependent synthesis of prostaglandin E-2 and nitric oxide in chondrogenically differentiated mesenchyrnal progenitor cells

Objective: Peroxynitrite (ONOO-) is formed in the inflamed and degenerating human joint. Peroxynitrite-modified collagen-II (PMC-II) was recently discovered in the serum of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Therefore we investigated the cellular effects of PMC-II on human mesenchymal progenitor cells (MPCs) as a model of cartilage and cartilage repair cells in the inflamed and degenerating joint. Design: MPCs were isolated from the trabecular bone of patients undergoing reconstructive surgery and were differentiated into a chondrogenic lineage. Cells were exposed to PMC-II and levels of the proinflammatory mediators nitric oxide (NO) and prostaglandin E-2 (PGE(2)) measured. Levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), phosphorylated mitogen activated protein kinases (MAPKs) and nuclear factor kappa B (NF-kappa B) activation were measured by enzyme linked immunosorbent assay (ELISA) together with specific MAPK and NF-kappa B inhibitors. Results: PMC-II induced NO and PGE(2) synthesis through upregulation of iNOS and COX-2 proteins. PMC-II also lead to the phosphorylation of MAPKs, extracellularly regulated kinase 1/2 (ERK1/2) and p38 [but not c-Jun NH2-terminal kinase (JNK1/2)] and the activation of proinflammatory transcription factor NF-kappa B. Inhibitors of p38, ERK1/2 and NF-kappa B prevented PMC-II induced NO and PGE(2) synthesis, NOS and COX-2 protein expression and NF-kappa B activation. Conclusion: iNOS, COX-2, NF-KB and MAPK are known to be activated in the joints of patients with OA and RA. PMC-II induced iNOS and COX-2 synthesis through p38, ERK1/2 and NF-KB dependent pathways suggesting a previously unidentified pathway for the synthesis of the proinflammatory mediators, NO and PGE(2), further suggesting that inhibitors of these pathways may be therapeutic in the inflamed and degenerating human joint. (c) 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Synthesis of alpha-galactooligosaccharides with alpha-galactosidase from Lactobacillus reuteri of canine origin

Crude cell-free extracts from Lactobacillus reuteri grown on cellobiose, maltose, lactose and raffinose were assayed for glycosidic activities. When raffinose was used as the carbon source, alpha-galactosidase was produced, showing the highest yield at the beginning of the stationary growth phase. A 64 kDa enzyme was purified by ultra- and gel filtration, and characterized for its hydrolytic and synthetic activity. Highest hydrolytic activity was found at pH 5.0 at 50 degreesC (K-M 0.55 mM, V-max 0.80 mumol min(-1) mg(-1) of protein). The crude cell-free extract was further used in glycosyl transfer reactions to synthesize oligosaccharides from melibiose and raffinose. At a substrate concentration of 23% (w/v) oligosaccharide mixtures were formed with main products being a trisaccharide at 26% (w/w) yield from melibiose after 8 h and a tetrasaccharide at 18% (w/w) yield from raffinose after 7 h. Methylation analysis revealed the trisaccharide to be 6' alpha-galactosyl melibiose and the tetrasaccharide to be stachyose. In both cases synthesis ceased when hydrolysis of the substrate reached 50%.

Expression of four beta-galactosidases from Bifidobacterium bifidum NCIMB41171 and their contribution on the hydrolysis and synthesis of galactooligosaccharides

This paper deals with two aspects tightly related to the enzymatic characteristics and expression of four beta-galactosidases (BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171. The growth patterns of this strain indicated a preference towards complex (i.e. lactose, galactooligosaccharides (GOSs)) rather than simple carbohydrates (i.e. glucose and galactose) and a collaborative action and synergistic relation of more than one beta-galactosidase isoenzyme for either lactose or GOS hydrolysis and subsequent assimilation. Native polyacrylamide gel electrophoresis analysis of protein extracts from cells growing on different carbohydrates (i.e. glucose, lactose or GOS) indicated that two lactose hydrolysing enzymes (BbgI and BbgIII) and one GOS hydrolysing enzyme (BbgII) were constitutively expressed, whereas a fourth lactose hydrolysing enzyme (BbgIV) was induced in the presence of lactose or different GOS fractions. Furthermore, the beta-galactosidase expression profiles of B. bifidum cells and the transgalactosylating properties of each individual isoenzyme, with lactose as substrate, clearly indicated that mainly three isoenzymes (BbgI, BbgIII and BbgIV) are implicated in GOS synthesis when whole B. bifidum cells are utilised. Two of the isoenzymes (BbgI and BbgIV) proved to have better transgalactosylating properties giving yields ranging from 42% to 47% whereas the rest (BbgI and BbgIII) showed lower yields (15% and 29%, respectively).

Synthesis of isomaltooligosaccharides and oligodextrans by the combined use of dextransucrase and dextranase

Batch syntheses of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase were performed with the aim of understanding the reaction mechanism and the parameters which affect product characteristics and molecular size. Both activities described for dextransucrase (dextran formation and acceptor reaction) achieved synthesis whilst the hydrolytic activity of dextranase regulated the product molecular size and acceptor availability. Depending on the reaction conditions, the product oligosaccharide mixtures contained mainly sugars (up to 36%) with degrees of polymerization (DP) varying between 10 and 60 together with lower concentrations of both lower and higher molecular weight sugars. Alterations in substrate and dextranase concentrations (50-400 mg ml(-1) and 2.5-46 U ml(-1), respectively) affected the molecular weight of IMO, the reaction rate and the formation of leucrose. This permitted manipulation of the product characteristics. It was found that higher substrate and dextranase concentrations gave rise to products with lower molecular sizes and a dextransucrase:dextranase ratio of 1: 1 or 1:2 appeared to produce a polymer with a molecular weight which is desirable for prebiotic use. (C) 2004 Elsevier Inc. All rights reserved.

Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase

A recycle ultrafiltration membrane reactor was used to develop a continuous synthesis process for the production of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase. A variety of membranes were tested and the parameters affecting reactor stability, productivity, and product molecular weight distribution were investigated. Enzyme inactivation in the reactor was reduced with the use of a non-ionic surfactant but its use had severe adverse effects on the membrane pore size and porosity. During continuous isomaltooligosaccharide synthesis, dextransucrase inactivation was shown to occur as a result of the dextranase activity and it was dependent mainly on the substrate availability in the reactor and the hydrolytic activity of dextranase. Substrate and dextranase concentrations (50-200 mg/mL(-1) and 10-30 U/mL(-1), respectively) affected permeate fluxes, reactor productivity, and product average molecular weight. The oligodextrans and isomaltooligosaccharides formed had molecular weights lower than in batch synthesis reactions but they largely consisted of oligosaccharides with a degree of polymerization (DP) greater than 5, depending on the synthesis conditions. No significant rejection of the sugars formed was shown by the membranes and permeate flux was dependent on tangential flow velocity. (C) 2004 Wiley Periodicals, Inc.

Synthesis of galacto-oligosaccharide from lactose using beta-galactosidase from Kluyveromyces lactis: studies on batch and continuous UF membrane-fitted bioreactors

A study of galacto-oligosaccharides (GOS) synthesis from lactose with beta-galactosidase from Kluyveromyces lactis (Maxilact(R) L2000) was carried out. The synthesis was performed using various initial lactose concentrations ranging from 220 to 400 mg/mL and enzyme concentrations ranging from 3 to 9 U/mL, and was investigated at 40degreesC and pH 7, in a stirred-tank reactor. In the experimental range examined, the results showed the amount of GOS formed depended on lactose concentration but not on enzyme concentration. Galactose was a competitive inhibitor, while glucose was a non-competitive inhibitor. In a further study, a laboratory-scale reactor system, fitted with a 10-kDa NMWCO composite regenerated cellulose membrane, was used in a continuous process. The reactor was operated in cross-flow mode. The effect of operating pressures on flux and productivity was investigated by applying different transmembrane pressures to the system. The continuous process showed better production performance compared to the batch synthesis with the same lactose and enzyme concentrations at 40degreesC, pH 7. Comparison of product structures from batch and continuous processes, analyzed by HPAEPAD and methylation analysis, showed similarities but differed from the structures found in a commercial GOS product (Vivinal(R)GOS). (C) 2004 Wiley Periodicals, Inc.

Regioselective synthesis of mannobiose and mannotriose by reverse hydrolysis using a novel 1,6-alpha-D-mannosidase from Aspergillus phoenicis

A novel 1,6-alpha-D-mannosidase was produced by Aspergillus phoenicis grown on a commercial manno-oligosaccharide preparation in liquid culture. The enzyme hydrolysed only alpha-D-Manp-(1 --> 6)-D-Manp and did not act on alpha-D-Manp-(1 --> 2)-D-Manp, or alpha-D-Manp-(1 --> 3)-D-Manp. The 1,6-alpha-D-mannosidase was used for synthesis of manno-oligosaccharides by reverse hydrolysis reaction. The highest yields, expressed as percentages (w/w) of total sugar, were similar to21% mannobiose and similar to5% mannotriose, and they were obtained with 45% (w/w) initial mannose concentration at pH 4.5 after 12 days incubation at 55 degreesC. The disaccharide and trisaccharide products were separated and their structures determined by methylation analysis. Only 1-6 linkages were found in both of them. (C) 2003 Elsevier B.V. All rights reserved.

New prodrugs derived from 6-aminodopamine and 4-aminophenol as candidates for melanocyte-directed enzyme prodrug therapy (MDEPT)

Two novel tyrosinase mediated drug delivery pathways have been investigated for the selective delivery of cytotoxic units to melanocytes from urea and thiourea prodrugs. The synthesis of these prodrugs is reported, as well as oximetry data that illustrate that the targets are substrates for tyrosinase. The stability of each of the prodrugs in (i) phosphate buffer and (ii) bovine serum is discussed, and the urea prodrugs are identified as lead candidates for further studies. Finally, HPLC studies and preliminary cytotoxicity studies in a melanotic and an amelanotic cell line, that illustrate the feasibility of the approach, are presented.

Synthesis and glycosidase inhibitory profiles of functionalised morpholines and oxazepanes

In this work libraries of morpholines and oxazepanes have been prepared via the reductive amination reaction between dialdehydes, derived from carbohydrates, and a range of amines. In this way, functionalised morpholines and oxazepanes have been prepared that include N-alkylated derivatives, disaccharide analogues, and ester containing derivatives. The abilities of these functionalised morpholines and oxazepanes to inhibit a broad panel of glycosidase enzymes, that are associated with a range of diseases, have been probed and in this way new inhibitors of a range of glycosidases, but particularly β-d-galactosidase derived from Bovine kidney, have been discovered. N-Alkyl morpholines demonstrated the best inhibition profiles for this enzyme and derivatives (15a)–(15d) acted as non-competitive inhibitors with IC50 values of 55.1–88.6 μM. Within this study, some preliminary structure–activity relationships are proposed, and it is demonstrated that N-substituted morpholines display better inhibitory profiles for the enzymes analysed than any of the N-substituted oxazepanes.

High yield production of a soluble bifidobacterial β-galactosidase (BbgIV) inE. coliDH5α with improved catalytic efficiency for the synthesis of prebiotic galactooligosaccharides

The bifidobacterial β-galactosidase (BbgIV) was produced in E. coli DH5α at 37 and 30 °C in a 5 L bioreactor under varied conditions of dissolved oxygen (dO2) and pH. The yield of soluble BbgIV was significantly (P < 0.05) increased once the dO2 dropped to 0–2% and remained at such low values during the exponential phase. Limited dO2 significantly (P < 0.05) increased the plasmid copy number and decreased the cells growth rate. Consequently, the BbgIV yield increased to its maximum (71–75 mg per g dry cell weight), which represented 20–25% of the total soluble proteins in the cells. In addition, the specific activity and catalytic efficiency of BbgIV were significantly (P < 0.05) enhanced under limited dO2 conditions. This was concomitant with a change in the enzyme secondary structure, suggesting a link between the enzyme structure and function. The knowledge generated from this work is very important for producing BbgIV as a biocatalyst for the development of a cost-effective process for the synthesis of prebiotic galactooligosaccharides from lactose.

Synthesis of novel hyperbranched polymers featuring oxazoline linear units and their application in fast-drying solvent-borne coating formulations

Novel acid-terminated hyperbranched polymers (HBPs) containing adipic acid and oxazoline monomers derived from oleic and linoleic acid have been synthesized via a bulk polymerization procedure. Branching was achieved as a consequence of an acid-catalyzed opening of the oxazoline ring to produce a trifunctional monomer in situ which delivered branching levels of >45% as determined by 1H and 13C NMR spectroscopy. The HBPs were soluble in common solvents, such as CHCl3, acetone, tetrahydrofuran, dimethylformamide, and dimethyl sulfoxide and were further functionalized by addition of citronellol to afford white-spirit soluble materials that could be used in coating formulations. During end group modification, a reduction in branching levels of the HBPs (down to 12–24%) was observed, predominantly on account of oxazoline ring reformation and trans-esterification processes under the reaction conditions used. In comparison to commercial alkyd resin paint coatings, formulations of the citronellol-functionalized hyperbranched materials blended with a commercial alkyd resin exhibited dramatic decreases of the blend viscosity when the HBP content was increased. The curing characteristics of the HBP/alkyd blend formulations were studied by dynamic mechanical analysis which revealed that the new coatings cured more quickly and produced tougher materials than otherwise identical coatings prepared from only the commercial alkyd resins.

Synthesis of prebiotic galactooligosaccharides from lactose using bifidobacterial β-galactosidase (BbgIV) immobilised on DEAE-Cellulose, Q-Sepharose and amino-ethyl agarose

The bifidobacterial β-galactosidase BbgIV was immobilised on DEAE-Cellulose and Q-Sepharose via ionic binding and on amino-ethyl- and glyoxal-agarose via covalent attachment, and was then used to catalyse the synthesis of galactooligosaccharides (GOS). The immobilisation yield exceeded 90 % using ionic binding, while it was low using aminoethyl agarose (25 – 28 %) and very low using glyoxal agarose (< 3 %). This was due to the mild conditions and absence of chemical reagents in ionic binding, compared to covalent attachment. The maximum GOS yield obtained using DEAE-Cellulose and Q-Sepharose was similar to that obtained using free BbgIV (49 – 53 %), indicating the absence of diffusion limitation and mass transfer issues. For amino-ethyl agarose, however, the GOS yield obtained was lower (42 – 44 %) compared to that obtained using free BbgIV. All the supports tried significantly (P < 0.05) increased the BbgIV operational stability and the GOS synthesis productivity up to 55 °C. Besides, six successive GOS synthesis batches were performed using BbgIV immobilised on Q-Sepharose; all resulted in similar GOS yields, indicating the possibility of developing a robust synthesis process. Overall, the GOS synthesis operation performance using BbgIV was improved by immobilising the enzyme onto solid supports, in particular on Q-Sepharose

Synthesis and biological analysis of novel glycoside derivatives of L-AEP, as targeted antibacterial agents

To develop targeted methods for treating bacterial infections, the feasibility of using glycoside derivatives of the antibacterial compound L-R-aminoethylphosphonic acid (L-AEP) has been investigated. These derivatives are hypothesized to be taken up by bacterial cells via carbohydrate uptake mechanisms, and then hydrolysed in situ by bacterial borne glycosidase enzymes, to selectively afford L-AEP. Therefore the synthesis and analysis of ten glycoside derivatives of L-AEP, for selective targeting of specific bacteria, is reported. The ability of these derivatives to inhibit the growth of a panel of Gram-negative bacteria in two different media is discussed. β-Glycosides (12a) and (12b) that contained L-AEP linked to glucose or galactose via a carbamate linkage inhibited growth of a range of organisms with the best MICs being <0.75 mg/ml; for most species the inhibition was closely related to the hydrolysis of the equivalent chromogenic glycosides. This suggests that for (12a) and (12b), release of L-AEP was indeed dependent upon the presence of the respective glycosidase enzyme.