Neuroprotective effects of a dendrimer-based glutamate carboxypeptidase inhibitor on superoxide dismutase transgenic mice after neonatal hypoxic-ischemic brain injury.

The result of a deprivation of oxygen and glucose to the brain, hypoxic-ischemic encephalopathy (HIE), remains the most common cause of death and disability in human neonates globally and is mediated by glutamate toxicity and inflammation. We have previously shown that the enzyme glutamate carboxypeptidase (GCPII) is overexpressed in activated microglia in the presence of inflammation in fetal/newborn rabbit brain. We assessed the therapeutic utility of a GCPII enzyme inhibitor called 2-(3-Mercaptopropyl) pentanedioic acid (2MPPA) attached to a dendrimer (D-2MPPA), in order to target activated microglia in an experimental neonatal hypoxia-ischemia (HI) model using superoxide dismutase transgenic (SOD) mice that are often more injured after hypoxia-ischemia than wildtype animals. SOD overexpressing and wild type (WT) mice underwent permanent ligation of the left common carotid artery followed by 50 min of asphyxiation (10% O2) to induce HI injury on postnatal day 9 (P9). Cy5-labeled dendrimers were administered to the mice at 6 h, 24 h or 72 h after HI and brains were evaluated by immunofluorescence analysis 24 h after the injection to visualize microglial localization and uptake over time. Expression of GCPII enzyme was analyzed in microglia 24 h after the HI injury. The expression of pro- and anti-inflammatory cytokines were analyzed 24 h and 72 h post-HI. Brain damage was analyzed histologically 7 days post-HI in the three randomly assigned groups: control (C); hypoxic-ischemic (HI); and HI mice who received a single dose of D-2MPPA 6 h post-HI (HI+D-2MPPA). First, we found that GCPII was overexpressed in activated microglia 24 h after HI in the SOD overexpressing mice. Also, there was an increase in microglial activation 24 h after HI in the ipsilateral hippocampus which was most visible in the SOD+HI group. Dendrimers were mostly taken up by microglia by 24 h post-HI; uptake was more prominent in the SOD+HI mice than in the WT+HI. The inflammatory profile showed significant increase in expression of KC/GRO following injury in SOD mice compared to WT at 24 and 72 h. A greater and significant decrease in KC/GRO was seen in the SOD mice following treatment with D-2MPPA. Seven days after HI, D-2MPPA treatment decreased brain injury in the SOD+HI group, but not in WT+HI. This reduced damage was mainly seen in hippocampus and cortex. Our data indicate that the best time point to administer D-2MPPA is 6 h post-HI in order to suppress the expression of GCPII by 24 h after the damage since dendrimer localization in microglia is seen as early as 6 h with the peak of GCPII upregulation in activated microglia seen at 24 h post-HI. Ultimately, treatment with D-2MPPA at 6 h post-HI leads to a decrease in inflammatory profiles by 24 h and reduction in brain injury in the SOD overexpressing mice.

Ferritin-supported palladium nanoclusters: selective catalysts for aerobic oxidations in water.

Confinement of nanometallic Pd within the core of a hyperthermophilic ferritin cage (from Pyrococcus furiosus) is reported. The resulting nanostructured hybrid catalysts can be used for highly specific aerobic oxidation of alcohols in water.

Probing the enantioselectivity of a diverse group of purified cobalt-centred nitrile hydratases.

In this study a diverse range of purified cobalt containing nitrile hydratases (NHases, EC 4.2.1.84) from Rhodopseudomonas palustris HaA2 (HaA2), Rhodopseudomonas palustris CGA009 (009), Sinorhizobium meliloti 1021 (1021), and Nitriliruptor alkaliphilus (iso2), were screened for the first time for their enantioselectivity towards a broad range of chiral nitriles. Enantiomeric ratios of >100 were found for the NHases from HaA2 and CGA009 on 2-phenylpropionitrile. In contrast, the Fe-containing NHase from the well-characterized Rhodococcus erythropolis AJ270 (AJ270) was practically aselective with a range of different α-phenylacetonitriles. In general, at least one bulky group in close proximity to the α-position of the chiral nitriles seemed to be necessary for enantioselectivity with all NHases tested. Nitrile groups attached to a quaternary carbon atom were only reluctantly accepted and showed no selectivity. Enantiomeric ratios of 80 and >100 for AJ270 and iso2, respectively, were found for the pharmaceutical intermediate naproxennitrile, and 3-(1-cyanoethyl)benzoic acid was hydrated to the corresponding amide by iso2 with an enantiomeric ratio of >100.

Characterisation of the substrate specificity of the nitrile hydrolyzing system of the acidotolerant black yeast Exophiala oligosperma R1.

The ;black yeast' Exophiala oligosperma R1 can utilise various organic nitriles under acidic conditions as nitrogen sources. The induction of a phenylacetonitrile converting activity was optimised by growing the strain in the presence of different nitriles and /or complex or inorganic nitrogen sources. The highest nitrile hydrolysing activity was observed with cells grown with 2-cyanopyridine and NaNO(3). The cells metabolised the inducer and grew with 2-cyanopyridine as sole source of nitrogen. Cell extracts converted various (substituted) benzonitriles and phenylacetonitriles. They usually converted the isomers carrying a substituent in the meta-position with higher relative activities than the corresponding para- or ortho-substituted isomers. Aliphatic substrates such as acrylonitrile and 2-hydroxy-3-butenenitrile were also hydrolysed. The highest specific activity was detected with 4-cyanopyridine. Most nitriles were almost exclusively converted to the corresponding acids and no or only low amounts of the corresponding amides were formed. The cells hydrolysed amides only with extremely low activities. It was therefore concluded that the cells harboured a nitrilase activity. The specific activities of whole cells and cell extracts were compared for different nitriles and evidence obtained for limitation in the substrate-uptake by whole cells. The conversion of 2-hydroxy-3-butenenitrile to 2-hydroxy-3-butenoic acid at pH 4 demonstrated the unique ability of cells of E. oligosperma R1 to hydrolyse aliphatic alpha-hydroxynitriles under acidic conditions. The organism could grow with phenylacetonitrile as sole source of carbon, energy and nitrogen. The degradation of phenylacetonitrile presumably proceeds via phenylacetic acid, 2-hydroxyphenylacetic acid, 2,5-dihydroxyphenylacetic acid (homogentisate), maleylacetoacetate and fumarylacetoacetate.

Cross-linked enzyme aggregates (CLEAs): stable and recyclable biocatalysts.

The key to obtaining an optimum performance of an enzyme is often a question of devising an effective method for its immobilization. In the present review, we describe a novel, versatile and effective methodology for enzyme immobilization as CLEAs (cross-linked enzyme aggregates). The method is exquisitely simple (involving precipitation of the enzyme from aqueous buffer followed by cross-linking of the resulting physical aggregates of enzyme molecules) and amenable to rapid optimization. We have shown it to be applicable to a wide variety of enzymes, including, in addition to a wide variety of hydrolases, lyases, e.g. nitrile hydratases and oxynitrilases, and oxidoreductases such as laccase and galactose oxidase. CLEAs are stable, recyclable catalysts exhibiting high catalyst productivities. Because the methodology is essentially a combination of purification and immobilization into one step, the enzyme does not need to be of high purity. The technique is also applicable to the preparation of combi-CLEAs, containing two or more enzymes, for use in one-pot, multistep syntheses, e.g. an oxynitrilase/nitrilase combi-CLEA for the one-pot conversion of benzaldehyde into (S)-mandelic acid, in high yield and enantiomeric purity.

Preparation, optimization, and structures of cross-linked enzyme aggregates (CLEAs).

The broad applicability of the cross-linking of enzyme aggregates to the effective immobilisation of enzymes is demonstrated and the influence of many parameters on the properties of the resulting CLEAs is determined. The relative simplicity of the operation ideally lends itself to high-throughput methodologies. The aggregation method was improved up to 100% activity yield for any enzyme. For the first time, the physical structures of CLEAs are elucidated.

Characterisation of nitrilase and nitrile hydratase biocatalytic systems.

Biocatalytic transformations converting aromatic and arylaliphatic nitriles into the analogous related amide or acid were investigated. These studies included synthesis of the beta-substituted nitrile 3-hydroxy-3-phenylpropionitrile, subsequent enrichment and isolation on this substrate of nitrile-degrading microorganisms from the environment, and a comparative study of enzymatic reactions of nitriles by resting cell cultures and enzymes. Each biocatalyst exhibited a distinctive substrate selectivity profile, generally related to the length of the aliphatic chain of the arylaliphatic nitrile and the position of substituents on the aromatic ring or aliphatic chain. Cell-free nitrilases generally exhibited a narrower substrate range than resting whole cells of Rhodococcus strains. The Rhodococcus strains all exhibited nitrile hydratase activity and converted beta-hydroxy nitriles (but did not demonstrate enantioselectivity on this substrate). The biocatalysts also mediated the synthesis of a range of alpha-hydroxy carboxylic acids or amides from aldehydes in the presence of cyanide. The use of an amidase inhibitor permits halting the nitrile hydratase/amidase reaction at the amide intermediate.

Chloroperoxidase-catalyzed enantioselective oxidations in hydrophobic organic media.

Chloroperoxidase from Caldariomyces fumago, a peroxidase that performs P450-like chemistry, was immobilized via covalent attachment into polyurethane foam as well as conjugated with a surfactant or polymer via colyophilization. The resulting preparations catalyzed enantio- and regioselective oxidations in hydrophobic organic media with tert-butyl hydroperoxide as the oxidant. Dried PUR-foam immobilized CPO mediated the selective oxidation of indole to 2-oxindole (regioselectivity: 99%) in water-saturated isooctane or 1-octanol. Thioanisole was converted into the corresponding (R)-sulfoxide (ee > 99%) in isooctane medium. The complexes of CPO with sodium octadecylsulphate or ethyl cellulose mediated the oxidation of thioanisole in water-immiscible organic media with variable enantioselectivity due to radical side-reactions. In the presence of alpha-tocopherol, acting as radical scavenger, the (R)-sulfoxide was formed with ee > 90%. The effect of the water activity on the catalytic activity of the complexes was investigated. The CPO complexes likewise mediated the regioselective oxidation of indole into 2-oxindole in water-saturated isooctane or 1-octanol and its kinetics were investigated. The reaction suffered from substrate inhibition when carried out in isooctane.

Efficient and selective aerobic oxidation of alcohols into aldehydes and ketones using ruthenium/TEMPO as the catalytic system.

The combination of RuCl2(PPh3)3 and TEMPO affords an efficient catalytic system for the aerobic oxidation of a variety of primary and secondary alcohols, giving the corresponding aldehydes and ketones, in >99% selectivity in all cases. The Ru/TEMPO system displayed a preference for primary vs secondary alcohols. Results from Hammett correlation studies (rho = -0.58) and the primary kinetic isotope effect (kH/kD = 5.1) for the catalytic aerobic benzyl alcohol oxidations are inconsistent with either an oxoruthenium (O=Ru) or an oxoammonium based mechanism. We postulate a hydridometal mechanism, involving a "RuH2(PPh3)3" species as the active catalyst. TEMPO acts as a hydrogen transfer mediator and is either regenerated by oxygen, under catalytic aerobic conditions, or converted to TEMPH under stoichiometric anaerobic conditions.

Facile enzymatic aldol reactions with dihydroxyacetone in the presence of arsenate.

Aldol reactions of in situ formed dihydroxyacetone arsenate with different aldehydes were catalyzed by bacterial D-fructose-1,6-bisphosphate aldolase (FruA). Aldolases from bacteria were found to be much more stable and active than FruA from rabbit muscle. Arsenate acts as a phosphate mimic and can, in principle, be used in catalytic amounts. The use of inorganic arsenate and dihydroxyacetone afforded high yields with hydrophobic aldehydes. Cosolvents increased the solubility of hydrophobic aldehydes and afforded higher reaction rates and enzyme stability. Insight is given, for the first time, in the influence of arsenate on the stereoselectivity of the aldol reaction.

Delayed cell death in neonatal mouse hippocampus from hypoxia-ischemia is neither apoptotic nor necrotic.

Hypoxic-ischemic (HI) injury in neonatal mice is associated with significant cell loss in hippocampus, striatum and deep layers of the cortex. The pattern of cell death in hippocampus after a moderate focal ischemic-global hypoxic insult is studied through morphologic changes in dying neurons at both the light and ultrastructural levels. Light microscopy at 24 h showed a number of injured neurons, as evidenced by dark, round, condensed nuclei, primarily in CA1 through CA3. Nuclei appeared punctate and cytoplasm vacuolated. Electron microscopy revealed that the punctate appearance of the nuclei corresponded to clumped chromatin. At 7 days after HI, injured neurons were shrunken and had a uniformly dark, angular appearance. While dying cells had an appearance consistent with apoptosis on light microscopy, cells were neither necrotic nor apoptotic at the ultrastructural level.

Selenium-catalyzed oxidations with aqueous hydrogen peroxide. 2. Baeyer-Villiger reactions in homogeneous solution.

Several diselenides were tested for catalytic activity in Baeyer-Villiger reactions with 60% aqueous hydrogen peroxide. Bis[3,5-bis(trifluoromethyl)phenyl] diselenide forms the corresponding 3,5-bis(trifluoromethyl)benzene seleninic acid in situ, which is a highly reactive and selective catalyst for the oxidation of carbonyl compounds in 1,1,1,3,3,3-hexafluoro-2-propanol, 2,2,2-trifluoroethanol, or dichloromethane.

Highly efficient synthesis of ampicillin in an "aqueous solution-precipitate" system: repetitive addition of substrates in a semicontinuous process.

The synthesis of ampicillin catalyzed by Escherichia coli penicillin acylase was optimized in an aqueous system with partially dissolved antibiotic nucleus 6-aminopenicillanic acid (6-APA). The yields of both 6-APA and acyl donor could be improved by repetitively adding substrates to the reaction, allowing the concentration of 6-APA to remain saturated throughout. In this reaction concept, with four subsequent additions of substrates, 97% conversion of 6-APA and 72% of D-(-)-phenylglycine methyl ester (D-PGM) to ampicillin was achieved. The synthetic potential of this concept was estimated using a mathematical model which showed that by increasing the amount of added substrates a nearly quantitative conversion of 6-APA and 85% conversion of acyl donor into ampicillin could be achieved.

Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme.

The Caldariomyces fumago chloroperoxidase was successfully expressed in Aspergillus niger. The recombinant enzyme was produced in the culture medium as an active protein and could be purified by a three-step purification procedure. The catalytic behavior of recombinant chloroperoxidase (rCPO) was studied and compared with that of native CPO. The specific chlorination activity (47 units/nmol) of rCPO and its pH optimum (pH 2.75) were very similar to those of native CPO. rCPO catalyzes the oxidation of various substrates in comparable yields and selectivities to native CPO. Indole was oxidized to 2-oxindole with 99% selectivity and thioanisole to the corresponding R-sulfoxide (enantiomeric excess >98%). Incorporation of (18)O from labeled H(2)18O(2) into the oxidized products was 100% in both cases.

Improving the catalytic performance of peroxidases in organic synthesis.

Peroxidases are ubiquitous enzymes that catalyze a variety of enantioselective oxygen-transfer reactions with hydrogen peroxide (H2O2). Although they have enormous potential, their industrial application is hampered by their high price and low operational stability. Recent developments, such as the controlled addition and in situ formation of the oxidant, protein engineering and the rational design of semi-synthetic peroxidases, aim to improve the operational stability of peroxidases.

Selective oxygen transfer catalysed by heme peroxidases: synthetic and mechanistic aspects.

The synthetic and mechanistic aspects of the use of heme peroxidases as functional mimics of the cytochrome P450 monooxygenases in oxygen-transfer reactions have been described. The chloroperoxidase from Caldariomyces fumago (CPO) is the catalyst of choice in sulfoxidation, hydroxylation and epoxidation on account of its high activity and enantioselectivity. Other heme peroxidases were less active by orders of magnitude; protein engineering has resulted in impressive improvements but even the most active mutant was still at least an order of magnitude less active than CPO. The 'oxygen-rebound' mechanisms of oxygen transfer mediated by heme enzymes - as originally conceived - have proved to be untenable. Dual pathway mechanisms, via oxoferryl species that insert oxygen as well as iron hydroperoxide species that insert OH(+), have been proposed that accommodate all of the known experimental data.

A four-step enzymatic cascade for the one-pot synthesis of non-natural carbohydrates from glycerol.

A total of four enzymatic steps were combined, in a one-pot reaction, to synthesize carbohydrates starting from glycerol. First, phosphorylation of glycerol by reaction with pyrophosphate in the presence of phytase at pH 4.0 in 95% glycerol afforded racemic glycerol-3-phosphate in 100% yield. The L-enantiomer of the latter underwent selective aerobic oxidation to dihydroxyacetone phosphate (DHAP) at pH 7.5 in the presence of glycerolphosphate oxidase (GPO) and catalase. Subsequently, fructose-1,6-bisphosphate aldolase catalyzed the aldol reaction of DHAP with butanal. Finally, dephosphorylation of the aldol adduct was mediated by phytase at pH 4 affording 5-deoxy-5-ethyl-D-xylulose in 57% yield from L-glycerol-3-phosphate. The phytase on/off-switch by pH was the key to controlling phosphorylation and dephosphorylation.

Nitric oxide synthase activity and inhibition after neonatal hypoxia ischemia in the mouse brain.

Despite the emergence of therapies for hypoxic-ischemic injury to the mature nervous system, there have been no proven efficacious therapies for the developing nervous system. Recent studies have shown that pharmacological blockade of neuronal nitric oxide synthase (nNOS) activity can ameliorate damage after ischemia in the mature rodent. We have previously shown that elimination of nNOS neurons, either by targeted disruption of the gene or by pharmacological depletion with intraparenchymal quisqualate, can decrease injury after hypoxia-ischemia. Using a simpler pharmacological approach, we studied the efficacy of a systemically administered NOS inhibitor, 7-nitroindazole, a relatively selective inhibitor of nNOS activity. Using multiple doses and concentrations administered after the insult, we found that there was only a trend for protection with higher doses of the drug. A significant decrease in NOS activity was seen at 18 h and 5 days in the cortex, and at 2 h and 18 h in the hippocampus after the hypoxia-ischemia. nNOS expression decreased and remained depressed for at least 18 h after the insult. When nNOS expression was normalized to MAP2 expression, a decrease was seen at 18 h in the cortex and at 2 and 18 h in the hippocampus. These data suggest that further inhibition of NOS activity at early timepoints may not provide substantial benefit. At 5 days after the insult, however, NOS activity and normalized nNOS expression returned to baseline or higher in the hippocampus, the region showing the most damage. These data suggest that delayed administration of nNOS inhibitor after hypoxic-ischemic injury might be beneficial.

Highly efficient immobilization of glycosylated enzymes into polyurethane foams.

Glycosylated enzymes, including aminoacylase from Aspergillus melleus, chloroperoxidase from Caldariomyces fumago, and phytase from Aspergillus ficuum, were covalently immobilized into polyurethane foams with very high enzyme loadings of up to 0.2 g protein per gram dry foam. The immobilization efficiency (retained activity) ranged from 100% at a low loading to 60% at high loadings. In contrast to many other immobilization methods no leaching of the enzyme from the support took place under the reaction conditions. In short, a universal method for the immobilization of enzymes from fungal sources was developed, affording a highly active, stable, and reusable biocatalyst.