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The immobilization of proteins on inorganic supports is attracting increasing interest since the realization of active surfaces finds application in enzyme-assisted catalysis, environmental sciences, and medical fields. In the present study, cytochrome c (cyt c) is adsorbed on silica nanoparticles (SNPs) and amino-functionalized silica nanoparticles (SNPs-APTES), which are prepared for this purpose and having a diameter of about 50 nm. The peroxidase activity of the protein is investigated under different experimental conditions, to evaluate the impact of differently charged surfaces on the catalytic activity of the biomolecule. The peroxidase activity of cyt c increases upon adsorption on SNPs, and it shows a linear behavior with nanoparticles concentration; on the other hand, the contact with increasing amounts of SNPs-APTES does not affect the catalytic activity of the protein. The kinetic profile of the oxidation reaction is altered for cyt c-SNPs sample, suggesting that upon adsorption, changes in the catalytic process take place. Moreover, we observe that the enhancement of peroxidase activity of cyt c-SNPs is almost completely inhibited in high-ionic-strength buffer: this indicates that the protein establishes electrostatic interactions with SNP. The spectroscopic properties of the adsorbed protein on the two different matrices are investigated by using fluorescence and Raman spectroscopies to account for the enzymatic activity of the hybrid materials. The fluorescence spectra of cyt c-silica bio-composites reveal that the adsorption on silica modifies the microenvironments of the emitting amino acid residues of the protein. Indeed, their fluorescence gains intensity and appears blue-shifted compared to that of the native protein; these modifications are more evident when cyt c is adsorbed on SNPs. Raman spectra suggest that both oxidation and spin state of heme iron change when cyt c is adsorbed on SNPs but not on SNPs-APTES. The spectroscopic data of biocomposite materials are discussed in terms of structural changes to account for the increment of peroxidase activity upon adsorption on the negatively charged surface of SNPs.

Nanosized hydrotalcite-like compounds (HTlc) with different chemical composition were prepared and used to study protein adsorption. Two soft proteins, myoglobin (Mb) and bovine serum albumin (BSA), were chosen to investigate the nature of the forces controlling the adsorption and how these depend on the chemical composition of the support. Both proteins strongly interact with HTlc exhibiting in most cases a Langmuir-type adsorption. Mb showed a higher affinity for Nickel Chromium (NiCr-HTlc) than for Nickel Aluminum (NiAl-HTlc), while for BSA no significant differences between supports were found. Adsorption experiments in the presence of additives showed that proteins exhibited different types of interactions onto the same HTlc surface and that the adsorption was strongly suppressed by the addition of disodium hydrogen phosphate (Na 2 HPO 4 ). Atomic force microscopy images showed that the adsorption of both proteins onto nanoparticles was followed by the aggregation of biocomposites, with a more disordered structure for BSA. Fluorescence measurements for adsorbed Mb showed that the inorganic nanoparticles induced conformational changes in the biomolecules; in particular, the interactions with HTlc surface quenched the tryptophan fluorescence and this process was particularly efficient for NiCr-HTlc. The adsorption of BSA onto the HTlc nanoparticles induced a selective quenching of the exposed fluorescent residues, as indicated by the blue-shift of the emission spectra of tryptophan residues and by the shortening of the fluorescence decay times.

The aza-Diels–Alder reaction between benzaldimines 1 and Danishefsky's diene in solvent-free conditions is reported for the first time. The reaction occurs rapidly at 30 °C and is catalyzed by porous α-zirconium hydrogen phosphate (p-αZrP) in the presence of sodium dodecyl sulfate (SDS). The yields were excellent. The recycling of the catalyst has also been investigated.

Hydrotalcite nanocrystals doped with Eu(III) ions were prepared by the microemulsion method. XRPD measurements revealed that europium ions are very likely incorporated in a low-symmetry hydrotalcite-type layer. TEM and AFM imaging indicated that nanocrystals have a 150 nm width and a plate shape. Luminescence measurements showed that the doped powder is luminescent and the Eu(III) ions are located in a low symmetry site.

The influence of methanol on the activity, stability and internal dynamics of myoglobin (Mb) has been investigated to obtain information about the effect of alcohol on protein behaviour. Spectroscopic investigations show that there are no significant changes in conformational structures of the protein within the range of alcohol concentrations where the catalytic activity ( k cat ) and thermal stability ( T m ) of Mb decrease. Neutron scattering measurements indicate that the characteristic times related to protein internal fluctuations increase when the methanol molar fraction in the mixtures increases. This suggests a direct relationship between the protein internal motion rate and the catalytic activity.

The adsorption of myoglobin (Mb) onto nanosized nickel aluminum hydrotalcite (NiAl-HTlc) surface was studied, and the structural properties of the resulting protein layer were analyzed by using FT-IR, Raman, and fluorescence spectroscopies. Upon adsorption onto the nanoparticle surface, the protein molecules maintained their secondary structure, while the tertiary structure was altered. The fluorescence spectra and anisotropy values of adsorbed Mb revealed that the emitting amino acid residues are affected by different microenvironments when compared to the native protein behavior. Moreover, the decrease of fluorescence decay times of tryptophan indicated the occurrence of interactions among the fluorophores and the constituents of the nanoparticles, such as the metal cations, which can take place when conformational changes of Mb occur. Raman spectra indicated that the interaction of Mb molecules with NiAl-HTlc nanoparticles modified the porphyrin core, changing the spin state of the heme iron from high spin (HS) to low spin (LS). The enzymatic activity of the nanostructured biocomposite was evaluated in the oxidation of 2-methoxyphenol by hydrogen peroxide and discussed on the basis of structural properties of adsorbed myoglobin.

A water-in-oil microemulsion made up of a cetyltrimethylammonium bromide/n-butanol/isooctane/MII and AlIII nitrate aqueous solution (MII = Mg or Ni or Zn) has been mixed with a microemulsion of the same composition but containing an ammonia solution instead of a metal nitrate solution. Collisions between the reverse micelles containing an MII and AlIII solution and those containing an NH3 solution form short-lived dimers that act as reaction vessels and control the nucleation and growth of MgAl, NiAl or ZnAl hydrotalcite-like compounds (HTlcs). The double water-in-oil microemulsion technique yields colloidal dispersions of nanoparticles whose size and shape were examined by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The colloidal particles were between 50 and 100 nm depending on the composition of the microemulsions (water-to-surfactant and oil-to-water molar ratios). The separation of the nanoparticles from the reaction medium produces a gel made up of surfactant-free particles. HTlc nanocrystals were recovered from the gel (reaction medium), washed, and dried. They were then studied by chemical and thermogravimetric analyses, X-ray powder diffraction, N2-adsorption and BET surface analysis and scanning electron microscopy (SEM). The AlIII/(MII + AlIII) molar ratio of the recovered HTlc nanocrystals was between 0.24 and 0.29, and the basal spacing was about 8 A, a value in agreement with the presence of charge compensating bromide anions in the interlayer region. The proposed technique allows for HTlc nanocrystals to be obtained with a relatively high surface area with values ranging between 50 and 110 m2/g. The SEM analyses show that the nanoparticles are aggregated to give rise to complex spherical or rod-shaped microstructures.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Lipases from Candida rugosa , Candida antartica B and Carica papaya are employed as the biocatalyst for the hydrolytic resolution of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane, in which excellent to good enantioselectivity without the formation of byproducts is obtained for the papaya lipase when using ( R , S )-2-fluoronaproxen methyl ester ( 1 ) and methyl ( R , S )-2-fluoro-2-(4-methoxyphenyl)propionate ( 2 ), but not methyl ( R , S )-2-fluoro-2-(naphth-1-yl)propionate ( 3 ) as the substrates. The thermodynamic analysis indicates that the enantiomer discrimination for the papaya lipase is driven by the difference in activation enthalpy for compound 1 , 2 or ( R , S )-naproxen methyl ester ( 4 ). The kinetic analysis also demonstrates that in comparison with ( S )- 4 , the insertion of the 2-fluorine moiety in ( R )- 1 has increased k 2 , but not K m , and consequently the lipase activity.

We investigated the adsorption and catalytic activity of myoglobin (Mb) immobilized on colloidal particles of zirconia covalently grafted with phosphoric (ZrO2-P) and benzenephosphonic acid (ZrO2-BP). The maximum adsorption was reached after 1 h of contact and was greater on a hydrophilic support, ZrO2-P, compared to a hydrophobic support, ZrO2-BP. The equilibrium isotherms fitted the Langmuir equation, suggesting the presence of a monolayer of protein molecules on the surface of the nanoparticles. The nanostructured biocomposites are active in the oxidation of 2-methoxyphenol (guaiacol) by hydrogen peroxide. The oxidation catalyzed by immobilized Mb followed a Michaelis-Menten kinetics, similar to that observed in the oxidation by free Mb. Furthermore, the catalytic efficiency is similar to that of free Mb and higher than that of "large-size" biocatalysts (with sizes larger than 1 mum). In the latter case, the kinetic parameters, k(cat) and K(M), indicate that this is mostly due to an increased affinity of the nano-biocomposite for the substrate. The activity of the nano-biocomposites decreases slightly as the amount of adsorbed protein increases. This is mainly due to the formation of a nonordered monolayer, which reduces the accessibility of the substrate to the active center.

The enzymatic hydrolysis of methyl 2-fluoro-2-arylpropionates was performed using lipases from Candida rugosa and Candida cylindracea (OF-360). A careful analysis of the reaction products revealed that racemic 2-hydroxy-2-arylpropionic acid and traces of 2-arylacrylic acid are formed, in addition to the expected 2-aryl-2-fluoropropionic acid. The presence of powerful electron-releasing groups in the aromatic ring of the substrate increase the amount of 2-hydroxypropionic acid. A mechanistic hypothesis has been formulated according to which the enzyme facilitates the elimination of fluoride ion from the hydrolysed acid with the formation of an α-carboxy-stabilized carbocation which provides 2-hydroxypropionic acids by nucleophilic attack of H2O and 2-arylacrylic acids by a β-elimination process.

Aqueous solutions of crude extract of lipase from Candida rugosa (C-CRL) and of a semipurified material obtained by treating C-CRL with propan-2-ol (PT-CRL) were used as a source of biomaterial for its adsorption onto the surface of layered micro-crystals of α-zirconium phosphate Zr(HPO4)2 and phosphonates such as Zr(C6H5PO3)2, Zr(HPO4)(C6H5PO3) and Zr(HOOCCH2PO3)(HPO4), which possess groups with different hydrophobic character anchored to the inorganic matrix. Several biocomposites have been obtained by changing the temperature and the time of equilibration of the various supports with the C-CRL and PT-CRL solutions. The biocomposites have shown different esterase activities and enantio-selectivities in the hydrolysis of p-nitrophenylacetate (p-NPA), ethyl butyrate and (±)-methyl-2-(4-chlorophenoxy) propionate as a function of the nature of the support and of the time and temperature of equilibration. These results have been interpreted on the basis of a selective adsorption of different isoforms of the enzyme. The biocomposites can be stored for more than 1 month at 4 °C and can be used for several cycles without a significant decrease in catalytic activity.

The lipase from Candida rugosa lipase (CRL) was used for the preparation of optically active primary allenic alcohols with axial chirality. The biocatalytic material used was the commercial CRL (C-CRL) and propan-2-ol-treated CRL (PT-CRL). The kinetic resolution of (±)-1 and (±)-3a–c in water using C-CRL resulted in either the absence of or low enantiomeric ratio, whereas PT-CRL increased the E-value. Under optimized conditions (temperature and medium used) (+)-S-3a and (−)-R-4 were isolated in excellent yields and high optical purity (o.p.). The resolution was carried out on multigram scale in water/n-hexane at 4 °C.

The reaction temperature, pH of the aqueous medium and use of an aqueous- n -hexane reaction medium markedly influenced the hydrolysis rate and enantioselectivity of (±)-4-acetoxy-[2,2]-paracyclophane with propan-2-ol-treated Candida rugosa lipase. The results have been justified on the basis of a possible conformational change in the enzyme as a consequence of the displacement of its polypeptide lid.

Racemic esters of 1-phenyl- and 1-(pyridyl)ethyl acetates 1a–c (R = Me, Ph) were subjected to hydrolysis in water in the presence of Candida rugosa lipase (CRL). 1-Pyridylethyl acetates (1, R = Me) are hydrolyzed by crude CRL (C-CRL) and isopropanol (isopropyl alcohol) treated CRL (PT-CRL) at very low rates, and the enantiorecognition was disappointing. By using 1-pyridylethyl benzoates (1, R = Ph) the results differed greatly: hydrolysis occurred much faster, and the enantiorecognition was good for 3- and 4-pyridyl derivatives and excellent for 2-pyridyl derivative. Analogous results were obtained by submitting the 1-phenylethanol esters 4 to enzymatic hydrolysis under the same experimental conditions. The hydrolysis of methyl o-acetoxybenzoates 7 (R = Me) gave quantitatively the deacetylated methyl o-hydroxybenzoates 9 (R = Me) by using either C-CRL or PT-CRL. A complete reversed selectivity is observed in the hydrolysis of phenyl o-acetoxybenzoates 7 (R = Ph) catalyzed by PT-CRL. Molecular modeling studies, aimed at probing the substrate specificity and the enantioselectivity of enzyme in terms of its three-dimensional structure is reported.

Racemic methyl 2-aryloxypropionates (±)-1 were subjected to hydrolysis in water and in a series of two-phase aqueous organic media in the presence of Candida rugosa lipase (CRL). The biocatalytic material used was the enzyme of commercial CRL purified by treatment with different alcohols. The purification of CRL and the reaction medium play an important role in the enantioselection of racemates (±)-1. While it is not possible to use the same protocol for all substrates, by combining the different ways of purifying the enzyme with the various reaction media, it is possible to achieve high enantioselectivities of racemic esters.

Colloidal aqueous dispersions of nanometric NiAl, ZnAl, NiCr, ZnCr, NiFe, and MgFe hydrotalcite-like compounds were prepared in a water/cetyltrimethylammonium bromide/n-butanol/isooctane microemulsion. Particle sizes were analyzed with different techniques, and the results confirm dimensions between 10 and 30 nm, except for ZnAl–HTlc (150–200 nm). A good colloidal stability of HTlc–NPs aqueous dispersions, investigated with DLS and Pz measurements, was obtained without the need for any stabilizing agent. SEM images clearly showed that the obtained HTlc posses a high tendency to spontaneously form homogeneous and dense stacking of plate-like HTlc crystals directly from aqueous solution, giving rise to the developing of functional materials in optical, electrical and magnetic fields.

A colloidal dispersion of zinc aluminum hydrotalcite nanoparticles (ZnAl-HTlc) has been used for in vitro experimental procedures in order to provide reliable data on their potential application in cellular delivery. Two different cell lines (HeLa tumor cells and MDCK normal cells) with a similar epithelial derivation have been used. Sedimentation studies performed in the presence of different constituents of the cell culture medium revealed the importance of serum components to stabilize the colloidal dispersions of nanosized ZnAl-HTlc. Cell viability assay showed for nanosized ZnAl-HTlc a higher cell growth inhibition on tumor cells compared to normal cells whereas LDH test showed the absence of toxicity for both cell lines. Cellular uptake experiments indicated a preferential internalization of ZnAl-HTlc nanoparticles in HeLa tumor cells. Adsorption study and steady state fluorescence measurements on the phenol red/HTlc hybrid were carried out in order to verify the possibility of using phenol red as fluorescent dye for ZnAl-HTlc nanoparticles. The observed spectral behavior indicated a strong interaction between the dye and the inorganic matrix and the preferential adsorption of the dye on the nanoparticle surface has been confirmed by the XRPD data. Fluorescence confocal imaging showed a different localization pattern of nanosized HTlc in the two cell lines and a higher fluorescence signals in tumor cells supporting the occurrence of more efficient internalization processes in the pathogen cell line as observed in the cellular uptake experiments.

The influence of short-chain alcohols and osmolytes on the structural properties, catalytic activity and thermal stability of myoglobin (Mb) has been investigated. Results show that in the presence of a cosolvent, the catalytic activity of Mb decrease as the concentration increase, starting when the concentration was low. Spectroscopic investigations show that changes in catalytic activity are not due to protein conformational changes. Otherwise, no significant correlation between the thermal stability and the catalytic activity is evident from the data. Our measurements show a correlation between the effect of cosolvents on catalytic activity and changes in the microstructure of solvent, as inferred from previous IR data on these mixtures.

Regioselectively fluoro-, trifluoromethyl- and trifluoromethoxy-substituted 3-methyleneindolines have been prepared using a four-step procedure involving metalation/bromination of fluorinated Boc-protected anilines, N -propargylation of the resulting o -bromoarylcarbamate and reductive radical cyclization of the product with tributyltin hydride/AIBN. 3-Methyleneindolines, as valuable, versatile intermediates, can be transformed into highly functionalized 3-substituted indoles by ene-type reactions using different enophiles. Thus, fluoro-, trifluoromethyl- and trifluoromethoxy-substituted diethyl 2-hydroxy-2-[(1 H -indol-3-yl)methyl]malonates, ethyl 2-hydroxy-3-(1 H -indol-3-yl)propionates and ethyl 2-hydroxy-3-(1 H -indol-3-yl)-2-trifluormethylpropionates were obtained in 77–86% yield by simply heating the corresponding tert -butyl 3-methyleneindoline-1-carboxylate with an equimolar amount of diethyl ketomalonate, ethyl glyoxalate and ethyl 3,3,3-trifluoropyruvate, respectively, at 100 °C, without solvent, for 0.5–4 h.

Regioselectively fluoro-, trifluoromethyl- and trifluoromethoxy-substituted 3-methyleneindolines have been prepared using a four-step procedure involving metalation/bromination of fluorinated Boc-protected anilines, N -propargylation of the resulting o -bromoarylcarbamate and reductive radical cyclization of the product with tributyltin hydride/AIBN. 3-Methyleneindolines, as valuable, versatile intermediates, can be transformed into highly functionalized 3-substituted indoles by ene-type reactions using different enophiles. Thus, fluoro-, trifluoromethyl- and trifluoromethoxy-substituted diethyl 2-hydroxy-2-[(1 H -indol-3-yl)methyl]malonates, ethyl 2-hydroxy-3-(1 H -indol-3-yl)propionates and ethyl 2-hydroxy-3-(1 H -indol-3-yl)-2-trifluormethylpropionates were obtained in 77–86% yield by simply heating the corresponding tert -butyl 3-methyleneindoline-1-carboxylate with an equimolar amount of diethyl ketomalonate, ethyl glyoxalate and ethyl 3,3,3-trifluoropyruvate, respectively, at 100 °C, without solvent, for 0.5–4 h.

The adsorption of myoglobin (Mb) onto phosphate grafted-zirconia (ZrO2-P) nanoparticles was studied in terms of conformational studies and thermal stability, determined by circular dichroism (CD), differential scanning calorimetry (DSC), and atomic force microscopy (AFM). The changes in protein structure have been correlated with the catalytic activity of free and adsorbed Mb. CD and DSC studies indicate marked rearrangements in Mb structure upon adsorption onto phosphate-grafted zirconia nanoparticles. These structural rearrangements of Mb could be responsible for the loss of catalytic activity observed for the adsorbed Mb. In particular, the conformational changes due to the adsorption process induced a reduction of kcat and KM. AFM measurements indicate that the interaction with the grafted-zirconia nanoparticles also affects the morphology of the bound protein, inducing the nucleation of prefibrillar-like aggregates.

Porous zirconium phosphate (P-ZrP) and zirconium benzenephosphonate (P-ZrBP) were prepared in the presence of an anionic surfactant acting as a template. Poorly crystalline materials with a P/Zr molar ratio equal to 2 and having a relatively high surface area and micro/mesoporosity have been obtained. The interaction of myoglobin with the two types of surfaces, the hydrophobic P-ZrBP and the hydrophilic P-ZrP, was investigated, and the adsorption isotherms were determined at different pH and temperature values. A model was proposed for the mechanism of the interaction of the protein with the surface based on the shape of the adsorption isotherm and the physical-chemical properties of myoglobin. The pH has been found to be an important parameter for determining the maximum adsorption capacity of P-ZrBP and P-ZrP for myoglobin molecules because of the changes that occur in the type and net charge of the protein surface as the pH of the medium changes. Protein binding affinity and capacity increase when the temperature is increased. This phenomenon occurs because myoglobin varies its conformation at high temperature with an increase in the exposed hydrophobic region. This process causes a stronger hydrophobic interaction between the protein and the adsorbent and reduces the repulsion between the adsorbed molecules. Studies on the activities of the obtained biocomposites are in progress.

Non-ionic water-in-oil (w/o) microemulsions of pentaoxyethylenenonylphenyl ether (Igepal-CO520)/cyclohexane/water at 25 °C have been used as reaction vessels to obtain zirconium hydrogen phosphate nanoparticles.

The adsorption and catalytic activity of myoglobin (Mb) on zirconium phosphonates (a-zirconium benzenephosphonate (alpha-ZrBP), a-zirconium carboxyethanephosphonate (alpha-ZrCEP), and a novel layered zirconium fluoride aminooctyl-N,N-bis(methylphosphonate) (ZrC8)) were investigated. The maximum adsorption was reached after 16 h of contact and was greater on hydrophobic supports such as alpha-ZrBP and ZrC8 compared to hydrophilic supports such as alpha-ZrCEP. The equilibrium adsorption isotherms fitted the Langmuir equation, suggesting the presence of a monolayer of protein molecules on the support surfaces. The catalytic activities of free Mb and of the obtained biocomposites were studied in terms of the oxidation of two aromatic substrates, o-phenylenediamine and 2-methoxyphenol (guaiacol), by hydrogen peroxide. The oxidation catalyzed by immobilized myoglobin followed the Michaelis-Menten kinetics, similar to oxidation by free Mb. The kinetic parameters, kcat and KM, were significantly affected by the adsorption process. Mb/alpha-ZrCEP was the most efficient biocatalyst obtained, probably because of the hydrophilic nature of the support. The effect of immobilization on the stability of Mb toward inactivation by hydrogen peroxide was also investigated, and an increased resistance was found. The biocomposites obtained can be stored at 4 degrees C for months without a significant loss of catalytic activity.

Biocomposites with enzymatic activity were obtained by adsorption of lipase from Candida rugosa on the surface of different layered zirconium phosphates and phosphonates such as α-zirconium hydrogenphosphate, solid dispersions of zirconium phosphate in silica, zirconium carboxyethanephosphonate, zirconium phosphate-carboxyethanephosphonate, zirconium benzenephosphonate, and zirconium phosphate-benzenephosphonate. All the supports were characterized for chemical composition, BET specific surface area, surface ion exchange capacity, and X-ray diffraction patterns. The adsorption process at 4 °C was studied as a function of time of equilibration of the support with the lipase solutions (0.5 mg/mL) and as a function of the amount of protein present in the equilibrating solution. The activities of biocomposites with the different supports, at different protein loadings, were obtained by determining the amount of acetic acid produced by catalyzed hydrolysis of p-nitrophenylacetate. The best results in terms of ...