The Gibbs energies of activation of the Menschutkin reactions of triethylamine with ethyl iodide ... more The Gibbs energies of activation of the Menschutkin reactions of triethylamine with ethyl iodide in 10 monoalcohols and nine dialcohols, were dissected into contributions from the initial state and transition state. To perform this study, the infinite dilution activity coefficient values of the solutes in the solvents, TO, were determined by the UNIFAC group-contribution method, using the modified Flory-Huggins equation in the combinatorial term. For triethylamine, the 7-values were calculated using group-interaction parameters from the VLE Parameter Table, due to the nonavailability of specific 7-UNIFAC interaction parameters for the relevant groups. For ethyl iodide, the 7-values were calculated using the group-interaction parameters from the specific 7-UNIFAC Parameter Table, except for the solvents diethylene glycol and triethylene glycol, where VLE parameters were used for the same reasons as explained above for the triethylamine. The results were compared with those obtained for the unimolecular decomposition of tert-butyl halides. For the Menschutkin reaction, we conclude that solvent disruption and reorganization to a state appropriate to solvating the molecular species is a dominant interaction mechanisim.
The mixed micelle formation in aqueous solutions between an anionic gemini surfactant derived fro... more The mixed micelle formation in aqueous solutions between an anionic gemini surfactant derived from the amino acid cystine (C(8)Cys)(2), and the phospholipids 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC, a micelle-forming phospholipid) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, a vesicle-forming phospholipid) has been studied by conductivity and the results compared with the ones obtained for the mixed systems with the single-chain surfactant derived from cysteine, C(8)Cys. Phospholipid-surfactant interactions were found to be synergistic in nature and dependent on the type of phospholipid and on surfactant hydrophobicity. Regular solution theory was used to analyse the gemini surfactant-DHPC binary mixtures and the interaction parameter, β(12), has been evaluated, as well as mixed micelle composition. The results have been interpreted in terms of the interplay between reduction of the electrostatic repulsions among the ionic head groups of the surfactants and steric hindrances arising from incorporation of the zwitterionic phospholipids in the mixed micelles.
In this work results concerning pressure and solvent effects on the Menschutkin reactions rates w... more In this work results concerning pressure and solvent effects on the Menschutkin reactions rates were determined. The reaction of triethylamine with ethyl iodide was followed conductimetrically at seven different pressures in the range 1–2000 bar at 40 °C in the pure solvents butan-l-ol and pentan-1-ol. Special attention was paid to pressure effects from which volumic properties of the solvent were determined.
ABSTRACT Naringinase, an enzyme complex that expresses α -l-rhamnosidase and β -d-glucosidase act... more ABSTRACT Naringinase, an enzyme complex that expresses α -l-rhamnosidase and β -d-glucosidase activities in native state, can be used to deglycosylate natural glycosides. The selective inactivation of one of these activities will allow the biosynthesis of different bioactive compounds in a simple, effective and cheap way. In this work, pressure and temperature were the tools used to selectively inactivate the activities expressed by naringinase. The main goal was the identification of pressure–temperature conditions to acquire conditions for the maximization of enzymatic hydrolysis of substrates with different numbers of glycosidic residues. α -l-Rhamnosidase was 32-fold more resistant against inactivation at 250 MPa than at atmospheric pressure. The best pressure condition to reduce β -d-glucosidase inactivation at 75°C was 173 MPa, while in the case of α -l-rhamnosidase inactivation at 85°C, it was above 250 MPa. Moreover, a selective inactivation of β -d-glucosidase activity of naringinase was attained, allowing an easy and cheap method with which to produce prunin and other expensive glycosides. The present work highlights the effect of high pressure on enzyme protection against thermal inactivation, demonstrating its potential as a powerful tool in biosynthesis.
Lipoamino acid-based micelles have been developed as delivery vehicles for the hydrophobic drug a... more Lipoamino acid-based micelles have been developed as delivery vehicles for the hydrophobic drug amphotericin B (AmB). The micellar solubilisation of AmB by a gemini lipoamino acid (LAA) derived from cysteine and its equimolar mixtures with the bile salts sodium cholate (NaC) and sodium deoxycholate (NaDC), as well as the aggregation sate of the drug in the micellar systems, was studied under biomimetic conditions (phosphate buffered-saline, pH 7.4) using UV-vis spectroscopy. Pure surfactant systems and equimolar mixtures were characterized by tensiometry and important parameters were determined, such as critical micelle concentration (CMC), surface tension at the CMC (γCMC), maximum surface excess concentration (Γmax), and minimum area occupied per molecule at the water/air interface (Amin). Rheological behaviour from viscosity measurements at different shear rates was also addressed. Solubilisation capacity was quantified in terms of molar solubilisation ratio (χ), micelle-water partition coefficient (KM) and Gibbs energy of solubilisation (ΔGs°). Formulations of AmB in micellar media were compared in terms of drug loading, encapsulation efficiency, aggregation state of AmB and in vitro antifungal activity against Candida albicans. The LAA-containing micellar systems solubilise AmB in its monomeric and less toxic form and exhibit in vitro antifungal activity comparable to that of the commercial formulation Fungizone.
The effect of pressure was studied in an enzymatic reaction with an immobilized biocatalyst. Nari... more The effect of pressure was studied in an enzymatic reaction with an immobilized biocatalyst. Naringinase immobilized by entrapment in calcium alginate beads was the biocatalyst used to catalyze, at high-pressure, the hydrolysis of naringin to naringenin. These molecules have great potential in the pharmaceutical industry due to their recognized anti-oxidant, anti-inflammatory, anti-carcinogenic, anti-hypertensive and hypocholesterolemic effects. At high-pressure, the influence of relevant parameters on naringinase catalytic activity such as temperature, substrate concentration, and biocatalyst reuse was studied. At 160 MPa, naringinase entrapped in Ca-alginate beads displayed higher activity, namely in the range of 35-40 • C, whereas the optimum, at atmospheric pressure, was 35 • C. The immobilized naringinase presented a Michaelis-Menten kinetic, with a 65% higher maximum initial rate (V ap max = 0.069 mM min −1), and a 70% lower K ap M (0.097 mM) at 160 MPa, as compared to kinetic parameters, at atmospheric pressure (V ap max = 0.042 mM min −1 and K ap M = 0.303 mM). A positive effect of pressure on naringin hydrolysis by immobilized naringinase in Ca-alginate beads was confirmed with a negative activation volume (V =) of −9 mL mol −1. The stability of immobilized naringinase was also evaluated at high-pressure.
The production of flavonoid glycosides by removing rhamnose from rutinosides can be accomplished ... more The production of flavonoid glycosides by removing rhamnose from rutinosides can be accomplished through enzymatic catalysis. Naringinase is an enzyme complex, expressing both α-L-rhamnosidase and β-D-glucosidase activities, with application in glycosides hydrolysis. To produce monoglycosylated flavonoids with naringinase, the expression of β-D-glucosidase activity is not desirable leading to the need of expensive methods for α-L-rhamnosidase purification. Therefore, the main purpose of this study was the inactivation of β-D-glucosidase activity expressed by naringinase keeping α-L-rhamnosidase with a high retention activity. Response surface methodology (RSM) was used to evaluate the effects of temperature and pH on β-D-glucosidase inactivation. A selective inactivation of β-D-glucosidase activity of naringinase was achieved at 81.5 • C and pH 3.9, keeping a very high residual activity of α-L-rhamnosidase (78%). This was a crucial achievement towards an easy and cheap production method of very expensive flavonoids, like prunin and isoquercetin starting from naringin and rutin, respectively.
The quaternization reaction between 2-amino-1-methylbenzimidazole and iodomethane was investigate... more The quaternization reaction between 2-amino-1-methylbenzimidazole and iodomethane was investigated in the gas phase and in liquid acetonitrile. Both experimental and theoretical techniques were used in this study. In the experimental part of this work, accurate second-order rate constants were obtained for this reaction in acetonitrile from conductivity data in the 293-323 K temperature range and at ambient pressure. From two different empirical equations describing the effect of temperature on reaction rates, thermodynamic functions of activation were calculated. In the theoretical part of this work, the mechanism of this reaction was investigated in the gas phase and in acetonitrile. Two different quantum levels (B3LYP/[6-311++G(3df,3pd
The hydrolysis of naringin, a bitter flavonone glycoside, with naringinase leads to reducing suga... more The hydrolysis of naringin, a bitter flavonone glycoside, with naringinase leads to reducing sugars (rhamnose and glucose), to prunin and to the aglycone, naringenin. To overcome the low solubility of naringin in the enzymatic reaction media, the effect of different solvents was studied, in order to improve the productivity and yield of the system. The effect of increasing concentration of
Naringinase is an enzyme complex, expressing α-l-rhamnosidase and β-d-glucosidase activities. The... more Naringinase is an enzyme complex, expressing α-l-rhamnosidase and β-d-glucosidase activities. The impact of high pressure and temperature on naringinase activity and stability were studied, in order to assess the potential of enzyme thermostability on glycosides hydrolyses. To a better understanding of these effects on naringinase enzyme complex, they were also evaluated over α-l-rhamnosidase and β-d-glucosidase activities, using specific substrates, p-nitrophenyl
Initially exploited in chemistry and physics, high-pressure technology has gained importance in v... more Initially exploited in chemistry and physics, high-pressure technology has gained importance in various fields. Flavonoids, namely naringin and naringenin, from citrus, are functional chemicals with important properties in the fields of healthcare, food and agriculture. Naringin, the principal bitter flavonone glycoside and the primary bitter component in grapefruit juice, can be hydrolysed by naringinase into tasteless naringenin.
Estimating the area of seabed surfaces from pictures or videos is an important problem in seafloo... more Estimating the area of seabed surfaces from pictures or videos is an important problem in seafloor surveys. This task is complex to achieve with moving platforms such as submersibles, towed or remotely operated vehicles (ROV), where the recording camera is typically not static and provides an oblique view of the seafloor. A new method for obtaining seabed surface area estimates is presented here, using the classical set up of two laser devices fixed to the ROV frame projecting two parallel lines over the seabed. By combining lengths measured directly from the image containing the laser lines, the area of seabed surfaces is estimated, as well as the camera's distance to the seabed, pan and tilt angles. The only parameters required are the distance between the parallel laser lines and the camera's horizontal and vertical angles of view. The method was validated with a controlled in situ experiment using a deep-sea ROV, yielding an area estimate error of 1.5%. Further applications and generalizations of the method are discussed, with emphasis on deep-sea applications.
The Gibbs energies of activation of the Menschutkin reactions of triethylamine with ethyl iodide ... more The Gibbs energies of activation of the Menschutkin reactions of triethylamine with ethyl iodide in 10 monoalcohols and nine dialcohols, were dissected into contributions from the initial state and transition state. To perform this study, the infinite dilution activity coefficient values of the solutes in the solvents, TO, were determined by the UNIFAC group-contribution method, using the modified Flory-Huggins equation in the combinatorial term. For triethylamine, the 7-values were calculated using group-interaction parameters from the VLE Parameter Table, due to the nonavailability of specific 7-UNIFAC interaction parameters for the relevant groups. For ethyl iodide, the 7-values were calculated using the group-interaction parameters from the specific 7-UNIFAC Parameter Table, except for the solvents diethylene glycol and triethylene glycol, where VLE parameters were used for the same reasons as explained above for the triethylamine. The results were compared with those obtained for the unimolecular decomposition of tert-butyl halides. For the Menschutkin reaction, we conclude that solvent disruption and reorganization to a state appropriate to solvating the molecular species is a dominant interaction mechanisim.
The mixed micelle formation in aqueous solutions between an anionic gemini surfactant derived fro... more The mixed micelle formation in aqueous solutions between an anionic gemini surfactant derived from the amino acid cystine (C(8)Cys)(2), and the phospholipids 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC, a micelle-forming phospholipid) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, a vesicle-forming phospholipid) has been studied by conductivity and the results compared with the ones obtained for the mixed systems with the single-chain surfactant derived from cysteine, C(8)Cys. Phospholipid-surfactant interactions were found to be synergistic in nature and dependent on the type of phospholipid and on surfactant hydrophobicity. Regular solution theory was used to analyse the gemini surfactant-DHPC binary mixtures and the interaction parameter, β(12), has been evaluated, as well as mixed micelle composition. The results have been interpreted in terms of the interplay between reduction of the electrostatic repulsions among the ionic head groups of the surfactants and steric hindrances arising from incorporation of the zwitterionic phospholipids in the mixed micelles.
In this work results concerning pressure and solvent effects on the Menschutkin reactions rates w... more In this work results concerning pressure and solvent effects on the Menschutkin reactions rates were determined. The reaction of triethylamine with ethyl iodide was followed conductimetrically at seven different pressures in the range 1–2000 bar at 40 °C in the pure solvents butan-l-ol and pentan-1-ol. Special attention was paid to pressure effects from which volumic properties of the solvent were determined.
ABSTRACT Naringinase, an enzyme complex that expresses α -l-rhamnosidase and β -d-glucosidase act... more ABSTRACT Naringinase, an enzyme complex that expresses α -l-rhamnosidase and β -d-glucosidase activities in native state, can be used to deglycosylate natural glycosides. The selective inactivation of one of these activities will allow the biosynthesis of different bioactive compounds in a simple, effective and cheap way. In this work, pressure and temperature were the tools used to selectively inactivate the activities expressed by naringinase. The main goal was the identification of pressure–temperature conditions to acquire conditions for the maximization of enzymatic hydrolysis of substrates with different numbers of glycosidic residues. α -l-Rhamnosidase was 32-fold more resistant against inactivation at 250 MPa than at atmospheric pressure. The best pressure condition to reduce β -d-glucosidase inactivation at 75°C was 173 MPa, while in the case of α -l-rhamnosidase inactivation at 85°C, it was above 250 MPa. Moreover, a selective inactivation of β -d-glucosidase activity of naringinase was attained, allowing an easy and cheap method with which to produce prunin and other expensive glycosides. The present work highlights the effect of high pressure on enzyme protection against thermal inactivation, demonstrating its potential as a powerful tool in biosynthesis.
Lipoamino acid-based micelles have been developed as delivery vehicles for the hydrophobic drug a... more Lipoamino acid-based micelles have been developed as delivery vehicles for the hydrophobic drug amphotericin B (AmB). The micellar solubilisation of AmB by a gemini lipoamino acid (LAA) derived from cysteine and its equimolar mixtures with the bile salts sodium cholate (NaC) and sodium deoxycholate (NaDC), as well as the aggregation sate of the drug in the micellar systems, was studied under biomimetic conditions (phosphate buffered-saline, pH 7.4) using UV-vis spectroscopy. Pure surfactant systems and equimolar mixtures were characterized by tensiometry and important parameters were determined, such as critical micelle concentration (CMC), surface tension at the CMC (γCMC), maximum surface excess concentration (Γmax), and minimum area occupied per molecule at the water/air interface (Amin). Rheological behaviour from viscosity measurements at different shear rates was also addressed. Solubilisation capacity was quantified in terms of molar solubilisation ratio (χ), micelle-water partition coefficient (KM) and Gibbs energy of solubilisation (ΔGs°). Formulations of AmB in micellar media were compared in terms of drug loading, encapsulation efficiency, aggregation state of AmB and in vitro antifungal activity against Candida albicans. The LAA-containing micellar systems solubilise AmB in its monomeric and less toxic form and exhibit in vitro antifungal activity comparable to that of the commercial formulation Fungizone.
The effect of pressure was studied in an enzymatic reaction with an immobilized biocatalyst. Nari... more The effect of pressure was studied in an enzymatic reaction with an immobilized biocatalyst. Naringinase immobilized by entrapment in calcium alginate beads was the biocatalyst used to catalyze, at high-pressure, the hydrolysis of naringin to naringenin. These molecules have great potential in the pharmaceutical industry due to their recognized anti-oxidant, anti-inflammatory, anti-carcinogenic, anti-hypertensive and hypocholesterolemic effects. At high-pressure, the influence of relevant parameters on naringinase catalytic activity such as temperature, substrate concentration, and biocatalyst reuse was studied. At 160 MPa, naringinase entrapped in Ca-alginate beads displayed higher activity, namely in the range of 35-40 • C, whereas the optimum, at atmospheric pressure, was 35 • C. The immobilized naringinase presented a Michaelis-Menten kinetic, with a 65% higher maximum initial rate (V ap max = 0.069 mM min −1), and a 70% lower K ap M (0.097 mM) at 160 MPa, as compared to kinetic parameters, at atmospheric pressure (V ap max = 0.042 mM min −1 and K ap M = 0.303 mM). A positive effect of pressure on naringin hydrolysis by immobilized naringinase in Ca-alginate beads was confirmed with a negative activation volume (V =) of −9 mL mol −1. The stability of immobilized naringinase was also evaluated at high-pressure.
The production of flavonoid glycosides by removing rhamnose from rutinosides can be accomplished ... more The production of flavonoid glycosides by removing rhamnose from rutinosides can be accomplished through enzymatic catalysis. Naringinase is an enzyme complex, expressing both α-L-rhamnosidase and β-D-glucosidase activities, with application in glycosides hydrolysis. To produce monoglycosylated flavonoids with naringinase, the expression of β-D-glucosidase activity is not desirable leading to the need of expensive methods for α-L-rhamnosidase purification. Therefore, the main purpose of this study was the inactivation of β-D-glucosidase activity expressed by naringinase keeping α-L-rhamnosidase with a high retention activity. Response surface methodology (RSM) was used to evaluate the effects of temperature and pH on β-D-glucosidase inactivation. A selective inactivation of β-D-glucosidase activity of naringinase was achieved at 81.5 • C and pH 3.9, keeping a very high residual activity of α-L-rhamnosidase (78%). This was a crucial achievement towards an easy and cheap production method of very expensive flavonoids, like prunin and isoquercetin starting from naringin and rutin, respectively.
The quaternization reaction between 2-amino-1-methylbenzimidazole and iodomethane was investigate... more The quaternization reaction between 2-amino-1-methylbenzimidazole and iodomethane was investigated in the gas phase and in liquid acetonitrile. Both experimental and theoretical techniques were used in this study. In the experimental part of this work, accurate second-order rate constants were obtained for this reaction in acetonitrile from conductivity data in the 293-323 K temperature range and at ambient pressure. From two different empirical equations describing the effect of temperature on reaction rates, thermodynamic functions of activation were calculated. In the theoretical part of this work, the mechanism of this reaction was investigated in the gas phase and in acetonitrile. Two different quantum levels (B3LYP/[6-311++G(3df,3pd
The hydrolysis of naringin, a bitter flavonone glycoside, with naringinase leads to reducing suga... more The hydrolysis of naringin, a bitter flavonone glycoside, with naringinase leads to reducing sugars (rhamnose and glucose), to prunin and to the aglycone, naringenin. To overcome the low solubility of naringin in the enzymatic reaction media, the effect of different solvents was studied, in order to improve the productivity and yield of the system. The effect of increasing concentration of
Naringinase is an enzyme complex, expressing α-l-rhamnosidase and β-d-glucosidase activities. The... more Naringinase is an enzyme complex, expressing α-l-rhamnosidase and β-d-glucosidase activities. The impact of high pressure and temperature on naringinase activity and stability were studied, in order to assess the potential of enzyme thermostability on glycosides hydrolyses. To a better understanding of these effects on naringinase enzyme complex, they were also evaluated over α-l-rhamnosidase and β-d-glucosidase activities, using specific substrates, p-nitrophenyl
Initially exploited in chemistry and physics, high-pressure technology has gained importance in v... more Initially exploited in chemistry and physics, high-pressure technology has gained importance in various fields. Flavonoids, namely naringin and naringenin, from citrus, are functional chemicals with important properties in the fields of healthcare, food and agriculture. Naringin, the principal bitter flavonone glycoside and the primary bitter component in grapefruit juice, can be hydrolysed by naringinase into tasteless naringenin.
Estimating the area of seabed surfaces from pictures or videos is an important problem in seafloo... more Estimating the area of seabed surfaces from pictures or videos is an important problem in seafloor surveys. This task is complex to achieve with moving platforms such as submersibles, towed or remotely operated vehicles (ROV), where the recording camera is typically not static and provides an oblique view of the seafloor. A new method for obtaining seabed surface area estimates is presented here, using the classical set up of two laser devices fixed to the ROV frame projecting two parallel lines over the seabed. By combining lengths measured directly from the image containing the laser lines, the area of seabed surfaces is estimated, as well as the camera's distance to the seabed, pan and tilt angles. The only parameters required are the distance between the parallel laser lines and the camera's horizontal and vertical angles of view. The method was validated with a controlled in situ experiment using a deep-sea ROV, yielding an area estimate error of 1.5%. Further applications and generalizations of the method are discussed, with emphasis on deep-sea applications.
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Papers by António Calado