The electropolymerization of tyramine has been investigated using glassy carbon electrodes modifi... more The electropolymerization of tyramine has been investigated using glassy carbon electrodes modified with six classes of carbon materials, namely carbon black, graphitized carbon, graphite, graphene oxide, chemical vapour deposition based multi-walled carbon nanotubes and arc discharged based multi-walled carbon nanotubes. These materials were characterized before and after electrodeposition of polytyramine (PT) by Raman spectroscopy, inductively coupled plasma optical emission spectrometry, cyclic voltammetry and amperometry. Previously, other groups have established that impurities present in carbon materials, can play a critical role in electrocatalytic activity. In this study, the presence of graphitic basal plane carbon, rather than metallic impurities, is believed to initiate the formation of a redox active PT film that mediates the oxidation of NADH. The importance of graphitic basal plane carbon was supported by examining the impact of the graphitization of carbon black over the range of 500 to 2000 8C. Graphitic basal plane carbon impurity was demonstrated to be highly active and important with respect to the electrodeposition of a PT film that possesses significant catalytic activity towards the oxidation of NADH.
Please cite this article as: Kazi SA, Campi EM, Hearn MTW, A convenient and efficient one pot syn... more Please cite this article as: Kazi SA, Campi EM, Hearn MTW, A convenient and efficient one pot synthesis of unsymmetrically substituted p-terphenyls via a phosphine-free double Suzuki crosscoupling protocol using 1,4-dibromo-2-nitrobenzene as the substrate, Tetrahedron (2018),
Ionic liquids are molten salts at ambient temperatures. Because of their attributes, they can be ... more Ionic liquids are molten salts at ambient temperatures. Because of their attributes, they can be successfully employed as 'green' solvents in a range of biocatalytic reactions with a variety of enzymes.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was e... more ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
Experimental procedures have been developed to provide at the laboratory scale an efficient and r... more Experimental procedures have been developed to provide at the laboratory scale an efficient and relatively inexpensive continuous flow processing system to undertake metathesis chemistry. Glass discs, coated with porous silica particles impregnated with Grubbs (I) generation or Hoveyda-Grubbs (II) generation catalysts through non-covalent interactions, have been successfully used as annular zones for in situ metathesis reactions. The supported catalysts were active for ring closing metathesis and self-metathesis reactions with a variety of substrates with nearly quantitative conversion depending on the substrate structure. Advantages of these procedures include the ability to conduct reactions efficiently at small (low mmole) scale under ambient laboratory conditions, thereby avoiding the need for high energy/thermal procedures. Multiple experiments can be undertaken during the same day if catalyst screening is required. Moreover, separation of starting materials from products can be achieved using the same unit operations, avoiding the need for additional purification steps, thereby reducing the total solvent consumption.
International Journal of Peptide Research and Therapeutics, 2003
The resolution of several structurally related synthetic peptides, derived from the loop 3 region... more The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin betaA-betaD subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n-octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge-state-specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n-octadecyl-silica involved distinguishable contributions from both electrophoretic mobility and chromatographic retention. Temperature effects were reflected as variations in both the electro-osmotic flow and the electrophoretic mobility of the peptides. When the separation forces acting on the peptides were synergistic with the electro-osmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, kappacec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their kappacec values increased slightly with increasing temperature. Moreover, when the content of organic modifier, acetonitrile, was sufficiently high, e.g. > 40% (v/v) and nonpolar interactions with the Hypersil n-octadecyl-silica sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by exploiting the mutual processes of electrophoretic mobility and electrostatic interaction. A linear relationship between the ln kappacec values and the volume fractions, psi, of the organic modifier over a limited range of psi-values, was established for the negatively charged peptides under these isocratic conditions. These findings thus provide useful guidelines in a more general context for the resolution and analysis of structurally related synthetic peptides using CEC methods.
The interaction thermodynamics of the thrombin receptor agonistic peptide (TRAP-1), H-Ser-Phe-Leu... more The interaction thermodynamics of the thrombin receptor agonistic peptide (TRAP-1), H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-OH, and a set of alanine scan substitution peptides, have been investigated with an noctadecylacrylic polymer-bonded porous silica (Sil-ODA 18) and water-acetonitrile mobile phases at temperatures ranging from 5 to 80 C in 5 C increments. The retention of these peptides on the Sil-ODA 18 stationary phase decreased as the water content in the mobile phase was lowered from 80% (v/v) to ca. 45% (v/v) and reached a minimum value for each peptide at a specific water-acetonitrile composition. Further decreases in the water content of the mobile phase led to increased retention. The magnitude of the changes in enthalpy of interaction, DH 0 assoc , changes in entropy of interaction, DS 0 assoc , and changes in heat capacity, DC 0 p , were found to be dependent on the molecular properties of the mobile phase, the temperature, the structure/mobility of the stationary phase, and the conformation and solvation state of the peptides. With water-rich mobile phases, the retention behaviour of the TRAP analogues was dominated by enthalpic processes, consistent with the participation of strong hydrogen bonding effects, but became dominated by entropic effects with acetonitrile-rich mobile phases as the temperature was increased. These changes in the retention behaviour of these TRAP peptides are consistent with the generation of water or acetonitrile clusters in the mobile phase depending on the volume fractions of the organic solvent as the Sil-ODA 18 stationary phase transitions from its crystalline to its isotropic state.
Publisher Summary Reversed phase high performance liquid chromatography (RP-HPLC) now plays a cri... more Publisher Summary Reversed phase high performance liquid chromatography (RP-HPLC) now plays a critical role in the analysis and purification of peptides and proteins from natural and synthetic sources. The extraordinary popularity of RP-HPLC for polypeptide analysis can be attributed to a number of factors hi9ghlighted in this chapter. Some of the potential of RP-HPLC in the purification of polypeptides and proteins is demonstrated by the selected samples. Despite the current wide usage of RP-HPLC techniques in protein sequencing studies and many other purification areas involving high resolution polypeptide separations, the selection of a particular chemically-bonded stationary phase and mobile phase composition has frequently been based on empirical criteria. Not uncommonly, such arbitrary selections may not adequately address the issues of optimal chromatographic resolution, band shape or solute recovery. This chapter documents recent theoretical developments which characterize the RP-HPLC of polypeptides and proteins, and describes recent advances in the evolution of fully mechanistic models which describe in physicochemical terms the nature of the interaction between biological macromolecules and biological and synthetic surfaces.
The photochemical dimerisation of resveratrol analogues in the solid state to generate chiral phe... more The photochemical dimerisation of resveratrol analogues in the solid state to generate chiral phenyl substituted cyclobutanes is described. NMR spectroscopic and X-ray crystallographic methods have confirmed that the dimerisation leads to the head to tail orientation of the phenyl group substituents in the cyclobutane derivative. Interestingly, the parent compound, resveratrol, in the solid state, did not form a cyclobutane dimer, but the O-acetyl analogues gave the corresponding cyclobutane dimers in high yield, suggesting that the close packing of molecules together with the electron density through the conjugated double bond of the resveratrol structure are important determinants for photodimerisation to occur in the solid state.
Impact of the substitution pattern of xylyl-bridged 1,4-bis(1,4,7triazacyclononane) ligands on th... more Impact of the substitution pattern of xylyl-bridged 1,4-bis(1,4,7triazacyclononane) ligands on the crystal structures of their zinc(II) complexes and their ability to bind to N-terminally tagged proteins q
High-performance liquid chromatography (HPLC) is a fully automated form of liquid chromatography ... more High-performance liquid chromatography (HPLC) is a fully automated form of liquid chromatography that allows separations of high speed, high resolution, and high sensitivity. HPLC, when coupled to mass spectrometry, can be used for the separation, detection, identification and quantification of a very large number of individual components in a sample, thus enabling challenging peptide and protein analyses in complex matrices. In this chapter, the major separation modes, including size-exclusion, reversed-phase, normal-phase, hydrophilic interaction, aqueous normal phase, hydrophobic interaction, ion-exchange, and affinity chromatography, and their applications to the analysis of peptides and proteins, are described. Method development approaches for analytical HPLC procedures and their upscaling to a preparative level are discussed in the context of analytical “green chemistry” and process analytical technologies. Various options for multidimensional HPLC, involving different separation modes in sequence for the efficient analysis and purification complex peptide and protein mixtures, are presented. Recognition of the opportunities for HPLC in systems biology, medical diagnostics, personalized medicine, and in the manufacture of biotherapeutics are currently the drivers of interdisciplinary research in sorbent development, surface chemistry, mathematical modeling of chromatographic processes, protein engineering, and bioinformatics. Keywords: peptides/proteins; chromatography; elution; gradient elution; high-performance liquid chromatography; isocratic elution; multidimensional HPLC; orthogonality; peak capacity; retention factor; resolution; selectivity
Determination of the extent of host cell protein (HCP) contamination is an essential pre-requisit... more Determination of the extent of host cell protein (HCP) contamination is an essential pre-requisite to validate the chromatographic purification of recombinant proteins. This study explores how different experimental conditions affect the HCP profiles generated during the immobilised metal ion affinity chromatographic (IMAC) purification with a Ni2+-1,4,7-triaza-cyclononane (tacn) Sepharose FF™ sorbent of the Bacillus halodurans N- and C-terminal His6-tagged xylanase A, expressed by Escherichia coli BL21(DE3) cells, and captured directly from cell lysates. Comparative studies were also carried out under identical loading, wash and elution conditions using nitrilotriacetic acid (NTA), also immobilised onto an agarose support and complexed with Ni2+ ions. High-resolution tandem mass spectrometry of the tryptic peptides derived from the proteins present in the IMAC flow-through, wash and elution fractions confirmed that the E. coli BL21(DE3) HCP profiles were dependent on the choice of adsorbent. With feedstocks containing the N- or C-terminal His6-tagged xylanase A, in several instances the same E. coli BL21(DE3) HCPs were found to co-elute with the tagged protein from either adsorbent, indicating a preferential ability of some HCPs to bind to both the IMAC resin and to the recombinant protein. This promiscuous behaviour has been found to be due to factors other than just the presence of histidine-rich motifs within the amino acid sequences of these HCPs. This case study demonstrates that the choice of protein expression and separation conditions impact on the levels of HCP contamination when different IMAC systems are employed.
Large-scale chromatography has been playing an important role in downstream treatment processing ... more Large-scale chromatography has been playing an important role in downstream treatment processing in biotechnology. In order to improve the productivity, the throughput of the chromatographic equipment was often increased by increasing the flow-rate and/or by increasing the column sample loading. This paper reports the results of a study on the impact of these and other operating parameters in affinity and ion-exchange chromatographic columns when used for protein purification. A sectional model was developed to predict protein adsorption processes in a packed column. The formulations of this mathematical model are presented in the Appendix. The present study was carried out with computer simulation based on this model and using data obtained from laboratory-scale columns. This model can simulate both the adsorption and washing stages of the protein purification process for both porous and non-porous particles. The effects of changing operating parameters were simulated and contour plots were generated for the easy identification of these effects. It was shown that both flow-rate and column loading can have a considerable impact on the processing rate and the yield of the column. As for the column capacity utilization, the impact of changing flow-rate is not significant at column loading of less than 80% in the test case. It was suggest that the present investigation provides a systematic predictive strategy which will greatly reduce the need for expensive, labour-intensive and time-consuming experimental work during process scale-up.
The electropolymerization of tyramine has been investigated using glassy carbon electrodes modifi... more The electropolymerization of tyramine has been investigated using glassy carbon electrodes modified with six classes of carbon materials, namely carbon black, graphitized carbon, graphite, graphene oxide, chemical vapour deposition based multi-walled carbon nanotubes and arc discharged based multi-walled carbon nanotubes. These materials were characterized before and after electrodeposition of polytyramine (PT) by Raman spectroscopy, inductively coupled plasma optical emission spectrometry, cyclic voltammetry and amperometry. Previously, other groups have established that impurities present in carbon materials, can play a critical role in electrocatalytic activity. In this study, the presence of graphitic basal plane carbon, rather than metallic impurities, is believed to initiate the formation of a redox active PT film that mediates the oxidation of NADH. The importance of graphitic basal plane carbon was supported by examining the impact of the graphitization of carbon black over the range of 500 to 2000 8C. Graphitic basal plane carbon impurity was demonstrated to be highly active and important with respect to the electrodeposition of a PT film that possesses significant catalytic activity towards the oxidation of NADH.
Please cite this article as: Kazi SA, Campi EM, Hearn MTW, A convenient and efficient one pot syn... more Please cite this article as: Kazi SA, Campi EM, Hearn MTW, A convenient and efficient one pot synthesis of unsymmetrically substituted p-terphenyls via a phosphine-free double Suzuki crosscoupling protocol using 1,4-dibromo-2-nitrobenzene as the substrate, Tetrahedron (2018),
Ionic liquids are molten salts at ambient temperatures. Because of their attributes, they can be ... more Ionic liquids are molten salts at ambient temperatures. Because of their attributes, they can be successfully employed as 'green' solvents in a range of biocatalytic reactions with a variety of enzymes.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was e... more ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
Experimental procedures have been developed to provide at the laboratory scale an efficient and r... more Experimental procedures have been developed to provide at the laboratory scale an efficient and relatively inexpensive continuous flow processing system to undertake metathesis chemistry. Glass discs, coated with porous silica particles impregnated with Grubbs (I) generation or Hoveyda-Grubbs (II) generation catalysts through non-covalent interactions, have been successfully used as annular zones for in situ metathesis reactions. The supported catalysts were active for ring closing metathesis and self-metathesis reactions with a variety of substrates with nearly quantitative conversion depending on the substrate structure. Advantages of these procedures include the ability to conduct reactions efficiently at small (low mmole) scale under ambient laboratory conditions, thereby avoiding the need for high energy/thermal procedures. Multiple experiments can be undertaken during the same day if catalyst screening is required. Moreover, separation of starting materials from products can be achieved using the same unit operations, avoiding the need for additional purification steps, thereby reducing the total solvent consumption.
International Journal of Peptide Research and Therapeutics, 2003
The resolution of several structurally related synthetic peptides, derived from the loop 3 region... more The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin betaA-betaD subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n-octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge-state-specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n-octadecyl-silica involved distinguishable contributions from both electrophoretic mobility and chromatographic retention. Temperature effects were reflected as variations in both the electro-osmotic flow and the electrophoretic mobility of the peptides. When the separation forces acting on the peptides were synergistic with the electro-osmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, kappacec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their kappacec values increased slightly with increasing temperature. Moreover, when the content of organic modifier, acetonitrile, was sufficiently high, e.g. > 40% (v/v) and nonpolar interactions with the Hypersil n-octadecyl-silica sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by exploiting the mutual processes of electrophoretic mobility and electrostatic interaction. A linear relationship between the ln kappacec values and the volume fractions, psi, of the organic modifier over a limited range of psi-values, was established for the negatively charged peptides under these isocratic conditions. These findings thus provide useful guidelines in a more general context for the resolution and analysis of structurally related synthetic peptides using CEC methods.
The interaction thermodynamics of the thrombin receptor agonistic peptide (TRAP-1), H-Ser-Phe-Leu... more The interaction thermodynamics of the thrombin receptor agonistic peptide (TRAP-1), H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-OH, and a set of alanine scan substitution peptides, have been investigated with an noctadecylacrylic polymer-bonded porous silica (Sil-ODA 18) and water-acetonitrile mobile phases at temperatures ranging from 5 to 80 C in 5 C increments. The retention of these peptides on the Sil-ODA 18 stationary phase decreased as the water content in the mobile phase was lowered from 80% (v/v) to ca. 45% (v/v) and reached a minimum value for each peptide at a specific water-acetonitrile composition. Further decreases in the water content of the mobile phase led to increased retention. The magnitude of the changes in enthalpy of interaction, DH 0 assoc , changes in entropy of interaction, DS 0 assoc , and changes in heat capacity, DC 0 p , were found to be dependent on the molecular properties of the mobile phase, the temperature, the structure/mobility of the stationary phase, and the conformation and solvation state of the peptides. With water-rich mobile phases, the retention behaviour of the TRAP analogues was dominated by enthalpic processes, consistent with the participation of strong hydrogen bonding effects, but became dominated by entropic effects with acetonitrile-rich mobile phases as the temperature was increased. These changes in the retention behaviour of these TRAP peptides are consistent with the generation of water or acetonitrile clusters in the mobile phase depending on the volume fractions of the organic solvent as the Sil-ODA 18 stationary phase transitions from its crystalline to its isotropic state.
Publisher Summary Reversed phase high performance liquid chromatography (RP-HPLC) now plays a cri... more Publisher Summary Reversed phase high performance liquid chromatography (RP-HPLC) now plays a critical role in the analysis and purification of peptides and proteins from natural and synthetic sources. The extraordinary popularity of RP-HPLC for polypeptide analysis can be attributed to a number of factors hi9ghlighted in this chapter. Some of the potential of RP-HPLC in the purification of polypeptides and proteins is demonstrated by the selected samples. Despite the current wide usage of RP-HPLC techniques in protein sequencing studies and many other purification areas involving high resolution polypeptide separations, the selection of a particular chemically-bonded stationary phase and mobile phase composition has frequently been based on empirical criteria. Not uncommonly, such arbitrary selections may not adequately address the issues of optimal chromatographic resolution, band shape or solute recovery. This chapter documents recent theoretical developments which characterize the RP-HPLC of polypeptides and proteins, and describes recent advances in the evolution of fully mechanistic models which describe in physicochemical terms the nature of the interaction between biological macromolecules and biological and synthetic surfaces.
The photochemical dimerisation of resveratrol analogues in the solid state to generate chiral phe... more The photochemical dimerisation of resveratrol analogues in the solid state to generate chiral phenyl substituted cyclobutanes is described. NMR spectroscopic and X-ray crystallographic methods have confirmed that the dimerisation leads to the head to tail orientation of the phenyl group substituents in the cyclobutane derivative. Interestingly, the parent compound, resveratrol, in the solid state, did not form a cyclobutane dimer, but the O-acetyl analogues gave the corresponding cyclobutane dimers in high yield, suggesting that the close packing of molecules together with the electron density through the conjugated double bond of the resveratrol structure are important determinants for photodimerisation to occur in the solid state.
Impact of the substitution pattern of xylyl-bridged 1,4-bis(1,4,7triazacyclononane) ligands on th... more Impact of the substitution pattern of xylyl-bridged 1,4-bis(1,4,7triazacyclononane) ligands on the crystal structures of their zinc(II) complexes and their ability to bind to N-terminally tagged proteins q
High-performance liquid chromatography (HPLC) is a fully automated form of liquid chromatography ... more High-performance liquid chromatography (HPLC) is a fully automated form of liquid chromatography that allows separations of high speed, high resolution, and high sensitivity. HPLC, when coupled to mass spectrometry, can be used for the separation, detection, identification and quantification of a very large number of individual components in a sample, thus enabling challenging peptide and protein analyses in complex matrices. In this chapter, the major separation modes, including size-exclusion, reversed-phase, normal-phase, hydrophilic interaction, aqueous normal phase, hydrophobic interaction, ion-exchange, and affinity chromatography, and their applications to the analysis of peptides and proteins, are described. Method development approaches for analytical HPLC procedures and their upscaling to a preparative level are discussed in the context of analytical “green chemistry” and process analytical technologies. Various options for multidimensional HPLC, involving different separation modes in sequence for the efficient analysis and purification complex peptide and protein mixtures, are presented. Recognition of the opportunities for HPLC in systems biology, medical diagnostics, personalized medicine, and in the manufacture of biotherapeutics are currently the drivers of interdisciplinary research in sorbent development, surface chemistry, mathematical modeling of chromatographic processes, protein engineering, and bioinformatics. Keywords: peptides/proteins; chromatography; elution; gradient elution; high-performance liquid chromatography; isocratic elution; multidimensional HPLC; orthogonality; peak capacity; retention factor; resolution; selectivity
Determination of the extent of host cell protein (HCP) contamination is an essential pre-requisit... more Determination of the extent of host cell protein (HCP) contamination is an essential pre-requisite to validate the chromatographic purification of recombinant proteins. This study explores how different experimental conditions affect the HCP profiles generated during the immobilised metal ion affinity chromatographic (IMAC) purification with a Ni2+-1,4,7-triaza-cyclononane (tacn) Sepharose FF™ sorbent of the Bacillus halodurans N- and C-terminal His6-tagged xylanase A, expressed by Escherichia coli BL21(DE3) cells, and captured directly from cell lysates. Comparative studies were also carried out under identical loading, wash and elution conditions using nitrilotriacetic acid (NTA), also immobilised onto an agarose support and complexed with Ni2+ ions. High-resolution tandem mass spectrometry of the tryptic peptides derived from the proteins present in the IMAC flow-through, wash and elution fractions confirmed that the E. coli BL21(DE3) HCP profiles were dependent on the choice of adsorbent. With feedstocks containing the N- or C-terminal His6-tagged xylanase A, in several instances the same E. coli BL21(DE3) HCPs were found to co-elute with the tagged protein from either adsorbent, indicating a preferential ability of some HCPs to bind to both the IMAC resin and to the recombinant protein. This promiscuous behaviour has been found to be due to factors other than just the presence of histidine-rich motifs within the amino acid sequences of these HCPs. This case study demonstrates that the choice of protein expression and separation conditions impact on the levels of HCP contamination when different IMAC systems are employed.
Large-scale chromatography has been playing an important role in downstream treatment processing ... more Large-scale chromatography has been playing an important role in downstream treatment processing in biotechnology. In order to improve the productivity, the throughput of the chromatographic equipment was often increased by increasing the flow-rate and/or by increasing the column sample loading. This paper reports the results of a study on the impact of these and other operating parameters in affinity and ion-exchange chromatographic columns when used for protein purification. A sectional model was developed to predict protein adsorption processes in a packed column. The formulations of this mathematical model are presented in the Appendix. The present study was carried out with computer simulation based on this model and using data obtained from laboratory-scale columns. This model can simulate both the adsorption and washing stages of the protein purification process for both porous and non-porous particles. The effects of changing operating parameters were simulated and contour plots were generated for the easy identification of these effects. It was shown that both flow-rate and column loading can have a considerable impact on the processing rate and the yield of the column. As for the column capacity utilization, the impact of changing flow-rate is not significant at column loading of less than 80% in the test case. It was suggest that the present investigation provides a systematic predictive strategy which will greatly reduce the need for expensive, labour-intensive and time-consuming experimental work during process scale-up.
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