Jana Charvátová

Porphyrin phosphonates: novel anionic receptors for saccharide recognition

Abstract Novel porphyrin phosphonates ( 1 – 3 ) exhibit binding of alkyl pyranosides in organic media (receptor 1 ), monosaccharides, selected disaccharides in water (receptors 2 and 3 ); effective binding for d (−) fructose, d (+) maltose and α- d -lactose were found; association constants for 1 (in acetonitrile) and for 2 and 3 (in water) are of the order 10 4 M −1 .

Novel porphyrin based receptors for saccharide recognition in water

Abstract A binding study of the porphyrin phosphonates ( 1 , 2 ), porphyrin–oligopeptide conjugates ( 3 – 5 ) and meso-tetrakis-( p -sulfonato-phenyl) porphyrin ( 7 ) to saccharides and alkyl pyranosides showed strong interaction in water and DMSO. The 1 H , 31 P NMR, IR-spectroscopy and SPR technique were used for the study of mechanisms of complexation and potential using of receptors as sensor part; participant of hydrogen bonds in porphyrin–saccharide complexes formation was demonstrated.

Capillary electrochromatographic separation of aromatic amino acids possessing peptides using porphyrin derivatives as the inner wall modifiers

Two different porphyrin derivatives (H2TPP(m-OPh)4 and Rh(III)TPP(m-OPh)4) were investigated with respect to their capability to help resolution of five model aromatic peptides in capillary electrophoresis/open tubular capillary electrochromatography. Though the main separation mechanism was preferentially based on the ionic properties of the separated analytes, involvement of particularly H2TTPP(m-OPh)4-peptide interactions at alkaline pH (8.0) was clearly demonstrated. In combination with Tris-phosphate buffer, a speed up of the separation was observed at pH 2.25 (particularly if Rh(III)TPP(m-OPh)4 was used as capillary coating); in spite of the speed up of the separation the selectivity of the system was sufficient and resulted in a complete separation of the five model peptides. It can be expected that Rh(III)TPP(m-OPh)4 capillary coating in combination with Tris-phosphate buffer can be generally used for a considerable speeding up of lengthy separations of peptides in acidic media with some decrease in the separation power of the system.

Separation of structurally related peptides by open-tubular capillary electrochromatography using (metallo)porphyrins as the adsorbed stationary phase.

Several (metallo)porphyrins, particularly the porphyrin derivative tetraphenylporphyrin, and complexes of porphyrin derivatives with metal ions (Zn2+, Cu2+, Ni2+, Co2+, Co3+) have been employed as the stationary phase physically adsorbed onto the inner fused-silica capillary surface for open-tubular capillary electrochromatography, and applied for the separation of structurally related peptides. Four octapeptides, derivatives of the B23-B30 fragment of the B-chain of human insulin with minor changes in their sequences (presence of lysine or ornithine in position B-29, presence or absence of phenylacetyl protecting group on the amino group of lysine/ornithine or N-terminal amino group of glycine), were studied as model analytes. Separations were performed both in alkaline (pH 9.0) and in acidic (pH 2.25) background electrolytes, and the changes in the migration/retention behaviour of the model set of peptides were investigated with respect to the porphyrin periphery/central metal atom and the charge of the octapeptides modified. The key moment of successful separation of these peptides seems to be the accessibility of functional groups of the peptides to the interaction with the modifiers tested herein.

Capillary electrochromatographic study of the interactions of porphyrin derivatives with amino acids and oligopeptides

Open-tubular capillary electrochromatography (OT-CEC) was used to study the interactions of synthetic (metallo)porphyrin derivatives (immobilized by physical adsorption to the fused-silica capillary wall) with three aromatic amino acids (phenylalanine, tyrosine, tryptophan), three aliphatic amino acids (beta-alanine, proline, valine) and two oligopeptides (diglycine, triglycine). The effective mobilities of amino acids and peptides measured in OT-CEC mode in the acid and alkaline background electrolytes (BGEs) were compared with those obtained by capillary zone electrophoresis (CZE) in the bare fused-silica capillary in the same BGEs. In this way the influence of the peripheral substituents and the character of the central metal atom in porphyrin derivatives on the interactions with amino acids and peptides in the acid and alkaline media was investigated. Three types of noncovalent interactions, axial ligation to the central metal atom, pi-pi stacking and electrostatic repulsion seem to take part in the interactions of analyzed amino acids and peptides with porphyrin derivatives, resulting in a better separation of these analytes by OT-CEC than by CZE.

Influencing electroosmotic flow and selectivity in open tubular electrochromatography by tetrakis(pentafluorophenyl)porphyrin as capillary wall modifier.

A physically adsorbed and covalently bonded porphyrin derivative, 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin, H2TPFPP, has been used as a fused-silica capillary wall modifier in open tubular capillary electrochromatography (OT-CEC), and its influence on the electroosmotic flow (EOF) velocity and on the selectivity of OT-CEC separations of a set of model aromatic carboxylic acids has been tested. Whereas most of the coatings of this category bring about an increase in selectivity with a concomitant slow down of the EOF, H2TPFPP coating, depending on pH of the background electrolyte used, resulted both in decreasing of EOF at pH 8.5 by 5% and in increasing of EOF by 10-43% at pH 6 and 5, respectively. The separation efficiency and the resolution of aromatic carboxylic acids separation in coated capillaries, namely in that one with covalent coating, were better than in the bare fused-silica capillary. The perspectives of H2TPFPP as capillary wall modifier are visualized in introducing well defined electroosmotic properties of materials used for miniaturized separation channels preparation in chip-based electromigration devices.

Proteomics of collagen peptides: A method to reveal minor changes in post-translationally modified collagen by HPLC and capillary electrophoresis

Abstract Depository effects in slowly metabolized proteins, typically glycation or the estimation of products arising from the reaction of unsaturated long chain fatty acid metabolites (possessing aldehydic groups) are very difficult to assess owing to their extremely low concentration in the protein matrix. Posttranslational nonenzymatic modifications of collagen with sugars and oxidation products of lipid metabolism in tail tendons of two groups of rats (controls and hypertriglyceridemics with a high fructose diet) were studied. Collagen (a mixture of type I and III) was digested by bacterial collagenase and separated by reversed‐phase HPLC; the profile obtained was divided into five fractions, which were further characterizated by capillary electrophoresis (CE) in a bare fused silica capillary (37/30 cm×75 µm I.D.). As the chromatographic and electromigration behaviour of individual peptides was considerably different, the combination of HPLC and CE appeared as a suitable approach capable of acceptable separation of complex peptide mixtures (over 150 peptide peaks were obtained on the electropherograms). This two‐stepped peptide mapping with subsequent statistical evaluation (linear regression analysis) was shown to represent a reliable approach for revealing posttranslational modifications in slowly metabolized model proteins in vivo.

Open-tubular electrochromatography of organic phosphates on a sapphyrin-modified capillary

Sapphyrin coating of the inner wall of the capillary results in a distinct interaction of the phosphate residue-possessing compounds as proven by a seven-membered model mixture of nucleoside mono- and diphosphates and ATP. Modification of the inner surface of the capillary not only alters the endoosmotic flow (as would have been expected) but brings about an electrochromatographic effect based on the interaction of tested phosphate moiety-bearing solutes with the immobilized sapphyrin layer. Elution of the sample can be achieved by using either 25 mM borate-acetate buffer in which monophosphates are not only separated from each other, but also selectively separated from di- and triphosphates (ATP). With the other two buffer systems tested, i.e. borate-phosphate and Tris-HCl, better selectivity (though smaller interaction with the capillary coating) was observed. The coating is relatively stable (can be used for 20 subsequent runs at least), simple to materialize, and in spite of a strong UV absorbancy of sapphyrin at the wavelength used (254 nm), decreases the limit of detection by no more than one order of magnitude as compared to the untreated capillary. Resolution factors (calculated to the preceding peak) are in most cases better in the electrochromatographic separation mode as compared to the separation in the untreated capillary, which reflects both the decrease in the electroosmotic flow and the interaction with the capillary wall coating.

Interaction of porphyrin and sapphyrin macrocycles with nucleobases and nucleosides Spectroscopic, quantum chemical and chromatographic investigation

Novel oligopyrrole macrocycle-based sorbents were prepared via amide covalent bonding of carboxylic acids of porphyrin and sapphyrin derivatives to 3-aminopropyl silica. Covalent attachment of Cu(II)-tetraphenylporphyrin formyl-derivative was performed by amine linkage. Prepared sorbents were characterized by Raman spectroscopy and elemental analysis. Retention behavior of nucleobases and nucleosides was investigated by HPLC. UV–VIS and 1 H NMR titrations were used to study the role of oligopyrrol macrocyclic receptors for selective recognition of adenine, cytosine, thymine and uracil and their nucleoside forms. The spectroscopic results were compared with quantum chemical calculations at semiempirical level and with the data obtained by HPLC measurements. Obtained data suggest that an aromatic – stacking interactions govern the HPLC separation. A different behavior was found for purine and pyrimidine bases. An introduction of Cu(II) into porphyrin macrocycle led to a dramatic increase of separation efficiency, probably due to an increase of interaction energy.

Comparison of the electrophoretic separation of proteins in capillaries with different inner diameter

Two fused-silica capillaries of considerably different inner diameter (75 and 10 microm) were used for the separation of a set of five standard proteins. The separations were run in acid pH (50 mM phosphate buffer, pH 2.5). Generally better separations (with minor tailing only) were obtained using a standard capillary [27 cm (20 cm effective length)x 75 microm I.D.] in comparison with a narrow bore capillary [27 cm (20 cm effective length)x 10 microm I.D.]. The conditions of the electrophoretic separation were the same for both types of capillaries (25 degrees C; 10 kV; positive polarity at the inlet). The sequence of the proteins was cytochrome c, albumin, transferrin followed by a partly resolved peak of catalase and chymotrypsinogen A. In the narrow bore capillary severe tailing was observed--tailing factor ranged from 2.11 to 5.54 or 1.67 to 2.53 depending on the concentration of the analytes injected (2 or 0.2 mg/ml of each test compound injected). The relative [delta(deltaG(0))] values of the interaction with the capillary wall in the small bore capillary (with cytochrome c taken as initial standard) ranged from -0.74 to -1.04 kJ/mol. The problem of assaying the speed of the endoosmotic flow (EOF) in both capillary types was thoroughly investigated using thiourea and dithiothreitol as EOF markers. It was revealed that if thiourea is used as the EOF marker, the obtained value was dependent on the concentration of the marker injected. Optimum conditions for the EOF determination in acid buffer were specified. The higher speed of the EOF in the narrow bore capillary (10 microm) as compared to the 75 microm I.D. capillary is discussed.