Lecturer in higher education since 1990 University lecturer, associate professor Responsibilities: teaching, vocational training, training courses, writing applications, organising student practical training, study trips, conferences. Research interests: educational technology ICT applications in education Artificial intelligence in education
Careful lymph node dissection from colorectal resection specimens is important procedure for canc... more Careful lymph node dissection from colorectal resection specimens is important procedure for cancer staging. Present study intended to assess the impact of surgical technique and patient’s obesity on this process. Number of lymph nodes harvested by manual dissection from resection specimens of 141 patients with rectal cancer and the rate of nodal metastases were analyzed and compared in different groups of patients selected by length of resection specimen and body mass index. The median and mean number of lymph nodes found per patient were 6 and 6,7. The shorter resection specimens (≤16 cm after formalin fixation) yielded significantly lower number of nodes than those with length > 16 cm (5.7 versus 7.9). Most significant reduction in mean number of lymph nodes was observed in obese patients with short specimens (4.8). This subset of patients presented the lowest rate of nodal metastases (38%). The surgical technique seems to be an important factor for lymph node recovery from rectal resections specimens. The patient’s obesity had an unfavourable impact on this procedure. Standardized surgery and histopathological examination are needed even in non-specialized centers to harvest adequate number of lymph nodes.
Abstract The measurement of enzyme activity in urine provides a sensitive assessment for renal t... more Abstract The measurement of enzyme activity in urine provides a sensitive assessment for renal tubular cell damage. The present study was undertaken to evaluate the clinical value of the determination of tubular brush-border-associated enzymes, alkaline phosphatase (AP), gamma-glutamyl transferase (GGT), leucine aminopeptidase (LAP), and dipeptidyl peptidase IV (DPP), of patients with normal graft function (NOR, n= 20), with acute tubular necrosis (ATN, n= ll), with an acute rejection episode (ARE, n= 17) after transplantation, and of healthy persons (n=20). The second urine of the morning was collected daily during the patients' stay in hospital. The enzyme activities were measured at 25 °C and were expressed as U/mmol creatinine. The enzymuria in NOR is higher than in healthy controls, but is still in the normal range. By 5 days after transplantation the initial increased excretion declines as the graft function improves. Elevated enzymuria (DPP 0.69 ± 0.56, AP 3.06 ± 3.24, GGT 4.16 ± 4.13, and LAP 1.39 ± 1.27) was observed during the rejection episodes. Two days before clinical diagnosis of rejection, the release of DPP-IV and GGT increases to double, and the AP and LAP increases to 3 times the value on the fourth day before rejection. Successful treatment of rejection coincided with a quick return by the third day of the rejection period to the previous enzyme distribution. In ATN no decrease of enzymuria occurs and the excretion is much higher than in ARE. Our method with the every day monitoring of kidney graft function offers the possibility for the early diagnosis of acute rejection.
We describe here a gradient HPLC procedure for the separation, and quantification by UV absorptio... more We describe here a gradient HPLC procedure for the separation, and quantification by UV absorption of renin tri- and tetradecapeptide substrates, angiotensins I, II, III, IV and V, angiotensin-derived peptides, and peptidase inhibitors including amastatin, bestatin, pepstatin, lisinopril, a renin peptide inhibitor, Z-Pro-prolinal, N-[1-(R,S)-carboxy-2-phenylethyl]-L-Ala-L-Ala-L-Phe-p-aminobenzoate, and phosphoramidon. Most peptides and peptidase inhibitors were baseline-resolved within 32 min. The overall intra- and inter-assay precisions ranged from 0.8 to 5.9 (n=6) and 2 to 13% (n=6), respectively. There was a linear relationship (correlation coefficients> or =0.9660) between peak height and peptide amount injected. In conclusion, the present method when combined with a peptidase-inhibitor paradigm can lead to the identification of renin-angiotensin system metabolizing enzymes, and when combined with radioimmunoassay can enhance the specificity of angiotensin measurement.
Direct and indirect reversed-phase high-performance liquid chromatographic methods were developed... more Direct and indirect reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of seventeen unnatural β-amino acids, including several β-3-homo amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases containing macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T column) and teicoplanin aglycone (Chirobiotic TAG column). The indirect method involved pre-column derivatization with two new chiral derivatizing agents, (1S,2S)-1,3-diacetoxy-1-(4-nitrophenyl)-2-propylisothiocyanate, (S,S)-DANI and (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester, (S)-NIFE. The different methods were compared in systematic chromatographic examinations. The effects of organic modifier, mobile phase composition, pH and flow rate on the separation were investigated.
Direct reversed-phase high-performance liquid chromatographic methods were developed for the sepa... more Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of eighteen unnatural β-amino acids, including several β-3-homo-amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases containing macrocyclic glycopeptide antibiotics such as teicoplanin (Chirobiotic T and T2), teicoplanin aglycone (Chirobiotic TAG), vancomycin (Chirobiotic V and V2), and ristocetin A (Chirobiotic R) as chiral selectors. The effects of the organic modifier, mobile phase composition and pH on the separations were investigated. A comparison of the separation performances of the macrocyclic glycopeptide stationary phases revealed that the Chirobiotic T2 column exhibited better selectivity than the Chirobiotic T column for the separation of β-3-homo-amino acid enantiomers; vancomycin or ristocetin A exhibited lower selectivity. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers, with the exception of the Chirobiotic R column, where the elution sequence R was observed.
The delta-opioid antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP-OH) or its C-terminal amide analogue was s... more The delta-opioid antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP-OH) or its C-terminal amide analogue was systematically modified topologically by substitution of each amino acid residue by all stereoisomers of the corresponding beta-methyl amino acid. The potency and selectivity (delta- vs mu- and kappa-opioid receptor) were evaluated by radioreceptor binding assays. Agonist or antagonist potency were assayed in the mouse vas deferens and in the guinea pig ileum. In the TIPP analogues containing L-beta-methyl amino acids the influence on delta-receptor affinity and on delta-antagonist potency is limited, the [(2S,3R)-beta-MePhe3]TIPP-OH analogue being among the most potent delta-antagonists reported. In the D-beta-methyl amino acid series, the [D-beta-MeTic2] analogues are delta-selective antagonists whereas [D-Tic2]TIPP-NH2 is a delta-agonist. NMR studies did not indicate any influence of the beta-methyl substituent on the conformation of the Tic residue. The [(2R,3S)-beta-MePhe3]TIPP-NH2 is a potent delta-agonist, its C-terminal carboxylic acid analogue being more delta-selective but displaying partial agonism in both the delta- and mu-bioassay. These results constitute further examples of a profound influence of beta-methyl substitution on the potency, selectivity, and signal transduction properties of a peptide.
The isocratic retention of enantiomers of beta-methyl amino acids (beta-methyltyrosine, beta-meth... more The isocratic retention of enantiomers of beta-methyl amino acids (beta-methyltyrosine, beta-methylphenylalanine, beta-methyl-tryptophan and beta-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was studied on a teicoplanin-containing chiral stationary phase at different temperatures and at different mobile phase compositions, using the reversed-phase mode. With variation of both mobile phase composition and temperature, almost baseline separations could be achieved for all four enantiomers of sterically hindered amino acids. The retention factors and selectivity factors for the enantiomers of all investigated compounds decreased with increasing temperature. The natural logarithms of the retention factors (ln k) of the investigated compounds depended linearly on the inverse of temperature (1/T). van 't Hoff plots afforded thermodynamic parameters, such as the apparent change in enthalpy (delta H degree), the apparent change in entropy (delta S degree) and the apparent change in Gibbs free energy (delta G degree) for the transfer of analyte from the mobile to the stationary phase. The thermodynamic constants (delta H degree, delta S degree and delta G degree) were calculated in order to promote an understanding of the thermodynamic driving forces for retention in this chromatographic system.
The search for new and effective chiral selectors capable of separating a wide variety of enantio... more The search for new and effective chiral selectors capable of separating a wide variety of enantiomeric compounds is an ongoing process. In the past decade, macrocyclic antibiotics have proved to be an exceptionally useful class of chiral selectors for the separation of enantiomers of biological and pharmacological importance by means of HPLC, TLC and electrophoresis. More chiral analytes have been resolved through the use of glycopeptides than with all the other macrocyclic antibiotics combined (ansamycins, thiostrepton, aminoglycosides, etc.). The glycopeptides avoparcin, teicoplanin, ristocetin A and vancomycin have been extensively used as chiral selectors in the form of chiral bonded phases in HPLC, and HPLC stationary phases based on these glycopeptides have been commercialized. Teicoplanin, vancomycin, their analogs and ristocetin A seem to be the most useful glycopeptide HPLC bonded phases for the enantioseparation of proteins and unusal native and derivatized amino acids. In fact, the macrocyclic glycopeptides are to some extent complementary to one another: where partial enantioresolution is obtained with one glycopeptide, there is a high probability that baseline or better separation can be obtained with another. This review sets out to characterize the physicochemical properties of these antibiotics and their application in the enantioseparations of amino acids. The mechanism of separation, the sequence of elution of the stereoisomers and the relation to the absolute configuration are also discussed.
Endomorphins-1 and -2 were substituted with all the beta-MePhe stereoisomers in their Phe residue... more Endomorphins-1 and -2 were substituted with all the beta-MePhe stereoisomers in their Phe residues to generate a conformationally constrained peptide set. This series of molecules was subjected to biological assays, and for beta-MePhe(4)-endomorphins-2, a conformational analysis was performed. Incorporation of (2S,3S)-beta-MePhe(4) resulted in the most potent analogues of both endomorphins with enhanced enzymatic stability. Their micro opioid affinities were 4-times higher than the parent peptides, they stimulated [(35)S]GTPgammaS binding, and they were found to be full agonists. NMR experiments revealed that C-terminal (2S,3S)-beta-MePhe in endomorphin-2 strongly favored the gauche (-) spatial orientation which implies the presence of the chi(1) = -60 degrees rotamer of Phe(4) in the binding conformer of endomorphins. Our results emphasize that the appropriate orientation of the C-terminal aromatic side chain of endomorphins is substantial for binding to the micro opioid receptor.
The recently discovered native endomorphins play an important role in opioid analgesia, but their... more The recently discovered native endomorphins play an important role in opioid analgesia, but their metabolic fate in the organism remains relatively little known. This paper describes the application of high-performance liquid chromatography combined with electrospray ionization mass spectrometry to identify the degradation products resulting from the incubation of endomorphins with proteolytic enzymes. The native endomorphin-1, H-Tyr-Pro-Trp-Phe-NH2 (1), and endomorphin-2, H-Tyr-Pro-Phe-Phe-NH2 (2), and an analog of endomorphin-2, H-Tyr-Pro-Phe-Phe-OH (3), were synthetized, and the levels of their resistance against carboxypeptidase A, carboxypeptidase Y, aminopeptidase M and proteinase A were determined. The patterns of peptide metabolites identified by this method indicated that carboxypeptidase Y first hydrolyzes the C-terminal amide group to a carboxy group, and then splits the peptides at the Trp3-Phe4 or Phe3-Phe4 bond. The remaining fragment peptides are stable against the enzymes investigated. Carboxypeptidase A degrades only analog 3 at the Phe3-Phe4 bond. Aminopeptidase M cleaves the peptides at the Pro2-Trp3 or Pro2-Phe3 bond. The C-terminal fragments hydrolyze further, giving amino acids and Phe-NH2-s while the N-terminal part displays a resistance to further aminopeptidase M digestion. Proteinase A exhibits a similar effect to carboxypeptidase Y: the C-terminal amide group is first converted to a carboxy group, and one amino acid is then split off from the C-terminal side.
Two approaches to the design of very active and highly selective delta opioid peptides were used ... more Two approaches to the design of very active and highly selective delta opioid peptides were used to obtain new deltorpin analogs with altered hydrophobic and stereoelectronic properties. Deltorphin I and II analogs were synthesized involving the substitution of Ile instead of Val at positions 5 and 6 in the address domain and 2-aminotetralin-2-carboxylic acid (Atc) instead of Phe in the message domain. The peptides were agonists in the subnanomolar range in the MVD assay and in the micromolar or higher range in the GPI assay, showing a very high selectivity for delta receptors. A very similar trend could be observed in radioreceptor binding assays in which selective tritiated opioid ligands were used. (R)- and (S)-Atc-deltoriphins exhibited similar Ki values in the binding assay, with almost complete loss of the stereospecificity of the binding. Conformational studies provided evidence for the little disturbance of the backbone conformational equilibrium when Phe3 is replaced by (S)- or (R)-Atc. The use of the Atc constraint gives additional evidence that, during its interaction with the delta receptor, the side chain of residue 3 adopts the trans conformation at chi 1.
The application of(S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester,(S)-NIFE, as a n... more The application of(S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester,(S)-NIFE, as a new chiral derivatizing agent for the resolution of compounds possessing an amino group is described. Its applicability is demonstrated by the resolution of proteinogenic amino acid enantiomers. The diastereomeric derivatives produced were separated by reversedphase high-performance liquid chromatography. The effects of pH, excess reagent and reaction time on the derivatization kinetics, and the effects of pH and the organic modifier on the separation, were investigated.
... Antal PØter1 Erika VØkes1 Anita A´ rki1 Dirk TourwØ2 Wolfgang Lindner3 ... The direct HPLC me... more ... Antal PØter1 Erika VØkes1 Anita A´ rki1 Dirk TourwØ2 Wolfgang Lindner3 ... The direct HPLC method relies on the use of a unique quinine-derived weak anion-exchanger (WAX) type CSP (Fig-ure 2). The amino acids analysed were used in N-2,4-dini-trophenyl (N-2,4-DNP ...
Careful lymph node dissection from colorectal resection specimens is important procedure for canc... more Careful lymph node dissection from colorectal resection specimens is important procedure for cancer staging. Present study intended to assess the impact of surgical technique and patient’s obesity on this process. Number of lymph nodes harvested by manual dissection from resection specimens of 141 patients with rectal cancer and the rate of nodal metastases were analyzed and compared in different groups of patients selected by length of resection specimen and body mass index. The median and mean number of lymph nodes found per patient were 6 and 6,7. The shorter resection specimens (≤16 cm after formalin fixation) yielded significantly lower number of nodes than those with length > 16 cm (5.7 versus 7.9). Most significant reduction in mean number of lymph nodes was observed in obese patients with short specimens (4.8). This subset of patients presented the lowest rate of nodal metastases (38%). The surgical technique seems to be an important factor for lymph node recovery from rectal resections specimens. The patient’s obesity had an unfavourable impact on this procedure. Standardized surgery and histopathological examination are needed even in non-specialized centers to harvest adequate number of lymph nodes.
Abstract The measurement of enzyme activity in urine provides a sensitive assessment for renal t... more Abstract The measurement of enzyme activity in urine provides a sensitive assessment for renal tubular cell damage. The present study was undertaken to evaluate the clinical value of the determination of tubular brush-border-associated enzymes, alkaline phosphatase (AP), gamma-glutamyl transferase (GGT), leucine aminopeptidase (LAP), and dipeptidyl peptidase IV (DPP), of patients with normal graft function (NOR, n= 20), with acute tubular necrosis (ATN, n= ll), with an acute rejection episode (ARE, n= 17) after transplantation, and of healthy persons (n=20). The second urine of the morning was collected daily during the patients' stay in hospital. The enzyme activities were measured at 25 °C and were expressed as U/mmol creatinine. The enzymuria in NOR is higher than in healthy controls, but is still in the normal range. By 5 days after transplantation the initial increased excretion declines as the graft function improves. Elevated enzymuria (DPP 0.69 ± 0.56, AP 3.06 ± 3.24, GGT 4.16 ± 4.13, and LAP 1.39 ± 1.27) was observed during the rejection episodes. Two days before clinical diagnosis of rejection, the release of DPP-IV and GGT increases to double, and the AP and LAP increases to 3 times the value on the fourth day before rejection. Successful treatment of rejection coincided with a quick return by the third day of the rejection period to the previous enzyme distribution. In ATN no decrease of enzymuria occurs and the excretion is much higher than in ARE. Our method with the every day monitoring of kidney graft function offers the possibility for the early diagnosis of acute rejection.
We describe here a gradient HPLC procedure for the separation, and quantification by UV absorptio... more We describe here a gradient HPLC procedure for the separation, and quantification by UV absorption of renin tri- and tetradecapeptide substrates, angiotensins I, II, III, IV and V, angiotensin-derived peptides, and peptidase inhibitors including amastatin, bestatin, pepstatin, lisinopril, a renin peptide inhibitor, Z-Pro-prolinal, N-[1-(R,S)-carboxy-2-phenylethyl]-L-Ala-L-Ala-L-Phe-p-aminobenzoate, and phosphoramidon. Most peptides and peptidase inhibitors were baseline-resolved within 32 min. The overall intra- and inter-assay precisions ranged from 0.8 to 5.9 (n=6) and 2 to 13% (n=6), respectively. There was a linear relationship (correlation coefficients> or =0.9660) between peak height and peptide amount injected. In conclusion, the present method when combined with a peptidase-inhibitor paradigm can lead to the identification of renin-angiotensin system metabolizing enzymes, and when combined with radioimmunoassay can enhance the specificity of angiotensin measurement.
Direct and indirect reversed-phase high-performance liquid chromatographic methods were developed... more Direct and indirect reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of seventeen unnatural β-amino acids, including several β-3-homo amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases containing macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T column) and teicoplanin aglycone (Chirobiotic TAG column). The indirect method involved pre-column derivatization with two new chiral derivatizing agents, (1S,2S)-1,3-diacetoxy-1-(4-nitrophenyl)-2-propylisothiocyanate, (S,S)-DANI and (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester, (S)-NIFE. The different methods were compared in systematic chromatographic examinations. The effects of organic modifier, mobile phase composition, pH and flow rate on the separation were investigated.
Direct reversed-phase high-performance liquid chromatographic methods were developed for the sepa... more Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of eighteen unnatural β-amino acids, including several β-3-homo-amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases containing macrocyclic glycopeptide antibiotics such as teicoplanin (Chirobiotic T and T2), teicoplanin aglycone (Chirobiotic TAG), vancomycin (Chirobiotic V and V2), and ristocetin A (Chirobiotic R) as chiral selectors. The effects of the organic modifier, mobile phase composition and pH on the separations were investigated. A comparison of the separation performances of the macrocyclic glycopeptide stationary phases revealed that the Chirobiotic T2 column exhibited better selectivity than the Chirobiotic T column for the separation of β-3-homo-amino acid enantiomers; vancomycin or ristocetin A exhibited lower selectivity. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers, with the exception of the Chirobiotic R column, where the elution sequence R was observed.
The delta-opioid antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP-OH) or its C-terminal amide analogue was s... more The delta-opioid antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP-OH) or its C-terminal amide analogue was systematically modified topologically by substitution of each amino acid residue by all stereoisomers of the corresponding beta-methyl amino acid. The potency and selectivity (delta- vs mu- and kappa-opioid receptor) were evaluated by radioreceptor binding assays. Agonist or antagonist potency were assayed in the mouse vas deferens and in the guinea pig ileum. In the TIPP analogues containing L-beta-methyl amino acids the influence on delta-receptor affinity and on delta-antagonist potency is limited, the [(2S,3R)-beta-MePhe3]TIPP-OH analogue being among the most potent delta-antagonists reported. In the D-beta-methyl amino acid series, the [D-beta-MeTic2] analogues are delta-selective antagonists whereas [D-Tic2]TIPP-NH2 is a delta-agonist. NMR studies did not indicate any influence of the beta-methyl substituent on the conformation of the Tic residue. The [(2R,3S)-beta-MePhe3]TIPP-NH2 is a potent delta-agonist, its C-terminal carboxylic acid analogue being more delta-selective but displaying partial agonism in both the delta- and mu-bioassay. These results constitute further examples of a profound influence of beta-methyl substitution on the potency, selectivity, and signal transduction properties of a peptide.
The isocratic retention of enantiomers of beta-methyl amino acids (beta-methyltyrosine, beta-meth... more The isocratic retention of enantiomers of beta-methyl amino acids (beta-methyltyrosine, beta-methylphenylalanine, beta-methyl-tryptophan and beta-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was studied on a teicoplanin-containing chiral stationary phase at different temperatures and at different mobile phase compositions, using the reversed-phase mode. With variation of both mobile phase composition and temperature, almost baseline separations could be achieved for all four enantiomers of sterically hindered amino acids. The retention factors and selectivity factors for the enantiomers of all investigated compounds decreased with increasing temperature. The natural logarithms of the retention factors (ln k) of the investigated compounds depended linearly on the inverse of temperature (1/T). van 't Hoff plots afforded thermodynamic parameters, such as the apparent change in enthalpy (delta H degree), the apparent change in entropy (delta S degree) and the apparent change in Gibbs free energy (delta G degree) for the transfer of analyte from the mobile to the stationary phase. The thermodynamic constants (delta H degree, delta S degree and delta G degree) were calculated in order to promote an understanding of the thermodynamic driving forces for retention in this chromatographic system.
The search for new and effective chiral selectors capable of separating a wide variety of enantio... more The search for new and effective chiral selectors capable of separating a wide variety of enantiomeric compounds is an ongoing process. In the past decade, macrocyclic antibiotics have proved to be an exceptionally useful class of chiral selectors for the separation of enantiomers of biological and pharmacological importance by means of HPLC, TLC and electrophoresis. More chiral analytes have been resolved through the use of glycopeptides than with all the other macrocyclic antibiotics combined (ansamycins, thiostrepton, aminoglycosides, etc.). The glycopeptides avoparcin, teicoplanin, ristocetin A and vancomycin have been extensively used as chiral selectors in the form of chiral bonded phases in HPLC, and HPLC stationary phases based on these glycopeptides have been commercialized. Teicoplanin, vancomycin, their analogs and ristocetin A seem to be the most useful glycopeptide HPLC bonded phases for the enantioseparation of proteins and unusal native and derivatized amino acids. In fact, the macrocyclic glycopeptides are to some extent complementary to one another: where partial enantioresolution is obtained with one glycopeptide, there is a high probability that baseline or better separation can be obtained with another. This review sets out to characterize the physicochemical properties of these antibiotics and their application in the enantioseparations of amino acids. The mechanism of separation, the sequence of elution of the stereoisomers and the relation to the absolute configuration are also discussed.
Endomorphins-1 and -2 were substituted with all the beta-MePhe stereoisomers in their Phe residue... more Endomorphins-1 and -2 were substituted with all the beta-MePhe stereoisomers in their Phe residues to generate a conformationally constrained peptide set. This series of molecules was subjected to biological assays, and for beta-MePhe(4)-endomorphins-2, a conformational analysis was performed. Incorporation of (2S,3S)-beta-MePhe(4) resulted in the most potent analogues of both endomorphins with enhanced enzymatic stability. Their micro opioid affinities were 4-times higher than the parent peptides, they stimulated [(35)S]GTPgammaS binding, and they were found to be full agonists. NMR experiments revealed that C-terminal (2S,3S)-beta-MePhe in endomorphin-2 strongly favored the gauche (-) spatial orientation which implies the presence of the chi(1) = -60 degrees rotamer of Phe(4) in the binding conformer of endomorphins. Our results emphasize that the appropriate orientation of the C-terminal aromatic side chain of endomorphins is substantial for binding to the micro opioid receptor.
The recently discovered native endomorphins play an important role in opioid analgesia, but their... more The recently discovered native endomorphins play an important role in opioid analgesia, but their metabolic fate in the organism remains relatively little known. This paper describes the application of high-performance liquid chromatography combined with electrospray ionization mass spectrometry to identify the degradation products resulting from the incubation of endomorphins with proteolytic enzymes. The native endomorphin-1, H-Tyr-Pro-Trp-Phe-NH2 (1), and endomorphin-2, H-Tyr-Pro-Phe-Phe-NH2 (2), and an analog of endomorphin-2, H-Tyr-Pro-Phe-Phe-OH (3), were synthetized, and the levels of their resistance against carboxypeptidase A, carboxypeptidase Y, aminopeptidase M and proteinase A were determined. The patterns of peptide metabolites identified by this method indicated that carboxypeptidase Y first hydrolyzes the C-terminal amide group to a carboxy group, and then splits the peptides at the Trp3-Phe4 or Phe3-Phe4 bond. The remaining fragment peptides are stable against the enzymes investigated. Carboxypeptidase A degrades only analog 3 at the Phe3-Phe4 bond. Aminopeptidase M cleaves the peptides at the Pro2-Trp3 or Pro2-Phe3 bond. The C-terminal fragments hydrolyze further, giving amino acids and Phe-NH2-s while the N-terminal part displays a resistance to further aminopeptidase M digestion. Proteinase A exhibits a similar effect to carboxypeptidase Y: the C-terminal amide group is first converted to a carboxy group, and one amino acid is then split off from the C-terminal side.
Two approaches to the design of very active and highly selective delta opioid peptides were used ... more Two approaches to the design of very active and highly selective delta opioid peptides were used to obtain new deltorpin analogs with altered hydrophobic and stereoelectronic properties. Deltorphin I and II analogs were synthesized involving the substitution of Ile instead of Val at positions 5 and 6 in the address domain and 2-aminotetralin-2-carboxylic acid (Atc) instead of Phe in the message domain. The peptides were agonists in the subnanomolar range in the MVD assay and in the micromolar or higher range in the GPI assay, showing a very high selectivity for delta receptors. A very similar trend could be observed in radioreceptor binding assays in which selective tritiated opioid ligands were used. (R)- and (S)-Atc-deltoriphins exhibited similar Ki values in the binding assay, with almost complete loss of the stereospecificity of the binding. Conformational studies provided evidence for the little disturbance of the backbone conformational equilibrium when Phe3 is replaced by (S)- or (R)-Atc. The use of the Atc constraint gives additional evidence that, during its interaction with the delta receptor, the side chain of residue 3 adopts the trans conformation at chi 1.
The application of(S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester,(S)-NIFE, as a n... more The application of(S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester,(S)-NIFE, as a new chiral derivatizing agent for the resolution of compounds possessing an amino group is described. Its applicability is demonstrated by the resolution of proteinogenic amino acid enantiomers. The diastereomeric derivatives produced were separated by reversedphase high-performance liquid chromatography. The effects of pH, excess reagent and reaction time on the derivatization kinetics, and the effects of pH and the organic modifier on the separation, were investigated.
... Antal PØter1 Erika VØkes1 Anita A´ rki1 Dirk TourwØ2 Wolfgang Lindner3 ... The direct HPLC me... more ... Antal PØter1 Erika VØkes1 Anita A´ rki1 Dirk TourwØ2 Wolfgang Lindner3 ... The direct HPLC method relies on the use of a unique quinine-derived weak anion-exchanger (WAX) type CSP (Fig-ure 2). The amino acids analysed were used in N-2,4-dini-trophenyl (N-2,4-DNP ...
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