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E Bayer

Publications and source records attributed to E Bayer.

At least 19 recordsLinked to original sources
PubMed1997

Conformational analysis of neuropeptide Y-[18-36] analogs in hydrophobic environments.

The interactive and conformational behavior of a series of neuropeptide Y-[18-36] (NPY-[18-36]) analogs in hydrophobic environments have been investigated using reversed-phase high-performance liquid chromatography (RP-HPLC) and circular dichroism (CD) spectroscopy. The peptides studied comprised a series of 16 analogs of NPY-[18-36], each containing a single D-amino acid substitution. The influence of these single L-->D substitutions on the alpha-helical conformation of the NPY-[18-36] analogs in different solvent environments was determined by CD spectroscopy. Retention parameters related to the hydrophobic contact area and the affinity of interaction were determined with an n-octadecyl (C18) adsorbent. Structural transitions for all peptides were manifested as significant changes in the hydrophobic binding domain and surface affinity between 4 degrees C and 37 degrees C. The results indicated that the central region of NPY-[18-36] (residues 23-33) is important for maintenance of the alpha-helical conformation. Moreover, L-->D amino acid residue substitutions within the N- and C-terminal regions, as well as Asn29 and Leu30, do not appear to affect the secondary structure of the peptide. These studies demonstrate that RP-HPLC provides a powerful adjunct for investigations into the induction of stabilized secondary structure in peptides upon their interaction with hydrophobic surfaces.

Amino Acid Sequence↗
PubMed1997

Use of Fmoc-N-(2-hydroxy-4-methoxybenzyl)amino acids in peptide synthesis.

The use of N,O-bisFmoc-N-(2-hydroxy-4-methoxybenzyl)amino acid derivatives in the synthesis of peptides with difficult sequences has already been described. With these amino acid derivatives the reversible protecting group 2-hydroxy-4-methoxybenzyl (Hmb) for the backbone amide bonds of peptide chains is introduced, and thus the aggregation due to hydrogen-bond interchain association is inhibited. This paper describes the synthesis and use of Fmoc-N-(2-hydroxy-4-methoxybenzyl)amino acid derivatives as an alternative means of introducing Hmb backbone protection. These new monoFmoc derivatives were obtained in higher yield than the bisFmoc derivatives. Coupling yields to the amino peptide resin were the same as those obtained with bisFmoc derivatives, under the TBTU HOBt/DIEA conditions. We also compared different syntheses of a difficult peptide with the Fmoc approach [triple coupling, capping, use of chaotropic agents, backbone protection using monoFmoc (Hmb)Ala] and with optimized Boc chemistry. Both the backbone protection and optimized Boc chemistry approaches gave the desired product in excellent yield and purity.

Amino Acid Sequence↗
PubMed1997

Elucidation of phase I and phase II metabolic pathways of rhein: species differences and their potential relevance.

Because of previously observed species differences in rhein tolerability, with rabbits being very susceptible to kidney disturbances, in vivo and in vitro biotransformation studies were performed to find out whether the differences in the undesired effects of rhein are associated with qualitative, species-dependent differences in its metabolism. First hints on species-dependent biotransformation profiles were obtained from in vivo experiments with 14C-labeled rhein in rat, rabbit, dog, and man. TLC-analysis of urine samples obtained after oral administration of 14C-rhein to rabbits revealed an additional, hydrophilic metabolite fraction in rabbit urine as compared with dog and human urine, all of which contain phenolic monoglucuronide and monosulfate as major metabolites. An investigation of urine samples (obtained from dogs, rabbits, rats, and human volunteers after oral application of unlabeled rhein) was conducted by means of mass spectrometric tandem techniques including on-line HPLC-MS/MS. In vitro experiments with subcellular liver fractions of rats and rabbits revealed the presence of three monohydroxylated metabolites of rhein, their quinoid oxidation products, and a bishydroxylated derivative of rhein. The hydroxylated phase I metabolites were detected as glucuronides in urine samples of all investigated species, whereas the quinoid product was present only in rabbit urine. Moreover, two regioisomeric phenolic glucuronides and sulfates or glucosides of rhein were found as major phase II metabolites in urine of all species. Furthermore, acyl glucuronides of rhein and monohydroxylated rhein and their respective isomeric acyl migration products were identified in human urine. In rabbit urine we discovered different bisglucuronides (bisphenolic glucuronide, mixed ether/ ester glucuronides), whereas in rats only the bisether/ether glucuronide was present. In addition, the investigations of dog and human urine showed the formation of two regioisomeric phenolic glucosides. With respect to a potential reactivity with endogenous macromolecules the quinoid metabolites as well as the bisester/ether glucuronides appear most relevant.

Animals↗
PubMed1995

A dodecapeptide comprising the extended chain-alpha 4 region of the restriction endonuclease EcoRI specifically binds to the EcoRI recognition site.

The restriction endonuclease EcoRI binds and cleaves DNA containing GAATTC sequences with high specificity. According to the crystal structure, most of the specific contacts of the enzyme to the DNA are formed by the extended chain region and the first turn of alpha-helix alpha 4 (amino acids 137-145). Here, we demonstrate that a dodecapeptide (WDGMAAGNAIER), which is identical in the underlined parts of its sequence to EcoRI amino acids 137-145, specifically binds to GAATTC sequences. The peptide inhibits DNA cleavage by EcoRI but not by BamHI, BclI, EcoRV, HindIII, PacI, and XbaI. DNA cleavage by XbaI is slowed down at sites that partially overlap with EcoRI sites. The peptide inhibits cleavage of GAATTC sites by ApoI, which recognizes the sequence RAATTY. It interferes with DNA methylation by the EcoRI methyltransferase but not by the BamHI methyltransferase. It competes with EcoRI for DNA binding. Based on these results, the DNA binding constant of the peptide to GAATTC sequences was calculated to be 3 x 10(4) M-1. DNA binding is not temperature-dependent, suggesting that binding of the peptide is entropy-driven. As the peptide does not show any nonspecific binding to DNA, its DNA binding specificity is similar to that of EcoRI, in spite of the fact that the affinity is much smaller. These results suggest that contacts to the phosphate groups in EcoRI mainly provide binding affinity, whereas the specificity of EcoRI is based to a large extent on sequence-specific base contacts.

Amino Acid Sequence↗
PubMed1995

Chemical phosphorylation of the peptides GGXA (X = S, T, Y): an evaluation of different chemical approaches.

An evaluation was made of the two methods most commonly used for phosphorylation of hydroxyamino acids in peptides, i.e. the tetrazole-catalysed phosphitylation by di-tert-butyl-N,N-diethylphosphoramidite followed by oxidation and the phosphorylation by dibenzylphosphochloridate. As model system the sequence GGXA (X = S, T, Y) was used which represents a random-coil sequence avoiding the influence on the reaction kinetics of secondary structure formation. In the case of serine- and threonine-containing peptides, both synthetic methods gave comparable yields of the desired phosphopeptides. The phosphorylation of tyrosine was achieved more favorably via the phosphoramidite method. However, phosphotyrosine peptides are most easily obtained by peptide synthesis using Fmoc-Tyr(PO3Me2)OH as building block. The dibenzylphosphochloridate method yields the expected phosphopeptides as the only peptide derivative and in addition, a great number of unidentified by-products which can be removed by ion-exchange chromatography. The phosphoramidite method consistently resulted in three peptide derivatives, i.e. the desired phosphopeptide, the phosphitylated peptide and a bridged derivative with two GGXA fragments linked through a phosphodiester bridge. The derivatives were characterised by RP and ion-exchange chromatography, 31P- and 1H-NMR spectroscopy, and ion-spray and electrospray mass spectrometry. Interestingly, even these mild ionisation techniques resulted in partial fragmentation. The observed fragmentation pathways seem to be a diagnostic tool for the identification of phosphorylation sites in peptides. Both the phosphorylated serine and threonine peptide lost phosphoric acid (98 mass units), the tyrosine peptide lost phenyl phosphate (174 mass units).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

N-terminal amino-acid sequence of beta-lactamase from Shigella flexneri UCSF-129.

A beta-lactamase (EC 3.5.2.6 penicillinase, penicillin amino beta-lactam-hydrolase) was purified from Shigella flexneri USCF-129 by an efficient two-stage procedure involving chromatography in Sephadex G-75 and HPLC on a C18-reverse phase column. The homogeneity of the purified enzyme was confirmed by capillary zone electrophoresis (CZE), HPLC electrospray mass spectrometry (LC-ESMS) and amino acid sequence analyses. The highly purified enzyme was a monomeric protein with a molecular mass of 28.903 +/- 2 Da, as determined by LC-ESMS. The amino acid sequence of the first 49 N-terminal residues of this beta-lactamase revealed 100% similarity with the mature forms of the plasmid coded Escherichia coli enzymes (plasmid pBR 322 and R6K) a TEM-type beta-lactamase.

Amino Acid Sequence↗
PubMed1994

Analysis of double-stranded oligonucleotides by electrospray mass spectrometry.

Double-stranded oligonucleotides of different lengths and chemical modification have been analyzed by ion spray mass spectrometry. The non-covalent-bonded duplexes can be detected. Therefore, ion spray mass spectrometry is a useful method for investigation of hybridizations of natural and chemically modified oligonucleotides. Since the exact mass of the double strand can be detected, this method can distinguish between specific and nonspecific interaction.

Base Sequence↗
PubMed1993

Optimization of capillary electrophoresis of mixtures of basic peptides and comparison with HPLC.

Fourteen synthetic, strongly basic hexadeca- to octadecapeptides, which differ only in one amino acid residue, were synthesized and characterized. In order to investigate the application of capillary electrophoresis for the separation of mixtures of closely related basic peptides, seven different mixtures, which represent possible byproducts during solid-phase synthesis, were analyzed by HPLC and different techniques of capillary electrophoresis. Peaks within the mixture were identified by single peak injection or on-line CE/MS. Utilization of micellar electrokinetic capillary electrophoresis offered the separation of complex mixtures. Comparing the electrophoretic mobilities with calculated correlations, we found that the separation of peptides of this length is influenced not only by the different size and charge but also by hydrophobicity and the orientation of the charge due to different conformations.

Amino Acid Sequence↗
PubMed1993

Separation of cardiac glycosides by micellar electrokinetic capillary electrophoresis.

The separation of mixtures of primary and secondary cardiac glycosides by micellar electrokinetic capillary electrophoresis modified by cyclodextrins, urea and sodium cholate proved to be suitable for the determination of these hydrophobic compounds. It was possible to distinguish the two anomeric cardenolides glucodigifucoside and glucodigiglucomethyloside with all three buffer systems. Electropherograms of crude plant cell extracts from Digitalis lanata were obtained with this method.

Carbohydrate Sequence↗
PubMed1993

Recognition of enantiomers by Chirasil-Val and oligopeptide analogues as studied by gas-phase calorimetry and 1H NMR spectroscopy in solution.

A series of oligopeptides, L-Valn-NHtBu (n = 1-4), were linked to poly(beta-methyl)siloxy-alpha-methylpropanoic acid copolymer; the resulting chirally modified polydimethylsiloxanes were used for the resolution of enantiomers by gas chromatography. Chiral recognition proved most effective for n = 1 (known as Chirasil-Val), as judged from the resolution factors (alpha) and the thermodynamic parameters delta delta H, delta delta S and chi = delta delta H/delta H', where delta H' = delta HChir -delta HSE30. Likewise, 1H NMR spectroscopy in carbon tetrachloride solution revealed a maximum chemical shift non-equivalence of the amide N-H signal of racemic N-TFA-amino acid methyl esters on addition of the chiral polymer, for n = 1. From circular dichroism spectroscopy of L-Valn-NH'Bu (n = 2, 3, 4 and 6) and polyoxyethylene-bound pivaloyl-L-Valn-Gly-NH-POE3000 (n = 2-8), it is concluded that the peptide moiety of the stationary phase mostly adopts the unfavourable random coil conformation, whereas the beta-sheet structure was only partially found and only for n > or = 6.

Amino Acid Sequence↗
PubMed1991

Chemically bonded phases for the reversed-phase high-performance liquid chromatographic separation of basic substances.

A chemically bonded phase with a peptide group (PB) for reversed-phase high-performance liquid chromatography (HPLC) is described. This packing was prepared by a two-stage modification of the surface of silica gel with mono- and trifunctional 3-aminopropylsilane and then with an appropriate derivative of a fatty acid. Packings prepared in this way were compared with standard C18 materials used in HPLC. Surface characteristics of the packings before and after chemical modification were determined by different physico-chemical methods, e.g., porosimetry, elemental analysis, 13C and 29Si cross-polarization magic angle spinning NMR and HPLC. Chromatographic properties of these packings were evaluated by comparison between log k' of one phase and log k' of a second phase for substances with different chemical natures. The PB packing was found to be especially useful for the separation of basic substances.

Amino Acid Sequence↗
PubMed1990

In vivo 19F nuclear magnetic resonance spectroscopy of trifluorinated neuroleptics in the rat.

In vivo 19F NMR measurements of trifluorinated neuroleptics in the rat brain were made using a 13 x 18 mm surface coil at 4.7 T. The signal of fluphenazine was obtained within 8-15 min from brains of living rats treated chronically with drug doses of 5-30 mg/kg. Following the intravenous injection of a single dose of trifluoperazine (30 mg/kg), brain levels could be monitored with a time resolution of 30 min. The data demonstrate the possibility of obtaining in vivo pharmacokinetics of fluorinated agents in the rat brain. 19F NMR is expected to become an important tool in neurochemical research.

Animals↗
PubMed1987

Generation of formaldehyde by N-demethylation of antipyrine. Detection of formaldehyde in bile by 13C-NMR spectroscopy.

The importance of NMR spectroscopy as a tool to investigate metabolic events in vitro and in vivo becomes more and more evident. Particularly 13C-NMR spectroscopy is able to deliver a wide range of information regarding the chemistry of xenobiotics in vivo. We studied the N-demethylation of N-methyl-13C-labelled antipyrine using an isolated perfused rat liver with a fluorocarbon suspension (FC 43) as oxygen carrier. Bile was collected in different fractions during the experiment. On the vascular side metabolite formation was monitored by continuous flow NMR spectroscopy. In bile the metabolic events were detected by standard NMR techniques. The bile spectra exhibit, among others, a signal at 84.2 ppm, indicating formaldehyde hydrate derived from the N-methyl group of antipyrine by an oxidative metabolic pathway. Neither formaldehyde hydrate nor other oxidation products could be detected in the vascular perfusate. The biliary excretion of considerable amounts of formaldehyde during the N-demethylation of antipyrine might have toxicological consequences for the intra- and extrahepatic bile ducts.

Animals↗
PubMed1984

Styrene metabolism in man: gas chromatographic separation of mandelic acid enantiomers in the urine of exposed persons.

D- and L-mandelic acid are separated by gas chromatography as isopropyl ester or isopropyl ester-isopropyl urethane on capillary columns, coated with Chirasil -Val. For its determination in urine the isopropyl ester procedure gives better results because other components of the urine do not cause interference. Thus D- and L-mandelic acid in the urine of exposed workers could be detected and verified by means of GC/MS. Occupational styrene exposure near the MAK-value (100 ppm) results in a L/D-enantiomer-ratio in urine of nearly 1.5.

Chromatography, Gas↗