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M Bienert

Publications and source records attributed to M Bienert.

98 records · Page 6Linked to original sources
PubMedDate not supplied

Evaluation of cotton as a carrier for solid-phase peptide synthesis.

Cotton as a new support for the solid-phase synthesis of peptides was evaluated under the conditions of both Boc/Bzl and Fmoc/But strategy. Syntheses of peptides containing only bifunctional amino acids gave comparable results, but peptides containing trifunctional amino acids are clearly advantageously synthesized using the Fmoc approach. The comparison of cotton with other planar carriers (paper or polypropylene membranes) and with classical pellicular ones has shown practical applicability of cotton as the replacement of more sophisticated carriers. N-Methylimidazole was shown to be the optimal acylation catalyst for the modification of the cellulose carrier. Stability of the ester bond to the cotton was studied and shown to be adequate for the syntheses of small to medium size peptides. A relatively low level of substitution of cotton carrier in connection with its ability to soak the solvent was used for the complete elimination of a synthetic vessel. Couplings can be performed by wetting the carrier with the coupling solution, and all solvents can be removed either by squeezing it together with a porous material (paper) or by centrifugation. This leads to a significant decrease of solvent consumption. The efficiency of solvent elimination by centrifugation was demonstrated by the synthesis of model peptide ACP 65-74 in which only one wash between coupling and deprotection and between deprotection and coupling was used and which afforded the product of the same quality as the product synthesized using the standard protocol. Bromophenol blue monitoring has been proven as the optimal way of monitoring the acylation reaction progress. Peptides directly bound to cotton were shown to be useful in enzyme-linked immunosorbent assay tests.

Acylation↗
PubMedDate not supplied

Instability of side-chain protecting groups during MALDI-TOF mass spectrometry of peptide fragments.

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), a well-suited method for the characterization of peptides and proteins, was used for analysis of protected peptide fragments. It is shown that acidic matrices, e.g. 2,5-dihydroxybenzoic acid, frequently used in MALDI-TOF MS of peptides, causes partial cleavage of acid-labile side-chain protecting groups. Because this effect is strongly related to the matrix used, the observed deprotection can be avoided by choosing an appropriate matrix such as 2,4,6-trihydroxyacetophenone or 2-amino-5-nitropyridine. The advantage of neutral matrix compounds for MALDI-TOF analysis of protected peptides is clearly demonstrated, confirming the potential of MALDI-TOF mass spectrometry.

Amino Acid Sequence↗
PubMedDate not supplied

Peptide destabilization by two adjacent D-amino acids in single-stranded amphipathic alpha-helices.

We recently described the local destabilizing effect of systematic double D-amino acid replacements for characterization of amphipathic helices in peptides. The objective of this study was to determine the destabilizing effect of two adjacent D-amino acids incorporated into the center of a single-stranded amphipathic alpha-helix by hydrogen exchange and guanidine hydrochloride denaturation studies in trifluoroethanol (TFE)/water. Data from guanidine hydrochloride titration experiments in the presence of 30% TFE suggest that double D-amino acid replacements at the center of the helix destabilize the secondary structure by 4.5 kJ/mol. While the exchange rate for one backbone proton was found to vary by a factor of 10 at the replacement position, the remaining backbone protons are not markedly influenced by double D-amino acid replacement. These results confirm the hypothesis that the energy of -4.5 kJ/mol per residue is a major contribution to the stability of helical peptides in water and in solvent mixtures of TFE/water.

Amino Acid Sequence↗
PubMedDate not supplied

Mass spectrometric sequencing of synthetic peptides containing alpha, alpha-dialkylated amino acid residues by MALDI post-source decay analysis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), a method well-suited for mass determination of biomolecules, has been used to analyze fragment ions generated by post-source decay (PSD) of synthetic peptaibols containing high proportions of the sterically hindered amino acids alpha-amino isobutyric acid (Aib) and isovaline (Iva). Since peptaibols do not have a free N-terminal amino group or side chains subject to protonation, the analyzed peptides saturnisporin SA III, trichotoxin A-50 and chrysospermin B were shown to provide preferred N-terminal and C-terminal a, b, and y fragments as sodium adduct. Additionally, a cleavage of the labile Aib-Probond was observed for all peptides investigated. The fragmentation pattern allowed confirmation of the primary structure and, therefore, demonstrated the usefulness of MALDI-PSD mass spectrometry for sequence analysis of the peptaibols.

Alkylation↗