Determination of Binding Constants of Polyethylene Glycol Vancomycin Derivatives to Peptide Ligands Using Affinity Capillary Electrophoresis

Vancomycin (Van) from Streptomyces orientalis has been derivatized with polyethylene glycol [PEG; PEG-550 (1), 750 (2), 1,100 (3), 2,000 (4), 5,000 (5), and 8,000 (6) g mol⁻¹] at the N-terminus of the glycopeptide backbone and their binding to d-Ala-d-Ala terminus peptides assessed using affinity capillary electrophoresis (ACE). Utilizing ACE, a plug of Van-PEG and non-interacting standards are injected and electrophoresed. Analysis of the change in the relative migration time ratio of the Van-PEG species, relative to the non-interacting standards, as a function of the concentration of peptide, yields a value for the binding constant (K _b). Values of K _b for N-acetyl-d-Ala-d-Ala, 7 to the Van-PEG derivatives are weaker than those for N _α,N _ε-diacetyl-Lys-d-Ala-d-Ala, 8 (for example, values of K _b for 7-1 and 8-1 are 1.8 and 47.7 × 10³ M⁻¹, respectively). These results demonstrate that derivatization of Van with PEG has little effect on the affinity of d-Ala-d-Ala peptide ligands to it. The findings further prove the versatility of ACE and its ability to estimate binding parameters of ligands to antibiotics.     

Hydrogen determination by means of the1H(19F, αγ)16 O and1H(15N, αγ)12C resonance reactions

Hydrogen surface contamination and depth profiles can be measured by the resonant nuclear reactions¹H(¹⁹F, )¹⁶O and¹H(¹⁵N, )¹²C. The method was applied to study hydrogen-implanted silicon, amorphous silicon layers and silicon oxide films produced by anodic oxidation.     

Time-resolved fluorescence of the bacteriophage T4 capsid protein gp23

The time-resolved fluorescence properties of the bacteriophage T4 capsid protein gp23 are investigated. The structural characteristics of this protein are largely unknown and can be probed by recording time-resolved and decay-associated fluorescence spectra and intensity decay curves using a 200 ps-gated intensified CCD-camera. Spectral and decay data are recorded simultaneously, which makes data acquisition fast compared to time-correlated single-photon counting. A red-shift of the emission maximum within the first nanosecond of decay is observed, which can be explained by the different decay-associated spectra of fluorescence lifetimes of the protein in combination with dipolar relaxation. In addition, iodide quenching experiments are performed, to study the degree of exposure of the various tryptophan residues. A model for the origin of the observed lifetimes of 0.032 +/- 0.003, 0.39 +/- 0.06, 2.1 +/- 0.1 and 6.8 +/- 0.8 ns is presented: the 32 ps lifetime can be assigned to the emission of a buried tryptophan residue, the 0.4 and 2.1 ns lifetimes to two partly buried residues, and the 6.8 ns lifetime to a single tryptophan outside the bulk of the folded gp23.     

Die Zahl der Spitzen und die Jacobi-Algebra einer isolierten Hyperflächensingularität

Let f{x_o,...,x_n} define a germ of a complex analytic hypersurface (X_o,0) with isolated singularity. We show that the number of cusps of the unfolded discriminant curve is an invariant of the Jacobian algebra {x,_o},...,x_n/(f/x_o,...,f/x_n) of f. Moreover we show that this number + 1 equals the sum of the Milnor numbers of (X_o,0) and of the polar curve of (X_o,0). Our result generalizes formulas of Iversen and Lê for plane curves to arbitrary dimensions.     

Partial-filling affinity capillary electrophoresis

Partial-filling affinity capillary electrophoresis (PFACE) is used to examine the binding interactions between two model biological systems: D-Ala-D-Ala terminus peptides to the glycopeptide antibiotic vancomycin (Van) from Streptomyces orientalis, and arylsulfonamides to carbonic anhydrase B (CAB, EC 4.2.1.1, bovine erythrocytes). Using these two systems, modifications in the PFACE technique are demonstrated including flow-through PFACE (FTPFACE), competitive flow-through PFACE (CFTPFACE), on-column ligand synthesis PFACE (OCLSPFACE), and multiple-step ligand injection PFACE (MSLIPFACE). In PFACE small plugs of sample are injected into the capillary column and an equilibrium is established between receptor and ligand during electrophoresis. Binding constants are then obtained by Scatchard analysis using changes in the migration time of the receptor/ligand on changing the concentration of the ligand/receptor. Data demonstrating the quantitative potential of these methods are presented. This review focuses on the unique capabilities of the different PFACE techniques as applied to two model biological systems.     

Zinc(II) Hydration in Aqueous Solution: A Raman Spectroscopic Investigation and An ab initio Molecular Orbital Study of Zinc(II) Water Clusters

Raman spectra of aqueous Zn(II)–perchlorate solutions were measured over broad concentration (0.50–3.54 mol-L^–1) and temperature (25–120°C) ranges. The weak polarized band at 390 cm^–1 and two depolarized modes at 270 and 214 cm^–1 have been assigned to ₁(a _1g), ₂(e _g), and ₅(f _2g) of the zinc–hexaaqua ion. The infrared-active mode at 365 cm^–1 has been assigned to ₃(f _1u). The vibrational analysis of the species [Zn(OH₂) ₂ ⁺ ] was done on the basis of O _h symmetry (OH₂ as point mass). The polarized mode ₁(a _1g)-ZnO₆ has been followed over the full temperature range and band parameters (band maximum, full width at half height, and intensity) have been examined. The position of the ₁(a _1g)-ZnO₆ mode shifts only about 4 cm^–1 to lower frequencies and broadens by about 32 cm^–1 for a 95°C temperature increase. The Raman spectroscopic data suggest that the hexaaqua–Zn(II) ion is thermodynamically stable in perchlorate solution over the temperature and concentration range measured. These findings are in contrast to ZnSO₄ solutions, recently measured by one of us, where sulfate replaces a water molecule of the first hydration sphere. Ab initio geometry optimizations and frequency calculations of [Zn(OH₂) ₂ ⁺ ] were carried out at the Hartree–Fock and second-order Møller–Plesset levels of theory, using various basis sets up to 6-31 + G*. The global minimum structure of the hexaaqua–Zn(II) species corresponds with symmetry T _h. The unscaled vibrational frequencies of the [Zn(OH₂) ₂ ⁺ ] are reported. The unscaled vibrational frequencies of the ZnO₆, unit are lower than the experimental frequencies (ca. 15%), but scaling the frequencies reproduces the measured frequencies. The theoretical binding enthalpy for [Zn(OH₂) ₂ ⁺ ] was calculated and accounts for ca. 66% of the experimental single-ion hydration enthalpy for Zn(II).Ab initio geometry optimizations and frequency calculations are also reported for a [Zn(OH₂) ₂ ¹⁸ ] (Zn[6 + 12]) cluster with 6 water molecules in the first sphere and 12 in the second sphere. The global minimum corresponds with T symmetry. Calculated frequencies of the zinc [6 + 12] cluster correspond well with the observed frequencies in solution. The ₁-ZnO₆ (unscaled) mode occurs at 388 cm^–1 almost in perfect correspondence to the experimental value. The theoretical binding enthalpy for [Zn(OH₂) ₂ ¹⁸ ] was calculated and is very close to the experimental single ion-hydration enthalpy for Zn(II). The water molecules of the first sphere form strong hydrogen bonds with water molecules in the second hydration shell because of the strong polarizing effect of the Zn(II) ion. The importance of the second hydration sphere is discussed.     

Synthesis of a phosphine-modified cyclodextrin and its rhodium complex

A bidentate ligand consisting of a 2-(diphenylphosphinoethyl)-thio moiety was attached to β-cyclodextrin at the 6-position. The corresponding norbornadiene Rh(I) complex in the form of the BF₄⁻ salt was prepared.     

Reaktionen cyclischer Oxalylverbindungen, 34. Mitt.: Synthese von Dibenzoylacet-N-carboxyalkylamiden und semiempirische Rechnungen zur Keto-Enol Tautomerie

Summary 4-Benzoyl-5-phenylfuran-2,3-dione (1) and the urethanes2 combine under loss of carbon monoxide yielding the open chain dibenzoylacetic acid derivatives3 and4. 3a, b only can be cyclized to the oxazinone5. The keto-enole tautomerism3 4 is further investigated with aid of semiempirical quantum chemical calculations, based upon the molecular geometry of3a, deduced from an X-ray study.

Frau Univ. Prof. Dr. Helga Wittmann mit den besten Wünschen zum 70. Geburtstag gewidmet.     

Untersuchungen von Reaktionsmechanismen durch Isotopenmarkierung,IX Zum Ringverengungsmechanismus von 1,4-Benzthiazin-2,3-4H-dion

By¹⁴C-labeling it is shown that the ring contraction reaction of 1,4-benzothiazin-2,3-4H-dione (1), leading via benzthiazol-2-carbonic acid (2) finally to benzthiazole (3), proceeds via hydrolytic opening of the thiolactone-bond in1, irrespectively of H⁺ or OH^– catalysis.