Wolfgang Poppitz

A rapid and efficient microwave-assisted synthesis of hydantoins and thiohydantoins

The present paper describes studies on the synthesis of the antiepileptic drug phenytoin, and of structurally related derivatives. First, the influence of the solvent has been investigated in the microwave-assisted synthesis of the drug, resulting in a yield improvement and a cleaner reaction. Second; a two-step reaction is described to synthesize selectively and in high yields phenytoin. The first step consists in microwave activation of the reaction of benzil with thiourea, the second step includes the conversion of the resulting 2-thiohydantoin to phenytoin using hydrogen peroxide. Moreover, microwave activation is a very convenient method for the synthesis of 3-alkylated phenytoin derivatives, resulting in a much more selective method than the previously reported procedure using alkylating agents

Influence of the amino acid sequence and nature of the cyclodextrin on the separation of small peptide enantiomers by capillary electrophoresis using randomly substituted and single isomer sulfated and sulfonated cyclodextrins.

The separation of dipeptide and tripeptide enantiomers using negatively charged single isomers as well as randomly sulfated and sulfonated cyclodextrins (CDs) was investigated with respect to the amino acid sequence of the peptides and the nature of the CDs. Standardized conditions concerning buffer pH and molarity, CD concentration, and separation voltage were applied. Compared to sulfobutylether‐β‐CD and heptakis‐(2,3‐dimethyl‐6‐sulfato)‐β‐CD, randomly sulfated β‐CD as well as the single isomer derivatives heptakis‐6‐sulfato‐β‐CD and heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐CD were the more universal CDs for enantioseparations. The enantiomer migration order depended to a greater extent on the CD than on the amino acid sequence of the peptide although small structural differences such as formation of a peptide amide or ester affected the chiral recognition by the randomly substituted CD derivatives. Using sulfobutylether‐β‐CD or heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐CD the DD enantiomers migrated before the LL enantiomers for most peptides while the opposite migration order, i.e. LL before DD, was observed when heptakis‐6‐sulfato‐β‐CD was applied as chiral selector.

Influence of the amino acid sequence and nature of the cyclodextrin on the separation of small peptide enantiomers by capillary electrophoresis using α‐, β‐, and γ‐cyclodextrin and the corresponding hydroxypropyl derivatives

The separation of the LL and DD enantiomers of dipeptides and tripeptides using α-, β-, and γ-cyclodextrins as well as the corresponding hydroxypropyl derivatives was studied with respect to the amino acid sequence of the peptides and the nature of the cyclodextrins. Standardized conditions regarding buffer pH and molarity, cyclodextrin concentration, and separation voltage were applied. α-Cyclodextrin, hydroxypropyl-α-cyclodextrin, β-cyclodextrin, and hydroxypropyl-p-cyclodextrin were the more universal cyclodextrins for enantioseparations of the investigated set of peptides compared to γ-cyclodextrin and hydroxypropyl-y-cyclodextrin. The enantiomer migration order depended both on the cyclodextrin and on the amino acid sequence of the peptide. Reversal of the enantiomer migration order upon increasing the buffer pH from 2.5 to 3.5 was observed in some cases using β-cyclodextrin.

Separation of dipeptide and tripeptide enantiomers in capillary electrophoresis by the cationic cyclodextrin derivative 2‐hydroxypropyltrimethyl‐ammonium‐β‐cyclodextrin and by neutral β‐cyclodextrin derivatives at alkaline pH

The enantioseparation of dipeptides and tripeptides by the cationic CD derivative 2-Hydroxy-3-trimethylammoniumpropyl-β-cyclodextrin (TMA-β-CD) was studied under acidic and alkaline conditions. Enantioresolutions could be achieved at pH 3.5 when the peptides were partially positively charged, at pH 5.3 close to the isoelectric point of the peptides when the compounds are uncharged, and at pH 9.5 where the peptides exist as anions. Although not as effective as under acidic run conditions, native β-CD and neutral CD derivatives were able to resolve the enantiomers of several peptides at pH 9.5. Analysis of the complexation constants and the mobilities of the analyte-CD complexes at pH 9.5 in the presence of TMA-β-CD and β-CD revealed identical chiral recognition of both CDs towards Ala-Phe, Ala-Tyr, and Asp-PheOMe. Despite identical enantiomer migration order compared to the other peptides, TMA-β-CD displayed opposite chiral recognition towards the enantiomers of Ala-Trp. In contrast to the other investigated peptides, the enantiomer migration order of Ala-Trp was determined by the mobility of the CD-peptide complex which was higher in the case of the stronger complexed enantiomer.

Catechol oxidase model compounds based on aminocarbohydrates: new structure types and investigations on the catalytic reaction

Abstract Recently, we reported the structure and properties of several copper(II) complexes with aminocarbohydrate-based ligands. Four of these complexes are capable of catalyzing the oxidation of 3,5-di- t butyl -catechol (dtbc) to the corresponding quinone. The present work contains new compounds of this ligand series of which most form different structure types than the previously described. Investigations on the influence of possible inhibitors like kojic and cinnamic acid, as well as simple ligands like chloride ions and of the pH-value on the catalytic reaction are investigated.

Complexation of metal ions by pseudotripeptides with different functionalized N-alkyl residues

Seven pseudotripeptides with the common structure Bz-His-ψ[CO−N(CH2)n-X]Gly-His-NH2 were synthesized on the solid phase using the Fmoc-strategy, trityl protection for both His residues and Boc-or-OBut-protection for N-aminoalkyl-and N-carboxyalkyl residues, respectively. Functionalized N-alkyl glycyl peptides were formed on the solid phase by amination of a bromoacetyl dipeptide. All seven pseudotripeptides are able to form chelate complexes with the metal ions Zn2+, Ni2+, Cu2+ and Co2+. The existence of monomeric 1∶1 complexes for these pseudopeptides was calculated from the MW estimated by MALDI-MS and from the isotope distribution pattern estimated by ESI.

Validated capillary gas chromatographic–mass spectrometric assay to determine 2-methylcitric acid I and II levels in human serum by using a pulsed splitless injection procedure

BACKGROUND Despite some clinical applications of 2-methylcitric acid (2-MCA) determination in urine and amniotic fluid, a diagnostic use of 2-MCA levels in serum is not common practice. This could be related to the complexity of the assay, or possibly to unawareness of other feasible clinical applications. METHODS The levels of the diastereomers 2-MCA I and II in human serum were determined by GC-MS based on a method using a pulsed splitless injection technique. A stable isotope dilution principle was modified considering the diastereomer ratio and impurities of the internal standard. Precision parameters as well as recovery rates of the assay were determined. Reference intervals for 2-MCA(total), 2-MCA I and II levels were obtained in 52 healthy volunteers (31 female, 21 male, mean age 41.7+/-14.4 years). RESULTS 2-MCA was readily detected in each sample of serum, as well as in urine, cerebrospinal fluid and amniotic fluid. The limit of detection was 10 nmol/l for 2-MCA(total). The internal standard showed a diastereomer ratio of 2-MCA II-d3 to 2-MCA I-d3 of 0.83+/-0.05, its chemical purity had to be corrected to 90.5+/-0.5%. In concentrations of 446, 750 and 1256 nmol/l 2-MCA(total), recovery rates of 98.5, 93.7 and 88% with a mean intra-assay RSD of 1.5% were determined. The day-to-day precision was 10% RSD (SD 40 nmol/l) for 2-MCA(total) obtained with a pooled serum sample at a concentration of 401 nmol/l 2-MCA(total) over a period of 5 months (n=17). The normal range for 2-MCA(total) in human serum was calculated as 81-266 nmol/l confirming previous findings. CONCLUSIONS The GC-MS assay using a pulsed splitless injection procedure ensures a good response to differing concentrations of 2-MCA in various specimens. Considering exact determination of the diastereomer ratio as well as the purity of the internal standard, the assay offers good precision and recovery for 2-MCA I and II levels in serum.

Novel building blocks for oligonuclear copper complexes derived from β‐ketoenamines of histidine

Condensation products of L-histidine with the 3-oxoenolethers diethyl-ethoxymethylene-malonate (1) and ethyl-ethoxymethylene-cyanoacetate (2) react with copper(II) as di-anionic ligands to give neutral 1:1 complexes Cu-His1 and Cu-His2. Both complexes crystallize as oligonuclear units, even from strongly donating solvents like N-methylimidazole (Meim) (Cu-His1) and pyridine (Cu-His2). X-ray structure analyses show supramolecular structures, formed of two (Cu-His1) or four (Cu-His2) formula units of the complex, which arrange to macrocycles by means of intermolecular coordination of the imidazole-N. Strong H-bridges result in a face-to-face orientation of the hydrophilic sites of two great rings. ESI-MS investigations in pyridine solution give evidence for the existence of dimeric, tetrameric and – in case of Cu-His2 – trimeric units, besides the monomeric adducts with one pyridine. In contrast to the dimeric or tetrameric (“cubane-like”) copper(II) complexes of amino alcohols and their β-ketoenamines, the complexes Cu-His1 and Cu-His2 show no significant spin coupling from room temperature down to 4 K. The complexes Cu-His1 and Cu-His2 give no electrochemically reversible CuII/I reduction in pyridine. However, the isolation of a stable diamagnetic copper(I) complex of the methylester derivative, CuI-HisMe1, supports the assumption, that similar histidine-derived copper complexes should display reversible redox behaviour and catalytic activity in reactions with O2.

Estimation of pseudopeptide metal ion complexation tendencies by special MS methods, HPCE and circular dichroism

To obtain more insight into catalytic mechanisms of metallo enzymes and specific metal complexation by proteins we use linear and cyclic pseudopeptides as mimetics. Knowledge about tendencies of complex formation of different ligands with selected transition metal ions is an indispensable prerequisite for the development of homo-and hetero-dinuclear metallo enzyme mimetics. Three pseudotripeptide ligands were investigated with respect to formation tendency and properties of complexes with the transition metal ions Cu2+, Zn2+ Ni2+, Co2+ and Mn2+. To study complexation tendencies we applied different methods. One of the important prerequisites for the application in a secreening of series of peptide ligands is the necessity for a minimal amount of substance. We used and compared certain masspectrometric methods for the estimation of a rank order of complexation of certain transition metal ions. We also applied spectrophotometric titration, circular dichroism measurements, capillary electrophoresis and pH-rate profile of catalytic activity in the attempt to evaluate complex formation tendencies. Except for the spectrophotometric pH-titration and the pH-profile of catalytic activity all methods, were applicable, but each method has its advantages and disadvantages depending on the separation effect of the ligand from the metal complex, and depending on the spectroscopic properties of ligand and complex. The results regarding complex formation are compared to each other. Comparison of pairs by MALDI-TOF-and ESI-MS allows an estimation of the rank order of complexation tendency of one ligand with different metal ions and requires the least amount of substance. The other investigated methods provided additional information on structural properties of the formed complexes; however either they required too much pseudopeptide ligand or were not applicable for all transition metal ions used in this study.

Chemical and functional characterization of metal-binding pseudotripeptides with different functionalized N-alkyl residues

Pseudotripeptide ligands with 4 different N-functionalized glycine residues were qualitatively, semiquantitatively and quantitatively tested for their complexation of the bivalent transition metal ions Zn2+, Cu2+, Co2+, Ni2+ and Mn2+. The functional side chains have different length and different groups available for complexation. MALDI-MS and ESI-MS were used for more qualitative or semiquantitative estimation of the complex formation tendencies. The found ranking differs by these two methods only for Zn2+ and Ni2+. For one of the pseudotripeptide ligands, the ligand L1, complex formation with certain transition metal was estimated quantitatively by potentiometric titration. The Zn-complex of that ligand polarizes bound water strongly, resulting in a low pK a -value. Complexes of pseudotripeptide ligand L1 with certain metal ions were tested for their hydrolytic activity. The pseudo first order rate constants of the hydrolysis of the substrates 4-nitrophenyl acetate and bis(4-nitrophenyl)phosphate were compared to complexes with the same metal ions formed with a very well studied ligand from the literature, the 1,4,7,10-tetraaza cyclododecane (cyclen). The hydrolysis of the phosphate ester occurs very slowly compared to the acetate ester. No correlation exists between the estimated pK a values of complexes formed from ligand L1 with different metal ions and the phosphate ester hydrolysis. The Ni ions give totally different hydrolytic activities for pseudotripeptide ligand L1 and cyclen. With one exception, the Ni-cyclen complex, all other complexes have only a low or moderate catalytic activity.