No abstract

An HPLC assay with fluorimetric detection of the arbutin metabolites hydroquinone glucuronide (2) and hydroquinone sulphate (6) in urine was developed and validated. Methylarbutin (4) and 6 were synthesised as reference substances. Compound 2 was prepared enzymatically from hydroquinone and uridine 5'-diphosphoglucuronic acid using the glucosyltransferase system of rat liver microsomes and enriched by two liquid-liquid and an additional solid phase extraction. Compound 2 as the main component of this purified product was identified by UV and fluorescence spectroscopy, by HPLC-MS, and by enzymatic hydrolysis to hydroquinone (5). The assay yields precise and accurate urine levels of 2, 5 and 6 in the concentration range expected after oral administration of recommended therapeutic doses of bearberry leaf extract. In a preliminary pharmacokinetic study on 3 volunteers the time-dependent renal excretion of arbutin metabolites 2, 5 and 6 was investigated after ingestion of an aqueous bearberry leaf extract containing an arbutin dose recommended by the German Kommission E. More than half of the administered dose of arbutin was excreted within 4 hours mainly in form of the metabolites 2 and 6 and more than 75 % of the total applied arbutin was excreted within 24 h. The elimination of 5 was negligible in 2 out of 3 volunteers. The excretion of this metabolite in the third test person reached 5.6 % of the total administered arbutin dose. The preliminary pharmacokinetic results confirm that renal elimination of toxicologically critical concentrations of the metabolite 5 will not be expected.

The total synthesis of thymosin p4 by means of classical methods using three protected fragments is described. For their syntheses the Z/tBu strategy and temporary phenyl ester protection of the C-terminal carboxyl were used. Various coupling procedures were applied in order to optimize the yields of the synthesis. The BOP/HOBt method proved to be very efficient for the coupling of larger fragments. The fragment condensation for the synthesis of protected thymosin p4 was performed by two different strategies. The deprotected thymosin p4 was pufiried by prep. HPLC on a RP-18 column. Applying the first synthetic strategy the 43-peptide was obtained in 12% overall yleld for the final steps of the synthesis, including two fragment condensations, two hydrogenations, deprotection, and purification. The second synthetic strategy afforded thymosin p4 in 4% overall yield (based on the final synthetic steps: two fragment condensations, two hydrogenations, deprotection, and purification). The purified products of both synthetic pathways were shown to be identical with the natural thymosin p4, isolated from calf thymus tissue, according to HPLC, capillary zone electrophoresis, SDS-PAGE, ion-spray mass spectrometry, and amino acid analysis.Thymosin p4, isolated in 1981 from calf thymus tissue by Low et al."], consists of 43 amino acids and has the following amino acid sequence: Ac-Ser-Asp-Lys-Pro-Asp-Met-Ala-

As part of our total synthesis of thymosin β4 an optimized synthesis of the C-terminal part of thymosin β4 is described. Side chain functional residues of the tridecapeptide are masked by tert-butyl and the α-amino residues are protected by Z groups. The fully protected peptide derivative was obtained by WSCI/HOBt coupling of three fragments representing the segments [31-35], [36-37] and [38-43].

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