Total Synthesis of α- and ß-Amanitin.

α-Amanitin and related amatoxins have been studied for more than six decades mostly by isolation from death cap mushrooms. The total synthesis, however, remained challenging due to unique structural features. α-Amanitin is a potent inhibitor of RNA polymerase II. Interrupting the basic transcription processes of eukaryotes leads to apoptosis of the cell. This unique mechanism makes the toxin an ideal payload for antibody-drug conjugates (ADCs). Only microgram quantities of toxins, when delivered selectively to tumor sites through conjugation to antibodies, are sufficient to eliminate malignant tumor cells of almost every origin. By solving the stereoselective access to dihydroxyisoleucine, a photochemical synthesis of the tryptathion precursor, solid-phase peptide synthesis, and macrolactamization we obtained a scalable synthetic route towards synthetic α-amanitin. This makes α-amanitin and derivatives now accessible for the development of new ADCs.

Comparison of Pro-Regenerative Effects of Carbohydrates and Protein Administrated by Shake and Non-Macro-Nutrient Matched Food Items on the Skeletal Muscle after Acute Endurance Exercise.

Physical performance and regeneration after exercise is enhanced by the ingestion of proteins and carbohydrates. These nutrients are generally consumed by athletes via whey protein and glucose-based shakes. In this study, effects of protein and carbohydrate on skeletal muscle regeneration, given either by shake or by a meal, were compared. 35 subjects performed a 10 km run. After exercise, they ingested nothing (control), a protein/glucose shake (shake) or a combination of white bread and sour milk cheese (food) in a randomized cross over design. Serum glucose (n = 35), serum insulin (n = 35), serum creatine kinase (n = 15) and myoglobin (n = 15), hematologic parameters, cortisol (n = 35), inflammation markers (n = 27) and leg strength (n = 15) as a functional marker were measured. Insulin secretion was significantly stimulated by shake and food. In contrast, only shake resulted in an increase of blood glucose. Food resulted in a decrease of pro, and stimulation of anti-inflammatory serum markers. The exercise induced skeletal muscle damage, indicated by serum creatine kinase and myoglobin, and exercise induced loss of leg strength was decreased by shake and food. Our data indicate that uptake of protein and carbohydrate by shake or food reduces exercise induced skeletal muscle damage and has pro-regenerative effects.

A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity.

BACKGROUND: Many studies of the eukaryotic transcription mechanism and its regulation rely on in vitro assays. Conventional RNA polymerase II transcription assays are based on radioactive labelling of the newly synthesized RNA. Due to the inefficient in vitro transcription, the detection of the RNA involving purification and gel electrophoresis is laborious and not always quantitative. RESULTS: Herein, we describe a new, non-radioactive, robust and reproducible eukaryotic in vitro transcription assay that has been established in our laboratory. Upon transcription, the newly synthesized RNA is directly detected and quantified using the QuantiGene assay. Alternatively, the RNA can be purified and a primer extension followed by PCR detection or qPCR quantification can be performed. When applied to assess the activity of RNA polymerase II inhibitors, this new method allowed an accurate estimation of their relative potency. CONCLUSIONS: Our novel assay provides a non-radioactive alternative to a standard in vitro transcription assay that allows for sensitive detection and precise quantification of the newly transcribed, unlabelled RNA and is particularly useful for quantification of strong transcriptional inhibitors like α-amanitin. Moreover, the method can be easily adapted to quantify the reaction yield and the transcription efficiency of other eukaryotic in vitro systems, thus providing a complementary tool for the field of transcriptional research.

Design and semisynthesis of novel fredericamycin A derivatives with an improved antitumor profile.

We report the design, semisynthesis, and biological activity of a series of fredericamycin (1) derivatives. Within this series compound 1e combines low nanomolar cytotoxic potency in vitro, increased tumor cell line selectivity, and in vivo activity in a human xenograft model.