Zyklisierungsreaktionen [alpha]-Ketoester-modifizierter Aminosäuren und Peptide

Die Modifizierung von Aminosäuren und Peptiden mit der α-Ketoesterfunktionalität bietet die Möglichkeit, eine Reihe verschiedener zyklischer Verbindungen auf unterschiedliche Art und Weise zu synthetisieren. In der vorliegenden Arbeit wird über die Herstellung, Zyklisierungsmethoden und weiterführende Reaktionen mit der α-Ketoestergruppe modifizierter Peptide sowie über mechanistische Untersuchungen berichtet. N-substituierte β-Alanine und Asparaginsäuren mit α-Ketoestermodifikation (A) bildeten auf photochemischem Weg die 3-Hydroxy-2-pyrrolidinone B (Schema A). Es konnte ein genauerer Einblick in den Mechanismus dieser reduktiven Photozyklisierung gewonnen werden. Das Keton wird mittels eines Wasserstofftransfers von der Alkoholkomponente des Esters reduziert, wobei der Alkohol zum Aldehyd oxidiert und abgespalten wird. Hinweise auf eine diradikalische Zwischenstufe und auf unterschiedliche Reaktionswege konnten ebenfalls erhalten werden. Modifizierte Asparaginsäurepeptide mit C-terminaler Aminosäure (C) zyklisierten unter basischen Bedingungen zu den γ-Lactamen D und E (Schema B). Das Verhältnis der gebildeten Diastereomere D und E hing vom sterischen Anspruch der C-terminalen Aminosäure, sowie vom Vorhandensein einer N-terminalen Aminosäure ab. Die höchste Selektivität mit 19:1 konnte beim modifizierten Tripeptid Gly-Asp*-Val beobachtet werden. Die Ausbildung einer Wasserstoffbrücke zwischen der Hydroxygruppe und dem C-terminalen Aminosäureester beim favorisierten Diastereomer D erklärt die gefundene Selektivität. Durch Änderungen der Zyklisierungsbedingungen wurde gezeigt, dass das Diastereomer E das kinetisch bevorzugte Produkt ist. Dies kann auf unterschiedlich große 1,3-diaxiale Wechselwirkungen und die Ausbildung einer Wasserstoffbrücke in den Übergangszuständen der Zyklisierung zurückgeführt werden. Die γ-Lactam-Einheit wurde in größere Peptide eingebaut, wobei modifizierte Asparaginsäure als Aminosäurederivat für Peptidkupplungen verwendet wurde. In zwei Folgeschritten ließ sich schließlich das Zyklisierungsprodukt bilden. Alternativ konnten die γ-Lactame selbst als building blocks eingesetzt werden. Auch Glutaminsäurepeptide, die mit der α-Ketoestergruppe versehen wurden, gingen Zyklisierungsreaktionen ein. Dabei wurden vorwiegend Prolinderivate erhalten, in einigen Fällen auch δ-Lactame. Von der Aminosäuresequenz abhängige Diastereoselektivitäten konnten nicht beobachtet werden. Die photochemische Umsetzung von Glutaminsäuretripeptiden mit α-Ketoestermodifikation lieferte Dehydroalaninpeptide. Die γ-Lactame G, Zyklisierungsprodukte der modifizierten Asparaginsäurepeptide F, konnten durch einen zweiten Ringschluss zu den bizyklischen Verbindungen H - K umgesetzt werden (Schema C). Dabei wurden sowohl Fünfringlactone, als auch Sechsringlactone bzw. -lactame mit dem γ-Lactam G verbunden. Die erhaltenen bizyklischen Verbindungen könnten Einsatz als Peptidmimetika finden. In der hier vorliegenden Arbeit ließ sich das große Potential von Aminosäuren und Peptiden mit α-Ketoestermodifikation für die Synthese verschiedener mono- und bizyklischer Peptidderivate zeigen. Dabei wurde ein vertiefter Einblick in zwei ungewöhnliche Ringschlussreaktionen gewonnen, die reduktive Photozyklisierung und die α-Ketoester-Amid-Zyklisierung

Bachem – Insights into Innovative and Sustainable Peptide Chemistry and Technology by the Leading Independent Manufacturer of TIDES

Since its foundation in 1971, Bachem has grown sustainably over the last 50 years and is excellently positioned as the leading company for the development and production of TIDES i.e. peptides and oligonucleotides. Bachem's success relies on its commitment to manufacturing high-quality active pharmaceutical ingredients (APIs) alongside its continual passion for innovative chemistry and technologies. This review aims at summarizing improvements in high-quality peptide manufacturing as well as recent advances towards sustainable and innovative technology in peptide chemistry, thereby reducing the environmental footprint

Lassen sich mit Social-Media-Analysen die Absatzprognosen für Vertrauensgüter verbessern? Wechselwirkungen bei der Kommunikation über Filme vor dem Kinostart

Die vorliegende Untersuchung präsentiert am Beispiel des deutschen Kinomarkts die Ergebnisse eines Screening-Ansatzes verschiedener Social-Media-Metriken und -Kanäle. Ziel der Studie ist es zum einen, jene Kommunikationskanäle zu identifizieren, die eine signifikante Absatzwirkung aufweisen. Weiterhin werden die Wechselwirkungen zwischen dem Kommunikationsumfang einzelner Kommunikationskanäle im Zeitverlauf untersucht. Zu diesem Zweck werden autokorrelative sowie kreuzverzögerte Effekte für einen Zeitraum von zwölf Wochen vor dem Kinostart synchron modelliert. Bislang werden Kommunikationsprozesse vor der Markteinführung in Erfolgsfaktorenmodellen für Medienprodukte – ebenso wie in der Diffusionsforschung generell – vernachlässigt. Der Ansatz erlaubt es, Pfadabhängigkeiten im zeitlichen Verlauf unterschiedlicher Kommunikationsangebote zu untersuchen und gibt einen Einblick in deren Wachstumsprozess. Durch Reduktion auf relevante Kommunikationsindikatoren und deren Ausprägung zu frühen Zeitpunkten lässt sich Social-Media-Kommunikation als zusätzliche erklärende Variable in Prognosemodelle für Medienprodukte und andere Vertrauensgüter integrieren.The study examines the pre-release promotional strategies of movies introduced in Germany. These strategies are analyzed with respect to various social media metrics and the channels of communication employed. It aims to identify those pre-release social media channels and indicators that significantly influence sales. Lagged and cross-lagged effects are modeled synchronously for a period of twelve weeks before the theatrical release. Until now, communication processes before the launch have been understudied in comparable success factor and diffusion models. The approach thus allows insights in path dependencies between different social media metrics and their growth process. This is of particular relevance for forecast models, as it allows focusing on relevant indicators and points in time, leading to more parsimonious models

Cdc123, a Cell Cycle Regulator Needed for eIF2 Assembly, Is an ATP-Grasp Protein with Unique Features

International audienceEukaryotic initiation factor 2 (eIF2), a heterotrimeric guanosine triphosphatase, has a central role in protein biosynthesis by supplying methionylated initiator tRNA to the ribosomal translation initiation complex and by serving as a target for translational control in response to stress. Recent work identified a novel step indispensable for eIF2 function: assembly of eIF2 from its three subunits by the cell proliferation protein Cdc123. We report the first crystal structure of a Cdc123 representative, that from Schizosaccharomyces pombe, both isolated and bound to domain III of Saccharomyces cerevisiae eIF2 gamma. The structures show that Cdc123 resembles enzymes of the ATP-grasp family. Indeed, Cdc123 binds ATP-Mg2+, and conserved residues contacting ATP-Mg2+ are essential for Cdc123 to support eIF2 assembly and cell viability. A docking of eIF2 alpha gamma onto Cdc123, combined with genetic and biochemical experiments, allows us to propose a model explaining how Cdc123 participates in the biogenesis of eIF2 through facilitating assembly of eIF2 gamma to eIF2 alpha

Stepwise assembly of the eukaryotic translation initiation factor 2 complex

The eukaryotic translation initiation factor 2 (eIF2) has key functions in the initiation step of protein synthesis. eIF2 guides the initiator tRNA to the ribosome, participates in scanning of the mRNA molecule, supports selection of the start codon, and modulates the translation of mRNAs in response to stress. eIF2 comprises a heterotrimeric complex whose assembly depends on the ATP-grasp protein Cdc123. Mutations of the eIF2γ subunit that compromise eIF2 complex formation cause severe neurological disease in humans. To this date, however, details about the assembly mechanism, step order, and the individual functions of eIF2 subunits remain unclear. Here, we quantified assembly intermediates and studied the behavior of various binding site mutants in budding yeast. Based on these data, we present a model in which a Cdc123-mediated conformational change in eIF2γ exposes binding sites for eIF2α and eIF2β subunits. Contrary to an earlier hypothesis, we found that the associations of eIF2α and eIF2β with the γ-subunit are independent of each other, but the resulting heterodimers are nonfunctional and fail to bind the guanosine exchange factor eIF2B. In addition, levels of eIF2α influence the rate of eIF2 assembly. By binding to eIF2γ, eIF2α displaces Cdc123 and thereby completes the assembly process. Experiments in human cell culture indicate that the mechanism of eIF2 assembly is conserved between yeast and humans. This study sheds light on an essential step in eukaryotic translation initiation, the dysfunction of which is linked to human disease

Towards Probing Conformational States of Y2 Receptor Using Hyperpolarized 129Xe NMR

G protein-coupled receptors can adopt many different conformational states, each of them exhibiting different restraints towards downstream signaling pathways. One promising strategy to identify and quantify this conformational landscape is to introduce a cysteine at a receptor site sensitive to different states and label this cysteine with a probe for detection. Here, the application of NMR of hyperpolarized 129Xe for the detection of the conformational states of human neuropeptide Y2 receptor is introduced. The xenon trapping cage molecule cryptophane-A attached to a cysteine in extracellular loop 2 of the receptor facilitates chemical exchange saturation transfer experiments without and in the presence of native ligand neuropeptide Y. High-quality spectra indicative of structural states of the receptor–cage conjugate were obtained. Specifically, five signals could be assigned to the conjugate in the apo form. After the addition of NPY, one additional signal and subtle modifications in the persisting signals could be detected. The correlation of the spectroscopic signals and structural states was achieved with molecular dynamics simulations, suggesting frequent contact between the xenon trapping cage and the receptor surface but a preferred interaction with the bound ligand

The C-terminal region of Net1 is an activator of RNA polymerase I transcription with conserved features from yeast to human

RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA) in all eukaryotes, accounting for the major part of transcriptional activity in proliferating cells. Although basal Pol I transcription factors have been characterized in diverse organisms, the molecular basis of the robust rRNA production in vivo remains largely unknown. In S. cerevisiae, the multifunctional Net1 protein was reported to stimulate Pol I transcription. We found that the Pol I-stimulating function can be attributed to the very C-terminal region (CTR) of Net1. The CTR was required for normal cell growth and Pol I recruitment to rRNA genes in vivo and sufficient to promote Pol I transcription in vitro. Similarity with the acidic tail region of mammalian Pol I transcription factor UBF, which could partly functionally substitute for the CTR, suggests conserved roles for CTR-like domains in Pol I transcription from yeast to human. Author summary The production of ribosomes, cellular factories of protein synthesis, is an essential process driving proliferation and cell growth. Ribosome biogenesis is controlled at the level of synthesis of its components, ribosomal proteins and ribosomal RNA. In eukaryotes, RNA polymerase I is dedicated to transcribe the ribosomal RNA. RNA polymerase I has been identified as a potential target for cell proliferation inhibition. Here we describe the C-terminal region of Net1 as an activator of RNA polymerase I transcription in baker's yeast. In the absence of this activator RNA polymerase I transcription is downregulated and cell proliferation is strongly impaired. Strikingly, this activator might be conserved in human cells, which points to a general mechanism. Our discovery will help to gain a better understanding of the molecular basis of ribosomal RNA synthesis and may have implications in developing strategies to control cellular growth

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline