Granulocyte colony stimulating factor (G-CSF) is a cytokine used to treat neutropenia. Different glycosylated and non-glycosylated variants of G-CSF for therapeutic application are currently generated by recombinant expression. Here, we describe our approaches to establish a first semisynthesis strategy to access the aglycone and O-glycoforms of G-CSF, thereby enabling the preparation of selectively and homogeneously post-translationally modified variants of this important cytokine. Eventually, we succeeded by combining selenocysteine ligation of a recombinantly produced N-terminal segment with a synthetic C-terminal part, transiently equipped with a side-chain-linked, photocleavable PEG moiety, at low concentration. The transient PEGylation enabled quantitative enzymatic elongation of the carbohydrate at Thr133. Overall, we were able to significantly reduce the problems related to the low solubility and the tendency to aggregate of the two protein segments, which allowed the preparation of four G-CSF variants that were successfully folded and demonstrated biological activity in cell proliferation assays.

Diselenide–selenoester ligations are increasingly used for the synthesis of proteins. Excellent ligation rates, even at low concentrations, in combination with mild and selective deselenization conditions can overcome some of the most severe challenges in chemical protein synthesis. Herein, the versatile multicomponent synthesis and application of a new ligation auxiliary that combines a photocleavable scaffold with the advantages of selenium‐based ligation strategies are presented. Its use was investigated with respect to different ligation junctions and describe a novel para‐methoxybenzyl deprotection reaction for the selenol moiety. The glycine‐based auxiliary enabled successful synthesis of the challenging target protein G‐CSF.

Der Granulozyten-Kolonie-stimulierende Faktor (G-CSF) ist ein Zytokin, das zur Behandlung von Neutropenie eingesetzt wird. Unterschiedlich glykosylierte und nicht glykosylierte Varianten von G-CSF werden für therapeutische Anwendungen derzeit durch rekombinante Expression hergestellt. Im Folgenden beschreiben wir unsere Herangehensweise zur Etablierung einer ersten Semisynthesestrategie für die Synthese des Aglykons und verschiedener O-Glykoformen von G-CSF, um die Herstellung von selektiv und homogen posttranslational modifizierten Varianten dieses wichtigen Zytokins zu ermöglichen. Letztendlich erreichten wir dies, durch die Selenoesterligation eines rekombinant hergestellten N-terminalen Segments mit einem synthetisch hergestellten C-terminalen Segment, welches vorübergehend mit einer lichtabspaltbaren Polyethylenglykol-(PEG) Gruppe an einer Seitenkette versehen wurde, bei niedrigen Konzentrationen. Die transiente PEGylierung ermöglichte dabei die quantitative enzymatische Verlängerung des Kohlenhydrates an Thr133. Insgesamt ist es uns gelungen, die Syntheseherausforderungen auf Grund der niedrigen Löslichkeit und der Aggregationstendenz der zwei Proteinsegmente, signifikant zu reduzieren. Dies ermöglichte uns die Herstellung von vier G-CSF Varianten, die erfolgreich gefaltet wurden und in Zellproliferationsexperimenten biologische Aktivität zeigten.

The physical parameters of point defects in silicon at 1100~ are obtained by the simulation of the back-side oxidation (BSO) experiments of Mizuo and Higuchi. The simulation yielded the equilibrium concentrations and diffusion coefficients of interstitials and vacancies at 1100~ Furthermore it is seen from this analysis that the nitride layers used in the BSO experiments do not absorb any point defects. The point defects are found to penetrate the silicon bulk without loss by interaction with imperfections. The oversaturation of interstitiais and undersaturation of vacancies are obtained by fitting the OED and ORD experiments of Mizuo and Higuehi. From the consideration of diffusion under Si3N4 layers in comparison to the diffusion under oxide layers a relation between the equilibrium concentrations of vacancies and interstitials has been obtained. These have been used in the simulations together with the results from gold diffusion experiments concerning the contributions to self-diffusion originating from interstitials and vacancies. The resulting set of physical parameters obtained from 1100~ (c~ q = 1.3 -1016 cm -3, c~ q = 9.1 -1015 cm -3, DI = 6.0 9 10 -0 cm 2 s -1, Dv = 5.3 -10 9 cm 2 s-l) is uniquely determined.

Post‐translational modifications affect protein biology under physiological and pathological conditions. Efficient methods for the preparation of peptides and proteins carrying defined, homogeneous modifications are fundamental tools for investigating these functions. In the case of mucin 1 (MUC1), an altered glycosylation pattern is observed in carcinogenesis. To better understand the role of MUC1 glycosylation in the interactions and adhesion of cancer cells, we prepared a panel of homogeneously O‐glycosylated MUC1 peptides by using a quantitative chemoenzymatic approach. Cell‐adhesion experiments with MCF‐7 cancer cells on surfaces carrying up to six differently glycosylated MUC1 peptides demonstrated that different glycans have a significant impact on adhesion. This finding suggests a distinct role for MUC1 glycosylation patterns in cancer cell migration and/or invasion. To decipher the molecular mechanism for the observed adhesion, we investigated the conformation of the glycosylated MUC1 peptides by NMR spectroscopy. These experiments revealed only minor differences in peptide structure, therefore clearly relating the adhesion behaviour to the type and number of glycans linked to MUC1.