Dipl.-Chem. Robert Zitterbart
Profil
Forschungsthemen1
Skalierbare, parallele & nachhaltige HPLC-freie Reinigung von Peptiden durch eine chemische Affinitätschromatographie
Quelle ↗Förderer: Bundesministerium für Wirtschaft und Energie Zeitraum: 09/2016 - 04/2018 Projektleitung: Dipl.-Chem. Robert Zitterbart, Prof. Dr. rer. nat. Oliver Seitz
Mögliche Industrie-Partner10
Stand: 26.4.2026, 19:48:44 (Top-K=20, Min-Cosine=0.4)
- 16 Treffer56.9%
- Engineering of New-Generation Protein Secretion SystemsP56.9%
- Engineering of New-Generation Protein Secretion Systems
- 16 Treffer56.9%
- Engineering of New-Generation Protein Secretion SystemsP56.9%
- Engineering of New-Generation Protein Secretion Systems
- 16 Treffer56.9%
- Engineering of New-Generation Protein Secretion SystemsP56.9%
- Engineering of New-Generation Protein Secretion Systems
- 12 Treffer53.6%
- EU: Bottom-Up Generation of atomicalLy Precise syntheTIc 2D MATerials for High Performance in Energy and Electronic Applications – A Multi-Site Innovative Training Action (ULTIMATE)P53.6%
- EU: Bottom-Up Generation of atomicalLy Precise syntheTIc 2D MATerials for High Performance in Energy and Electronic Applications – A Multi-Site Innovative Training Action (ULTIMATE)
- 18 Treffer53.6%
- EU: Bottom-Up Generation of atomicalLy Precise syntheTIc 2D MATerials for High Performance in Energy and Electronic Applications – A Multi-Site Innovative Training Action (ULTIMATE)P53.6%
- Integrated Self-Assembled SWITCHable Systems and Materials: Towards Responsive Organic Electronics – A Multi-Site Innovative Training Action (iSwitch)P48.8%
- EU: Bottom-Up Generation of atomicalLy Precise syntheTIc 2D MATerials for High Performance in Energy and Electronic Applications – A Multi-Site Innovative Training Action (ULTIMATE)
- 21 Treffer53.6%
- EU: Bottom-Up Generation of atomicalLy Precise syntheTIc 2D MATerials for High Performance in Energy and Electronic Applications – A Multi-Site Innovative Training Action (ULTIMATE)P53.6%
- EU: Simulation in Multiscale Physical and Biological Systems (STIMULATE)P48.9%
- EU: Bottom-Up Generation of atomicalLy Precise syntheTIc 2D MATerials for High Performance in Energy and Electronic Applications – A Multi-Site Innovative Training Action (ULTIMATE)
- 12 Treffer53.6%
- EU: Bottom-Up Generation of atomicalLy Precise syntheTIc 2D MATerials for High Performance in Energy and Electronic Applications – A Multi-Site Innovative Training Action (ULTIMATE)P53.6%
- EU: Bottom-Up Generation of atomicalLy Precise syntheTIc 2D MATerials for High Performance in Energy and Electronic Applications – A Multi-Site Innovative Training Action (ULTIMATE)
- DYnamic control in hybrid plasmonic NAnopores: road to next generation multiplexed single MOlecule detectionP53.5%
- DYnamic control in hybrid plasmonic NAnopores: road to next generation multiplexed single MOlecule detection
- 20 Treffer52.8%
- EU: Monomer Sequence Control in Polymers: Toward Next-Generation Precision Materials (EURO-SEQUENCES)P52.8%
- EU: Monomer Sequence Control in Polymers: Toward Next-Generation Precision Materials (EURO-SEQUENCES)
- 21 Treffer52.8%
- EU: Monomer Sequence Control in Polymers: Toward Next-Generation Precision Materials (EURO-SEQUENCES)P52.8%
- EU: Monomer Sequence Control in Polymers: Toward Next-Generation Precision Materials (EURO-SEQUENCES)
Publikationen23
Top 25 nach Zitationen — Quelle: OpenAlex (BAAI/bge-m3 embedded für Matching).
Chemical Science · 62 Zitationen · DOI
The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (<i>via</i> the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His<sub>6</sub> unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, <i>i.e.</i> the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2-6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins which were obtained in 8-33% overall yield with 90-98% purity despite the omission of HPLC purification.
Angewandte Chemie International Edition · 28 Zitationen · DOI
Analysis of postranslationally modified protein domains is complicated by an availability problem, as recombinant methods rarely allow site-specificity at will. Although total synthesis enables full control over posttranslational and other modifications, chemical approaches are limited to shorter peptides. To solve this problem, we herein describe a method that combines a) immobilization of N-terminally thiolated peptide hydrazides by hydrazone ligation, b) on-surface native chemical ligation with self-purified peptide thioesters, c) radical-induced desulfurization, and d) a surface-based fluorescence binding assay for functional characterization. We used the method to rapidly investigate 20 SH3 domains, with a focus on their phosphoregulation. The analysis suggests that tyrosine phosphorylation of SH3 domains found in Abl kinases act as a switch that can induce both the loss and, unexpectedly, gain of affinity for proline-rich ligands.
Chemical Science · 21 Zitationen · DOI
Hundreds of peptides can be synthesized by automated parallel synthesizers in a single run. In contrast, the most widely used peptide purification method - high-pressure liquid chromatography (HPLC) - only allows one-by-one processing of each sample. The chromatographic purification of many peptides, therefore, remains a time-consuming and costly effort. Catch-and-release methods can be processed in parallel and potentially provide a remedy. However, no such system has yet provided a true alternative to HPLC. Herein we present the development of a side-reaction free, reductively cleavable linker. The linker is added to the target peptide as the last building block during peptide synthesis. After acidic cleavage from synthetic resin, the linker-tagged full-length peptide is caught onto an aldehyde-modified solid support by rapid oxime ligation, allowing removal of all impurities lacking the linker by washing. Reducing the aryl azide to an aniline sensitizes the linker for cleavage. However, scission does not occur at non-acidic pH enabling wash out of reducing agent. Final acidic treatment safely liberates the peptide by an acid-catalysed 1,6-elimination. We showcase this first-in-class reductively cleavable linker system in the parallel purification of a personalized neoantigen cocktail, containing 20 peptides for cancer immunotherapy within six hours.
Journal of Peptide Science · 20 Zitationen · DOI
On December 12th, 2023, the European Commission took regulatory action to amend Annex XVII of REACH, imposing restrictions on the use of N,N-dimethylformamide (DMF) within the EU market owing to its high toxicity. Historically, DMF has been widely considered the gold standard for solid-phase peptide synthesis (SPPS). Being urgent to propose alternative solvents, we tested the suitability of non-hazardous neat and mixed solvents. Notably, binary solvent mixtures containing dimethyl sulfoxide as one of the solvent partners demonstrated high efficacy in solubilizing reagents while maintaining the desired swelling characteristics of common resins. A series of binary solvent mixtures were tested in automated SPPS, both at room temperature and high temperature, employing the PurePep® Chorus synthesizer, which enabled controlled induction heating between 25 and 90°C with oscillation mixing. The performances were assessed in challenging peptide sequences, i.e., ACP (65-74), and in longer and aggregating sequences like SARS-CoV-2 RBM (436-507) and β-amyloid (1-42). Furthermore, as part of the proposed sustainable approach to minimize the utilization of hazardous solvents, we coupled the novel PurePep EasyClean catch-and-release purification technology. This work, addressing regulatory compliance, emphasizes the crucial role of green chemistry in advancing safer and more environmentally friendly practices in SPPS.
Journal of Peptide Science · 14 Zitationen · DOI
In contrast to peptide synthesis, peptide purification by high performance liquid chromatography (HPLC) cannot be easily varied in number and scale, which results in production restraints. Catch‐and‐release purification of peptides may help to ease HPLC‐related limitations and provides thus an alternative, allowing for fast protocols with great potential for parallelization and scale‐up. This work depicts a unique combination of base‐labile cleavable linkers with oxime‐based and hydrazone‐based ligation chemistry. For the first time, aminooxy or hydrazine functionalities are presented on site of the linker molecules. Aldehyde‐functionalized agarose beads are used for immobilization on the solid phase, allowing a rapid and high yielding purification protocol. In this experimental set‐up, many organic solvents or chaotropic reagents are accepted during immobilization, facilitating the dissolution of potentially hydrophobic or aggregation‐prone peptides. Feasibility of the system is demonstrated with six peptides ranging from 15 to 31 residues in length, including very hydrophobic and thus challenging sequences like the palmitoyl modified liraglutide and an aggregation prone beta‐amyloid fragment. Additionally, limitations of this system and the use of base‐labile linkers are discussed and demonstrated.
Bioconjugate Chemistry · 14 Zitationen · DOI
We describe an unprecedented solid phase peptide synthesis (SPPS) of short peptide-based multimetal tags designated as elemental tags for the quantification of biomolecules via inductively coupled plasma mass spectrometry (ICP-MS). The macrocyclic chelator 1,4,7,10-tetraazacyclododecane N,N′,N″,N‴-tetra acetic acid (DOTA) was attached to the side chain of N-α-(9-fluorenylmethoxycarbonyl)-l-lysine (Fmoc-Lys-OH) and metalated with a lanthanide to provide a building block for Fmoc-based SPPS. Thereby, in contrast to existing strategies for the synthesis of DOTA–peptide conjugates, an already metalated DOTA-amino acid was used as a building block for SPPS. The DOTA-lanthanide complex was stable throughout the whole SPPS, even during the final cleavage in concentrated trifluoroacetic acid. This indicates that the strategy to first metalate the Fmoc-Lys(DOTA)-OH and to utilize the metal coordination to protect the carboxyl groups of DOTA offers an alternative to conventional synthetic routes using tert-butyl protected DOTA. Several small peptides containing up to four metal ions were synthesized, among them peptides carrying defined metal sequences consisting of two different lanthanides. The peptides were N-terminally maleimide-functionalized, thus introducing a moiety for conjugation to thiol-bearing biomolecules. The final objective of this work was the signal enhancement in ICP-MS-based DNA quantification assays. To evaluate the performance of the multimetal peptide tags in assay, they were applied to label thiol-modified 15mer DNA oligonucleotide probes. These served as reporter probes in a model sandwich-type hybridization assay. Thereby, we found that the ICP-MS signal increased linearly with the number of lanthanide ions attached to the reporter probe.
Chemistry - A European Journal · 12 Zitationen · DOI
Dinitroacetylene and other nitroacetylenes are attractive stoichiometric precursors to high energy-density materials, but suffer from high reactivity and thermal instability. Herein, we report that nitroacetylenes can be dramatically stabilized in the form of their dicobalt hexacarbonyl complexes. In particular, we describe the syntheses and characterization of the first two transition-metal complexes of nitroalkynes, [μ-1-nitro-2-(trimethylsilyl)ethyne-1,2-diyl]bis(tricarbonylcobalt)(Co-Co) and [μ-1-nitroethyne-1,2-diyl]bis(tricarbonylcobalt)(Co-Co). The chemistry of these compounds reveals their potential as reaction partners in [2+2+2] cyclotrimerizations, furnishing nitroindane, nitrotetralin, and trinitrobenzene products. The X-ray crystal structure of 1,3,5-trinitro-2,4,6-tris(trimethylsilyl)benzene presents a distorted, yet planar, aromatic ring.
Synthesis · 11 Zitationen · DOI
Exploratory studies of the CpCo-mediated [2+2+2] cycloaddition of alkynes to the 2,3-double bond of benzo[b]furans (and some benzo[b]thiophenes) are presented, with the general aim to access morphinan substructures. The basic feasibility of constructing Co-complexed tetrahydrophenanthro[4,5-bcd]furans (and -thiophenes) in moderate to good yields is demonstrated, with complete to extensive diastereoselectivity. Limitations are the apparent necessity for bulky (silylated) monoalkynes, the lack of regioselectivity in the cocyclization with unsymmetrical alkynes, and the sensitivity of the ligands, both complexed and uncomplexed, with respect to ring opening and rearrangement.
Journal of Peptide Science · 6 Zitationen · DOI
The chemical synthesis of proteins typically involves the solid-phase peptide synthesis of unprotected peptide fragments that are stitched together in solution by native chemical ligation (NCL). The process is slow, and throughput is limited because of the need for repeated high performance liquid chromatography purification steps after both solid-phase peptide synthesis and NCL. With an aim to provide faster access to functional proteins and to accelerate the functional analysis of synthetic proteins by parallelization, we developed a method for the high performance liquid chromatography-free synthesis of proteins on the surface of microtiter plates. The method relies on solid-phase synthesis of unprotected peptide fragments, immobilization of the C-terminal fragment and on-surface NCL with an unprotected peptide thioester in crude form. Herein, we describe the development of a suitable immobilization chemistry. We compared (i) formation of nickel(II)-oligohistidine complexes, (ii) Cu-based [2 + 3] alkine-azide cycloaddition and (iii) hydrazone ligation. The comparative study identified the hydrazone ligation as most suitable. The sequence of immobilization via hydrazone ligation, on-surface NCL and radical desulfurization furnished the targeted SH3 domains in near quantitative yield. The synthetic proteins were functional as demonstrated by an on-surface fluorescence-based saturation binding analysis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
International Journal of Peptide Research and Therapeutics · 5 Zitationen · DOI
Abstract Introduction The growing need for sustainable practices in pharmaceutical manufacturing has stimulated advancements in peptide synthesis. This study focuses on applying green chemistry principles to the synthesis of the Glucagon-Like Peptide-1 analog liraglutide. Material and Methods The safer coupling reagent 1-tert-butyl-3-ethylcarbodiimide (TBEC) was tested in combination with eco-friendly binary solvents such as dimethyl sulfoxide and butyl acetate to propose novel and sustainable solid-phase synthetic and purification strategies of liraglutide. Results Two synthetic strategies were developed for liraglutide production. The first strategy was based on a “direct synthesis”, incorporating a lipidated lysine building block into the peptide sequence, achieving 86% HPLC purity after catch-and-release purification. The second strategy based on “catch-lipidation-and-release” approach, allowed to obtain the peptide precursor without the lipid moiety, which was later linked during a controlled lipidation step. This latter strategy yielded purities exceeding 90% and reduced reliance on preparative HPLC. TBEC minimizes hazardous byproducts, such as hydrogen cyanide, and enhances solvent compatibility, achieving crude purities and yields comparable to conventional syntheses. Conclusion This work underscores the potential of green chemistry to align pharmaceutical innovation with environmental responsibility. In particular our findings highlight the effectiveness of TBEC and green solvent systems optimizing scalable and sustainable SPPS processes and improving resource efficiency. Thus, we propose a viable pathway to produce the therapeutic peptide ingredient liraglutide significantly reducing the environmental impact while maintaining high efficiency and quality of the synthesis.
Angewandte Chemie · 5 Zitationen · DOI
Abstract Mangelnde Verfügbarkeit erschwert die Analyse posttranslational modifizierter Proteindomänen, denn rekombinante Methoden vermitteln nur selten die gewünschte Ortsspezifität. Die Totalsynthese ermöglicht zwar eine nahezu vollkommene Kontrolle über posttranslationale und andere Modifikationen, jedoch bleiben chemische Methoden auf kürzere Peptide beschränkt. Zur Lösung dieses Problems präsentieren wir hier eine Methode, die a) Immobilisierung N‐terminaler Thio‐Peptidhydrazide über Hydrazonverknüpfung, b) native chemische Ligation auf der Oberfläche mit selbstgereinigten Peptidthioestern c) radikalisch induzierte Entschwefelung und d) einen oberflächenbasierten Fluoreszenzbindungs‐Assay zur funktionellen Charakterisierung kombiniert. Die Methode wurde zur schnellen Untersuchung von 20 SH3‐Domänen verwendet. Die Analyse lässt schließen, dass Tyrosinphosphorylierung der SH3‐Domänen in Abl‐Kinasen als Schalter fungiert, der sowohl einen Verlust als auch einen Gewinn an Affinität für prolinreiche Liganden induzieren kann.
Research Square · DOI
TLQIRGRERFEMYRELNEALELK #6 ID Crude IH-SPPS Flow-SPPS + HPLC IH-SPPS + PEC IH-SPPS + PEC + HPLC ID SequenceRapid parallel production of high-quality synthetic peptide sequences by solid-phase peptide synthesis (SPPS) and subsequent purification is a challenging yet essential pursuit.Neoantigen peptides, in particular, with medium to long lengths used in cancer immunotherapies need to be manufactured rapidly and with high purity.However, conventional batchsynthesis is slow and purification with sequential high-pressure liquid chromatography (HPLC) offers low throughput, leading to extended production times and potential product loss requiring repeated synthesis.Shorter and more reliable production timelines are essential to ensure the rapid delivery of life saving personalized medicine to patients.This study presents a PurePep solution to improve neoantigen production for cancer therapy.The integrated approach includes induction heating (IH) for rapid automated peptide synthesis followed by automated orthogonal PurePep EasyClean (PEC ) purification on the PurePep Chorus synthesizer.
Proceedings of the 35th European Peptide Symposium · DOI
The Cambridge Structural Database · DOI
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
The Cambridge Structural Database · DOI
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
edoc Publication server (Humboldt University of Berlin) · DOI
Protein-Arrays sind das Mittel der Wahl, um eine Vielzahl von Proteinen parallel zu untersuchen. Ziele dieser Untersuchungen sind meistens Proteininteraktionsnetzwerke zu entdecken oder besser verstehen zu können. Bisher wurden die benötigten Proteine fast ausschließlich mit biologischen Methoden gewonnen. Diese bieten allerdings keinen generellen Zugang zu posttranslational-modi-fizierten (PTM)-Proteinen. Somit war es bisher nicht möglich den Einfluss von PTMs auf Protein-Protein-Interaktionen (PPIs) im Arrayformat zu untersuchen. Die chemische Synthese kann dagegen Proteine mit ortsspezifischen PTMs liefern. Daher ist es verwunderlich, dass bislang noch keine Berichte über chemisch hergestellte PTM-Protein-Arrays existieren, besonders da PTMs meist entscheidend für proteomische Interaktionsnetzwerke sind. In der vorliegenden Arbeit wird eine Methodik beschrieben, die es ermöglicht PTM-modifizierte Protein-Domänen-Arrays auf der Oberfläche zu synthetisieren und zu analysieren. Mit der Methodik wurden 20 SH3-Domänen synthetisiert und 64 PPIs gemessen. Neben vier Hefe-SH3-Domänen wurden je acht humane (Phospho)SH3-Domänen der Abl- und Arg(Abl2)-Tyrosinkinase synthetisiert und funktionell untersucht. Es wurde gefunden, dass die Ligandenspezifität von Abl-SH3-Domänen durch Phosphorylierung feinreguliert wird. Je nach Phosphorylierungsmustern wurde die Affinität für spezifische Liganden erhöht oder erniedrigt. Der Ursprung dieser Phosphoregulierung wurde für die Abl-SH3-Domäne mit Hilfe der NMR-Spektroskopie und durch Zellexperimente versucht zu entschlüsseln und weiter validiert.
The Cambridge Structural Database · DOI
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
The Cambridge Structural Database · DOI
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
The Cambridge Structural Database · DOI
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
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Stammdaten
Identität, Organisation und Kontakt aus HU-FIS.
- Name
- Dipl.-Chem. Robert Zitterbart
- Titel
- Dipl.-Chem.
- Fakultät
- Mathematisch-Naturwissenschaftliche Fakultät
- Institut
- Institut für Chemie
- Arbeitsgruppe
- Organische und Bioorganische Chemie III
- Telefon
- +49 30 2093-7488
- HU-FIS-Profil
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- 26.4.2026, 01:14:34