Jörg Rademann

Serine phosphorylation is an essential switch for regulating the interactions and functions of many disease-related proteins. Accordingly, stable phosphoserine mimetics constitute chemical tools to study the effects of these post-translational modifications. In this work, we present the synthesis and characterization of a novel structural analog of phosphoserine, L-3-(pentafluorophosphato-difluoromethyl)-alanine. The hyper-fluorinated amino acid was synthetically accessed in six steps starting from commercially available N-Boc-L-serine methyl ester. The protected PF₅ amino acid and peptides derived thereof were inhibitors of the serine protein phosphatase PPP2CA, demonstrating its activity as functional phosphoserine mimetic.

Kurzzusammenfassung: Die Phosphorylierung von Proteinen ist eine der wichtigsten posttranslationalen Modifikationen, die Struktur, Stabilität und Aktivität von mehr als 13.000 menschlichen Proteinen beeinflusst. In dieser Arbeit wurde das Phosphotyrosin‐Mimetikum Pentafluorphosphato‐difluormethyl‐phenylalanin (PF ₅ CF ₂ Phe) genetisch codiert und in drei verschiedene Proteine eingebaut. Durch das Screening zweier Bibliotheken orthogonaler Aminoacyl‐tRNA‐Synthetasen konnten Enzyme identifiziert werden, die eine effiziente und spezifische Inkorporation von PF ₅ CF ₂ Phe in rotes Fluoreszenzprotein (RFP) über die Unterdrückung des Amber‐Stoppcodons ermöglichten. Zwei Modellproteine, humanes Ubiquitin (Ubq) und die B1‐Immunoglobulin‐Bindungsdomäne des Streptokokken‐Proteins G (GB1), wurden mit PF ₅ CF ₂ ‐Mutationen versehen und auf potenzielle Interaktionspartner hin untersucht. Während das native GB1 keine Bindung an Protein‐Tyrosin‐Phosphatasen (PTP) zeigte, war PF ₅ ‐GB1, das PF ₅ CF ₂ Phe an Position 17 trägt, ein potenter Inhibitor der Phosphatasen PTP1B und SHP2. PF ₅ ‐Ubq wurde hergestellt und in das erste Beispiel eines Proteins überführt, welches das bekannteste Phosphotyrosin‐Mimetikum, Phosphono‐Difluormethyl‐Phenylalanin (PO ₃ CF ₂ Phe), trägt. Angesichts des wachsenden Bedarfs die Funktionen komplexer Phosphorylierungsmuster zu verstehen, eröffnet die genetische Codierung von PF ₅ CF ₂ Phe zur Herstellung von Phosphoprotein‐Mimetika einzigartige Möglichkeiten für präzise Funktionsstudien, bei denen spezifische und homogene Proteinmodifikationen erforderlich sind.

Protein phosphorylation is one of the most important posttranslational modifications altering the structure, stability, and activity of more than 13 000 human proteins. In this work, the phosphotyrosine mimetic pentafluorophosphato‐difluoromethyl‐phenylalanine (PF ₅ CF ₂ Phe) was genetically encoded and incorporated into three different proteins. Screening two libraries of orthogonal aminoacyl‐tRNA synthetases identified enzymes enabling the efficient and specific incorporation of PF ₅ CF ₂ Phe into red fluorescent protein (RFP) via amber stop codon suppression. Two model proteins, human ubiquitin (Ubq) and the B1 immunoglobulin‐binding domain of streptococcal protein G (GB1), were prepared with PF ₅ CF ₂ Phe mutations and investigated for potential interaction partners. While native GB1 showed no binding to protein tyrosine phosphatases (PTP), PF ₅ ‐GB1, with PF ₅ CF ₂ Phe at position 17, was a strong inhibitor of the phosphatases PTP1B and SHP2. PF ₅ ‐Ubq was produced and converted into the first example of a protein carrying the most prominent phosphotyrosine mimetic, phosphono‐difluoromethyl phenylalanine (PO ₃ CF ₂ Phe). With increasing need in the biosciences to delineate the functions of complex phosphorylation patterns, genetic encoding of PF ₅ CF ₂ Phe yielding phosphoprotein mimetics opens unique opportunities for precise functional studies where site‐specific and homogeneous protein modifications are required.

In this work, we present the synthesis and characterization of a novel structural analog of phosphoserine, L-3-(pentafluorophosphato-difluoromethyl)-alanine. The hyper-fluorinated, permanently anionic amino acid residue was synthetically accessed in six steps starting from commercially available N-Boc-L-serine methyl ester and showed biological activity as an inhibitor of the protein serine phosphatase PPP2CA, demonstrating its potential as phosphoserine biomimetic.

Heparan sulfate (HS) interactions with interleukin 8 (IL‐8) are crucial for immune system response. The structural features of the HS and the environmental entities, such as metal ions, can regulate these interactions. However, it is challenging to evaluate the effect of each parameter on the interactions because of low accessibility to well‐defined saccharides and the lack of characteristic features to be determined by analytical tools. We evaluated the effect of the HS structural features on IL‐8 binding affinity utilizing electrochemical impedance spectroscopy (EIS) and X‐ray photoelectron spectroscopy (XPS). We showed that the metal ions Ca²⁺ and Mg²⁺ dissimilarly mediate the interactions of HS and IL‐8 in structure‐dependent manner of the HS. We showed that in all glycans, a positive synergistic effect on IL‐8 binding was observed. For several glycans, the presence of ions resulted in a dramatic increase in the affinity to IL‐8, while for other glycans, a milder effect was observed. This demonstrated that both structural motifs and environmental features are crucial for maintaining the interactions between the HS and IL‐8.

Von Diazopeptiden wurden schon früher berichtet, sie haben aber aufgrund ihrer hohen Reaktivität bis heute kein breites Einsatzgebiet gefunden. Wir berichten hier von der Herstellung, den Eigenschaften und Anwendungen chemisch stabiler interner Diazopeptide. Peptidylphosphoranylidenester und ‐amide reagieren mit Triflylazid hauptsächlich zu neuartigen 4,5‐disubstituierten 1,2,3‐Triazolylpeptiden. Nonaflylazid hingegen liefert Diazopeptide, die als Amide bei pH 1–14 und als Ester bei pH 1–8 chemisch stabil sind. Somit sind durch Festphasenpeptidsynthese hergestellte Diazopeptide auch während der finalen Entschützung mit 95 % Trifluoressigsäure stabil. Diazopeptide mit der Erkennungssequenz von Caspase‐3 wurden als spezifische, kovalente und irreversible Inhibitoren dieses Enzyms bei niedrigen nanomolaren Konzentrationen identifiziert. Ein fluoreszierendes Diazopeptid drang in lebende Zellen ein und ermöglichte mikroskopische Visualisierung und Quantifizierung von Apoptose durch Durchflusszytometrie. Diazopeptide stellen somit eine neue Klasse aktivitätsbasierter Sonden und Enzyminhibitoren zum Einsatz in der chemischen Biologie und medizinischen Chemie dar.

Diazo peptides have been described earlier, however, due to their high reactivity have not been broadly used until today. Here, we report the preparation, properties, and applications of chemically stable internal diazo peptides. Peptidyl phosphoranylidene‐esters and amides were found to react with triflyl azide primarily to novel 3,4‐disubstituted triazolyl‐peptides. Nonaflyl azide instead furnished diazo peptides, which are chemically stable from pH 1–14 as amides and from pH 1–8 as esters. Thus, diazo peptides prepared by solid phase peptide synthesis were stable to final deprotection with 95 % trifluoroacetic acid. Diazo peptides with the recognition sequence of caspase‐3 were identified as specific, covalent, and irreversible inhibitors of this enzyme at low nanomolar concentrations. A fluorescent diazo peptide entered living cells enabling microscopic imaging and quantification of apoptotic cells via flow cytometry. Thus, internal diazo peptides constitute a novel class of activity‐based probes and enzyme inhibitors useful in chemical biology and medicinal chemistry.

Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a K_D of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well.