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Movement of the Rieske domain of the iron–sulfur protein is essential for intramolecular electron transfer within complex III2 (CIII2) of the respiratory chain as it bridges a gap in the cofactor chain towards the electron acceptor cytochrome c. We present cryo-EM structures of CIII2 from Yarrowia lipolytica at resolutions up to 2.0 Å under different conditions, with different redox states of the cofactors of the high-potential chain. All possible permutations of three primary positions were observed, indicating that the two halves of the dimeric complex act independently. Addition of the substrate analogue decylubiquinone to CIII2 with a reduced high-potential chain increased the occupancy of the Qo site. The extent of Rieske domain interactions through hydrogen bonds to the cytochrome b and cytochrome c1 subunits varied depending on the redox state and substrate. In the absence of quinols, the reduced Rieske domain interacted more closely with cytochrome b and cytochrome c1 than in the oxidized state. Upon addition of the inhibitor antimycin A, the heterogeneity of the cd1-helix and ef-loop increased, which may be indicative of a long-range effect on the Rieske domain.
The human growth factor receptor MET is a receptor tyrosine kinase involved in cell proliferation, migration, and survival. MET is also hijacked by the intracellular pathogen Listeria monocytogenes. Its invasion protein, internalin B (InlB), binds to MET and promotes the formation of a signaling dimer that triggers the internalization of the pathogen. Here, we use a combination of structural biology, modeling, molecular dynamics simulations, and in situ single-molecule Förster resonance energy transfer (smFRET) experiments to elucidate the early events in MET activation by Listeria. Simulations show that InlB binding stabilizes MET in a conformation that promotes dimer formation. smFRET identifies the organization of the in situ signaling dimer. Further MD simulations of the dimer model are in quantitative agreement with smFRET. We accurately describe the structural dynamics underpinning an important cellular event and introduce a powerful methodological pipeline applicable to studying the activation of other plasma membrane receptors.
The family of scaffold attachment factor B (SAFB) proteins comprises three members and was first identified as binders of the nuclear matrix/scaffold. Over the past two decades, SAFBs were shown to act in DNA repair, mRNA/(l)ncRNA processing and as part of protein complexes with chromatin-modifying enzymes. SAFB proteins are approximately 100 kDa-sized dual nucleic acid-binding proteins with dedicated domains in an otherwise largely unstructured context, but whether and how they discriminate DNA and RNA binding has remained enigmatic. We here provide the SAFB2 DNA- and RNA-binding SAP and RRM domains in their functional boundaries and use solution NMR spectroscopy to ascribe DNA- and RNA-binding functions. We give insight into their target nucleic acid preferences and map the interfaces with respective nucleic acids on sparse data-derived SAP and RRM domain structures. Further, we provide evidence that the SAP domain exhibits intra-domain dynamics and a potential tendency to dimerize, which may expand its specifically targeted DNA sequence range. Our data provide a first molecular basis of and a starting point towards deciphering DNA- and RNA-binding functions of SAFB2 on the molecular level and serve a basis for understanding its localization to specific regions of chromatin and its involvement in the processing of specific RNA species.
Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyse biomolecules in situ by subtomogram averaging (STA). Specimen thickness is a key factor affecting cryo-ET data quality. Cells that are too thick for transmission imaging can be thinned by cryo-focused-ion-beam (cryo-FIB) milling. However, optimal specimen thickness for cryo-ET on lamellae has not been systematically investigated. Furthermore, the ions used to ablate material can cause damage in the lamellae, thereby reducing STA resolution. Here, we systematically benchmark the resolution depending on lamella thickness and the depth of the particles within the sample. Up to ca. 180 nm, lamella thickness does not negatively impact resolution. This shows that there is no need to generate very thin lamellae and thickness can be chosen such that it captures major cellular features. Furthermore, we show that gallium-ion-induced damage extends to depths of up to 30 nm from either lamella surface.
Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identified a new subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterized a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and showed that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a new subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.
Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with amino acids modeled in their standard protonation state the structure diverges far from its starting conformation. In comparison, MD simulations performed with pre-determined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein-water and protein-protein interactions of the structure much better. The results suggest it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to launching any MD simulations. Furthermore, the combined approach of protonation state prediction and MD simulations can provide valuable information on the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states currently remains a challenge, we introduce an approach of combining pKa prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions, but also the atomic modeling of density data.
All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals
(2023)
Excitable cells can be stimulated or inhibited by optogenetics. Since optogenetic actuation regimes are often static, neurons and circuits can quickly adapt, allowing perturbation, but not true control. Hence, we established an optogenetic voltage-clamp (OVC). The voltage-indicator QuasAr2 provides information for fast, closed-loop optical feedback to the bidirectional optogenetic actuator BiPOLES. Voltage-dependent fluorescence is held within tight margins, thus clamping the cell to distinct potentials. We established the OVC in muscles and neurons of Caenorhabditis elegans, and transferred it to rat hippocampal neurons in slice culture. Fluorescence signals were calibrated to electrically measured potentials, and wavelengths to currents, enabling to determine optical I/V-relationships. The OVC reports on homeostatically altered cellular physiology in mutants and on Ca2+-channel properties, and can dynamically clamp spiking in C. elegans. Combining non-invasive imaging with control capabilities of electrophysiology, the OVC facilitates high-throughput, contact-less electrophysiology in individual cells and paves the way for true optogenetic control in behaving animals.
Die vorliegende Dissertation befasst sich mit der Synthese und Untersuchung funktioneller Materialien für die Modifizierung von Grenz- und Oberflächen. Einen wichtigen Einfluss auf die Bildung der untersuchten, hochgeordneten Strukturen hat das Konzept der Selbstanordnung, dessen Grundlage schwache Wechselwirkungen sind. Ihre Ausbildung erfordert das Vorliegen geeigneter, funktioneller Gruppen in den Präkursoren und damit die Nutzung der vielfältigen Möglichkeiten der chemischen Synthese zur Bereitstellung maßgeschneidert funktionalisierter Moleküle. Den fünf Projekten dieser Arbeit gemeinsam ist daher die Synthese und Untersuchung für den jeweiligen Anwendungszweck geeigneter, dipolarer Präkursor-Moleküle, die zur Ausbildung funktioneller Koordinationspolymere (CPs) bzw. Metall-organischer Gerüstverbindungen (MOFs) und selbstanordnender Monolagen (SAMs) genutzt werden können. In Zusammenarbeit mit Kooperationspartnern wurden auf dieser Grundlage Untersuchungen zur Anwendbarkeit der erhaltenen Materialien in der Sensorik und zur Oberflächenfunktionalisierung durchgeführt.
Im ersten Projekt dieser Dissertation erfolgte die Untersuchung der Bildungs- und Phasenumwandlungsreaktionen von zehn verschiedenen Kupfer-Terephthalat Koordinationspolymeren. Neben bereits bekannten Koordinationspolymeren konnten so auch drei bisher literaturunbekannte CPs hergestellt und ihre Strukturen durch Kooperationspartner gelöst bzw. Strukturvorschläge gemacht werden. Die Identifikation und Auseinandersetzung mit strukturstabilisierenden Wechselwirkungen schließen dieses Projekt ab und bilden die Grundlage für die Untersuchung der Synthese und Stabilität abgeleiteter, komplexerer Koordinationspolymere.
Im Fokus des zweiten Projekts steht 𝛽-Cu2(bdc)(OH)2, ein Kupfer-Terephthalat Koordinationspolymer, dessen Kristallstruktur zuvor nicht bekannt war, im vorliegenden Projekt aber durch Kooperationspartner auf Basis des Röntgenpulverdiffraktogramms des Materials gelöst werden konnte. Der Vergleich der analytischen Daten von 𝛽-Cu2(bdc)(OH)2 mit der Literatur zeigte gute Übereinstimmungen u. a. der Diffraktogramme und IR-Spektren mit dem in der Literatur als SURMOF-2 bezeichneten, oberflächengebundenen Schichtmaterial. Aufgrunddessen kann davon ausgegangen werden, dass es sich bei SURMOF-2 um 𝛽-Cu2(bdc)(OH)2 handelt, und folglich dessen Kristallstrukturlösung die beiden bisher in der Literatur vorhandenen Strukturvorschläge für SURMOF-2 ersetzt.
Im Rahmen des dritten Projekts sollten für die Sensorik anwendbare, MOF-basierte Dünnschichtsysteme hergestellt werden. Das Sensorkonzept, das auf der Änderung des dielektrischen Verhaltens der MOFs bei Einlagerung dipolarer Analytmoleküle beruht, erfordert den Einsatz dipolarer Liganden in den entsprechenden Koordinationsnetzwerken. Hierfür wurden mehrere teilweise dipolare pillar-Liganden synthetisiert und diese für den Aufbau von Kupfer(II)terephthalat-basierten pillared-layer MOFs eingesetzt. Im Rahmen des Projekts konnten so auf Grundlage der Erkenntnisse aus Projekt 1 und in Zusammenarbeit mit Kooperationspartnern neue pillared-layer MOFs hergestellt und ihre Kristallstrukturen gelöst werden. Die abschließend durch Kooperationspartner erfolgte Abscheidung dünner, oberflächengebundener Schichten dieser MOFs und erste Untersuchungen hinsichtlich ihrer Eignung für die geplante Sensorikanwendung runden das Projekt ab.
Im vierten Projekt sollte eine geeignete, in situ abspaltbare Schutzgruppe für die Thiolgruppe etabliert und ihr Einfluss auf die Bildung von Terphenylthiolat-SAMs untersucht werden. Diese Voraussetzung erfüllt die im Rahmen dieser Arbeit am Beispiel von CH3-, F- und CF3-terminierten Terphenylthiolen etablierte 3,4-Dimethoxybenzyl-Gruppe, die sich durch den Zusatz von Trifluoressigsäure in der Abscheidungslösung in situ abspalten lässt. Zum Vergleich wurden von Kooperationspartner Monolagen aus den entsprechenden freien Thiolen abgeschieden und untersucht. Schichtdicken, Packungsdichten, Kippwinkel und Elementarzellen von Monolagen aus freien und geschützten Terphenylthiolen zeigen gute Übereinstimmungen. Im Gegensatz zu anderen, ebenfalls in situ abspaltbaren Gruppen hat die Anwesenheit der 3,4-Dimethoxybenzyl-Gruppe folglich keinen negativen Einfluss auf die Struktur und Qualität der gebildeten Monolagen.
In Fortführung des vorangegangenen Projekts wurde im abschließenden Projekt in Zusammenarbeit mit Kooperationspartnern der Einfluss verschiedener Kopfgruppen (H-, CH3-, F-, CF3- und SF5-) und der Länge des aromatischen Rückgrats (Phenyl-, Biphenyl- und Terphenyl-) auf die Ladungstransporteigenschaften der entsprechenden SAMs untersucht. Mit Ausnahme einiger Benzolthiole, lieferten alle betrachteten Präkursoren hochgeordnete, dicht gepackte Schichten aus aufrecht angeordneten Molekülen. Wie erwartet korreliert die Austrittsarbeit der modifizierten Oberflächen mit dem Dipolmoment der jeweiligen Kopfgruppe, wobei der Effekt der SF5-Gruppe mit einer erzielten Austrittsarbeit von annähernd 6 eV besonders hervorzuheben ist. Den Erwartungen entsprechend, sinkt die elektrische Stromdichte bei gleichbleibender Kopfgruppe mit steigender Moleküllänge. Die Stromdichte ist außerdem von der Kopfgruppe abhängig und nimmt von CH3- über H-, CF3- und SF5- bis hin zu F- ab, korreliert aber folglich nicht mit der Austrittsarbeit oder dem Dipolmoment.
Vertebrate life depends on renal function to filter excess fluid and remove low-molecular-weight waste products. An essential component of the kidney filtration barrier is the slit diaphragm (SD), a specialized cell-cell junction between podocytes. Although the constituents of the SD are largely known, its molecular organization remains elusive. Here, we use super-resolution correlative light and electron microscopy to quantify a linear rate of reduction in albumin concentration across the filtration barrier. Next, we use cryo-electron tomography of vitreous lamellae from high-pressure frozen native glomeruli to analyze the molecular architecture of the SD. The resulting densities resemble a fishnet pattern. Fitting of Nephrin and Neph1, the main constituents of the SD, results in a complex interaction pattern with multiple contact sites between the molecules. Using molecular dynamics flexible fitting, we construct a blueprint of the SD, where we describe all interactions. Our architectural understanding of the SD reconciles previous findings and provides a mechanistic framework for the development of novel therapies to treat kidney dysfunction.
Vertebrate life depends on renal function to filter excess fluid and remove low-molecular-weight waste products. An essential component of the kidney filtration barrier is the slit diaphragm (SD), a specialized cell-cell junction between podocytes. Although the constituents of the SD are largely known, its molecular organization remains elusive. Here, we use super-resolution correlative light and electron microscopy to quantify a linear rate of reduction in albumin concentration across the filtration barrier under no-flow conditions. Next, we use cryo-electron tomography of vitreous lamellae from high-pressure frozen native glomeruli to analyze the molecular architecture of the SD. The resulting densities resemble a fishnet pattern. Fitting of Nephrin and Neph1, the main constituents of the SD, results in a complex interaction pattern with multiple contact sites between the molecules. Using molecular dynamics simulations, we construct a blueprint of the SD that explains its molecular architecture. Our architectural understanding of the SD reconciles previous findings and provides a mechanistic framework for the development of novel therapies to treat kidney dysfunction.
The EMT-transcription factor ZEB1 has been intensively studied in solid cancers, where it is expressed at the invasive front and in cancer-associated fibroblasts (CAFs). In tumour cells, ZEB1 has been involved in multiple steps of cancer progression including stemness, metastasis and therapy resistance, yet its role in the tumour-microenvironment is largely unknown. Here, the role of Zeb1 in CAFs was investigated using mouse models reflecting different tumour stages in immunocompetent fibroblast specific Zeb1 KO mice. Fibroblast-specific depletion of Zeb1 accelerated tumour growth in the inflammation driven AOM/DSS tumour initiation model, reduced tumour growth and invasion in the sporadic AOM/P53 model and reduced liver metastasis in a progressed orthotopic transplantation model. Immunohistochemical and single cell RNA-sequencing analysis showed that Zeb1 ablation resulted in attenuated expression of the myofibroblast marker aSMA and reduced ECM deposition, indicating a shift among fibroblast subpopulations. Modulation of CAFs was furthermore associated with increased inflammatory signaling in fibroblasts resulting in immune infiltration into primary tumours and exaggerated inflammatory signaling in T cells, B cells and macrophages. These changes in the tumour microenvironment were associated with increased efficacy of immune checkpoint inhibition therapy. In summary, Zeb1 expression in CAFs was identified as a potential target to block immunosuppression and metastatic dissemination in colon cancer.
A sustainable strategy for O-trifluoromethylation of electron-deficient phenols by combining electrochemical synthesis with flow technology is presented. The reaction is optimized by screening experiments to establish a fast and efficient flow protocol. Simultaneous anodic oxidation of Langlois reagent and the phenols in a micro flow cell leads to direct preparation of trifluoromethyl ethers in yields up to 90%. This one-step protocol is tolerant of several functional groups, shows good regioselectivity and works without any chemical oxidants and catalysts by using electrical current as an inexpensive and sustainable reagent.
HER2 belongs to the ErbB sub-family of receptor tyrosine kinases and regulates cellular proliferation and growth. Different from other ErbB receptors, HER2 has no known ligand. Activation occurs through heterodimerization with other ErbB receptors and their cognate ligands. This suggests several possible activation paths of HER2 with ligand-specific, differential response, which so far remained unexplored. Using single-molecule tracking and the diffusion profile of HER2 as a proxy for activity, we measured the activation strength and temporal profile in live cells. We found that HER2 is strongly activated by EGFR-targeting ligands EGF and TGFα, yet with a distinguishable temporal fingerprint. The HER4-targeting ligands EREG and NRGβ1 showed weaker activation of HER2, a preference for EREG, and a delayed response to NRGβ1. Our results indicate a selective ligand response of HER2 that may serve as a regulatory element. Our experimental approach is easily transferable to other membrane receptors targeted by multiple ligands.
The title compound, di-μ3-chlorido-tetra-μ2-chlorido-tetrakis(diethyl ether-κO)bis(1,1-dimethylethyl)tetramagnesium, [Mg4(C4H9)2Cl6(C4H10O)4], features an Mg4Cl6 open-cube cluster. The two four-coordinate Mg2+ ions show an almost tetrahedral coordination, whereas the two six-coordinate Mg2+ ions have their ligands in an octahedral environment. The Mg—Cl bond lengths differ depending on the coordination number (2 or 3) of the bridging μ-Cl− ligands. There are few comparable structures deposited in the Cambridge Structural Database.
HER2 belongs to the ErbB sub-family of receptor tyrosine kinases and regulates cellular proliferation and growth. Different from other ErbB receptors, HER2 has no known ligand. Activation occurs through heterodimerization with other ErbB receptors and their cognate ligands. This suggests several possible activation paths of HER2 with ligand-specific, differential response, which has so far remained unexplored. Using single-molecule tracking and the diffusion profile of HER2 as a proxy for activity, we measured the activation strength and temporal profile in live cells. We found that HER2 is strongly activated by EGFR-targeting ligands EGF and TGFα, yet with a distinguishable temporal fingerprint. The HER4-targeting ligands EREG and NRGβ1 showed weaker activation of HER2, a preference for EREG, and a delayed response to NRGβ1. Our results indicate a selective ligand response of HER2 that may serve as a regulatory element. Our experimental approach is easily transferable to other membrane receptors targeted by multiple ligands.
The TOM complex is the main entry point for precursor proteins into mitochondria. Precursor proteins containing targeting sequences are recognized by the TOM complex and imported into the mitochondria. We have determined the structure of the TOM core complex from Neurospora crassa by single-particle cryoEM at 3.3 Å resolution, showing its interaction with a bound presequence at 4 Å resolution, and of the TOM holo complex including the Tom20 receptor at 6-7 Å resolution. TOM is a transmembrane complex consisting of two β-barrels, three receptor subunits and three short transmembrane subunits. Tom20 has a transmembrane helix and a receptor domain on the cytoplasmic side. We propose that Tom20 acts as a dynamic gatekeeper, guiding precursor proteins into the pores of the TOM complex. We analyze the interactions of Tom20 with other TOM subunits, present insights into the structure of the TOM holo complex, and suggest a translocation mechanism.
Cell-free (CF) synthesis with highly productive E. coli lysates is a convenient method to produce labeled proteins for NMR studies. Despite reduced metabolic activity in CF lysates, a certain scrambling of supplied isotope labels is still notable. Most problematic are conversions of 15N labels of the amino acids L-Asp, L-Asn, L-Gln, L-Glu and L-Ala, resulting in ambiguous NMR signals as well as in label dilution. Specific inhibitor cocktails suppress most undesired conversion reactions, while limited availability and potential side effects on CF system productivity need to be considered. As alternative route to address NMR label conversion in CF systems, we describe the generation of optimized E. coli lysates with reduced amino acid scrambling activity. Our strategy is based on the proteome blueprint of standardized CF S30 lysates of the E. coli strain A19. Identified lysate enzymes with suspected amino acid scrambling activity were eliminated by engineering corresponding single and cumulative chromosomal mutations in A19. CF lysates prepared from the mutants were analyzed for their CF protein synthesis efficiency and for residual scrambling activity. The A19 derivative “Stablelabel” containing the cumulative mutations asnA, ansA/B, glnA, aspC and ilvE yielded the most useful CF S30 lysates. We demonstrate the optimized NMR spectral complexity of selectively labeled proteins CF synthesized in “Stablelabel” lysates. By taking advantage of ilvE deletion in "Stablelabel", we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin.
This thesis investigates the structure of the translocase of the outer membrane (TOM) complex in mitochondria, focusing on the TOM holo complex through single-particle electron cryo-microscopy (cryoEM) complemented by mass spectrometry and computational structure prediction. Mitochondria, crucial for energy production in eukaryotic cells, import most of their proteins from the cytoplasm. These proteins enter through the TOM complex, which in its core form consists of a membrane-embedded homodimer of Tom40 pores, two Tom22 cytoplasmic receptors, and six small TOM stabilizing subunits (Tom7, Tom6, and Tom5). The holo complex includes two additional subunits, Tom70 and Tom20, whose stoichiometry and positioning are less understood due to their easy dissociation during isolation of the complex. CryoEM analysis revealed the high-resolution structure of the Neurospora crassa TOM core complex at 3.3 Å, containing all core subunits, and the presence of a central phospholipid causing the Tom40 dimer to tilt to 20°. Furthermore, a 4 Å resolution map indicated the binding of a precursor protein as it transitions through the translocation barrel. Finally, at 6-7 Å resolution, the structure of the TOM holo complex highlighted Tom20's flexibility as it interacts with the core complex, emphasizing its role in protein translocation. This work provides significant insights into the architecture and functioning of the TOM complex, contributing to the understanding of mitochondrial protein import mechanisms.
Optimierung der Synthese eines neuen photolabil geschützten Nitroxid-Spin-Labels für RNA und DNA
(2023)
Im Rahmen dieser Arbeit konnte, ausgehend von den günstigen Ausgangsverbindungen Desoxyadenosin und Phthalsäureanhydrid, ein neues photolabil geschütztes Nukleotid 1 und sein Dummypartner 2 synthetisiert werden. Positiv zu bemerken ist, dass einige Schritte im Vergleich zu ähnlichen literaturbekannten Reaktionen in Einfachheit, Reinheit oder Ausbeute verbessert wurden. So konnte die Ausbeute der wichtigen Umwandlung des Amins 46 zum Iodid 47 durch den Ersatz des vorherigen DCM/DIM Gemisches durch reines DIM von 15 % auf akzeptable 50 % erhöht werden, was nicht nur Zeit, sondern auch zukünftige Chemikalienmengen einspart. Nicht nur hierbei, sondern auch bei der Nitrierung zu 61 oder auch der Oxidierung zu 63 war es von äußerster Wichtigkeit eine korrekte Temperaturkontrolle durchzuführen, da es sonst zu hohen Ausbeuteverlusten durch ungewollte Nebenreaktionen kommen konnte. Eine sehr interessante Beobachtung war die Kontrolle der Suzuki Miyaura-Kreuzkupplung durch die Anwendung verschieden starker Basen. Während schwache Basen wie KOAc nur zur Miyaura-Borilierung führten, begünstigten starke Basen wie K3PO4 die Suzuki Miyaura-Kreuzkupplung. Die Zusammenführung des Zucker Bausteins 36 und des Isoindolin-Bausteins 37 funktionierte sehr gut, sodass das Nukleotid 1 durch die Schützung der exozyklischen Amingruppe und Phosphorylierung des 3´-OH dargestellt werden konnte.
Die Synthese der 14mer DNA bzw. RNA Sequenzen mit den neuen Nukleotiden 1 und 2 funktionierten mit zufriedenstellenden Ausbeuten, nur die Abspaltung der Pac-Gruppe benötigte etwas harschere Bedingungen von 50 °C in 32 % Ammoniak über Nacht. Die photolabile Schutzgruppe in Strang (V) mDNA-Tetramethyl konnte nun abgespalten und das Nitroxid an Luft reoxidiert werden. Anhand von EPR-Spektren und einer HPLC Analyse ergab sich jedoch eine Abspalteffizienz von nur 70 %. Dies bedeutet, dass für künftige PELDOR Messungen eine Aufreinigung des Spaltgemisches zur Isolierung des Radikal-Strangs von Nöten ist.
Anhand des Schmelzpunkts der verschiedenen Duplexe wurde anschließend die mögliche Anwendung des Nukleotids weiter analysiert. Hierbei stellte sich heraus, dass der Benzolring sowohl in 2 als auch in 1 eine erhebliche Destabilisierung des Duplex erzeugte. Somit ist das neue photolabil geschützte Nukleotid als EPR Sonde in der Mitte von Sequenzen nur bedingt geeignet. Zukünftige Experimente könnten das neue Spin Label nicht in der Mitte, sondern an den Enden der Sequenzen ähnlich anderer Arbeiten[92,94,164] einbauen, wo die Destabilisierung eine geringere Auswirkung hat, oder die Sequenz für eine bessere Stabilisierung verlängern.[164] Bei ausreichenden Duplexstabilitäten könnten hiermit dann PELDOR Messungen durchgeführt werden. Ähnlich starre, sterisch anspruchsvolle Nukleotide zeigten auch ähnliche Schmelzpunkte für ihre Duplexe[120], dennoch wurden sie für weitere Markierungsexperimente verwendet. Hierbei handelte es sich jedoch nicht um EPR Sonden, sondern um Fluoreszenzmarker. Da auch das neue Spin-Label 1 ein großes π-System besitzt, könnte eine komplett neue Herangehensweise die Anwendung als Fluoreszenzmarker sein. Genaue Absorptionsmessungen müssten noch durchgeführt werden, jedoch zeigte das Spin Label sehr stark fluoreszierende Eigenschaften unter der UV-Lampe während der Säulenchromatographie. Hierbei würde die Synthese um einiges kürzer ausfallen, da das EPR-aktive Nitroxid nicht mehr benötigt wird und geschützt werden muss, was Zeit und Chemikalien spart.
Zusammenfassend wurde über eine 22-stufige Synthese ein neues photolabil geschütztes Spin-Label synthetisiert, in ein 14mer integriert, erfolgreich entschützt und mittels EPR-Spektroskopie vermessen. Schmelzpunktmessungen zeigten jedoch eine große Destabilisierung und deuten darauf hin, dass 1 und Nukleotide mit ähnlich Benzolringen nur eingeschränkt als EPR-aktive Nukleotide geeignet sind.