NMR Spectroscopic Investigations and Method Development in Photocatalysis and Medicinal Chemistry

Nowadays, photocatalysis is one of the major topics in chemistry with tremendous success in synthetic applications. But to sustain this growth, a profound understanding of the underlying mechanisms is required. In recent years, NMR spectroscopy has established itself as a powerful method, which can give insights into every aspect of a photoreaction ranging from the initial photoexcitation by photo-CIDNP (nanosecond time scale) to various downstream processes such as intermediate formation and their sequence in the reaction as well as aggregations, unproductive pathways and solvent effects (second to hour time scale). But despite the recent success of NMR spectroscopy in photochemistry, there is still much room for improvement in terms of new experimental setups and approaches. The aim of this thesis was the introduction of a new method for NMR, which combines illumination, UV/Vis spectroscopy and NMR as well as the investigation of photocatalytic reactions to further increase the applicability scope of NMR spectroscopy in the research field of photochemistry. In addition, apart from photochemical investigations, ligand based NMR methods for drug discovery are surveyed, which probe ligand-receptor interactions of Histamine-H2 receptor (H2R) antagonists in the presence of the H2R incorporated in SF9 cell membranes. The second chapter presented an elaborate introduction of NMR in combination with illumiantion. First, it discussed and compared commonly applied illumination sources in illuminated NMR spectroscopy. Furthermore, the chapter surveyed in situ and ex situ illumination approaches and illumination apparatus as well as special purpose illumination setups. In a second part, case studies of photo-NMR in the research fields of photocatalysis, photoswitches, photopolymerizations and various new concepts for in situ illumination NMR were presented in detail. In chapter three, a fully automated triple combination of UV/Vis spectroscopy, NMR spectroscopy and in situ illumination (UVNMR-illumination) was developed. This optical fiber based setup merges two major, analytical methods for the investigation of (photo)chemical reactions; i.e. it enables the time-resolved, simultaneous detection of diamagnetic (NMR) and paramagnetic (UV/VIS) species in the same (photo)system. Therefore, this UVNMR-illumination setup eliminates issues that arise from separated UV/Vis – NMR systems such as concentration and temperature. Furthermore, the setup also provides the same reaction vessel, light intensity, convection and diffusion. In addition, the setup is portable and can be used at every solution NMR spectrometer, independent of the magnetic field or the installed probe (including cryo-probes). The applicability of the setup was tested for a large temperature range (180 – 323 K), which can be exploited to modulate exchange rates; e.g. it was shown that a spiropyran based photoswitch, which has a fast thermal back isomerization and photodegrades under prolonged illumination, could be easily stabilized at low temperatures (180 K). As a result, the otherwise short lived isomer could be easily assigned, while also gaining quantitative information and absorbance spectra. In addition, the UVNMR-illumination setup was put to the test in photocatalysis for the investigation of a consecutive photoinduced electron transfer (ConPET) process. Since ConPET processes rely on the presence of stable radical species (paramagnetic), they presented an ideal system to demonstrate the potential of the UVNMR-illumination device. Here, during preliminary investigations of the reaction it was indeed possible, to acquire simultaneous reaction profiles of paramagnetic and diamagnetic species under the same conditions, for the first time. Chapter four follows up on the preliminary ConPET investigations, which were presented in chapter three. The studies focus on the mechanistic investigation of the first, proposed ConPET process; the visible light mediated dehalogenation of aryl halides with perylene bisimide (PDI). The mechanistic foundation of ConPET is the ability of the photocatalyst to utilize the energy of two photons for a chemical reaction and was recently confirmed for the ConPET reaction with rhodamine 6G as photocatalyst. But the PDI promoted ConPET was recently challenged by a new proposal, which suggest a reaction of PDI with the substrate to yield a new photocatalytic species. Chapter four continued the research of the contested PDI ConPET process by means of extensive UVNMR-illumination, in situ NMR illumination and UV/Vis spectroscopic investigations, which lead to new mechanistic insights resulting in a new mechanistic proposal for the reaction. First, UVNMR-illumination measurements detected an electron-donor-acceptor (EDA) complex of PDI and triethylamine (TEA) prior to illumination indicating that NMR can be very sensitive for the detection of EDAs in some cases. Second, combined results of NMR, photo-CIDNP and UV/Vis spectroscopy showed that PDI is effectively turned into its radical anion (PDI•-) once the light is turned on. Combined UVNMR-illumination reaction profiles revealed unexpected paramagnetic relaxation enhancements due to the formation of the stable PDI•-, which lead to a pseudo signal increase of slowly relaxing compounds. As a result it could be shown that in this case, the residual solvent signal can be used as an indirect tracer to track the evolution and relative amount of the PDI•-. In addition, the presence of an initiating phase that preceded product formation was detected. UVNMR-illumination reaction measurements showed that this initiating phase persisted although the maximum amount of PDI•- was already formed (PDI•- was supposed to promote the reaction). Hence, it could be concluded that the radical anion concentration and product formation do not correlate. In detail examinations of the initiating phase by means of UV/Vis spectroscopy and in situ NMR illumination revealed that PDI can also interact with itself and does not require TEA to generate PDI•-. In addition, pre-irradiation experiments revealed that PDI can interact with itself beyond the formation of PDI•- by physical interactions. Presumably, these interactions result in PDI/PDI•- stacks/aggregates because the observed effects could be reversed by the addition of oxygen. Furthermore, this pre-irradiation of PDI got rid of the initiating phase before the product formation, which indicated that the interactions of PDI with itself result in the formation of the actual photocatalytic species. These combined results led to a new mechanistic proposal, which deviates from the two existing proposals for the PDI promoted dehalogenation of aryl halides by light. This new proposal suggests that PDI can interact with itself to yield a new catalytic photoactive species under illumination; i.e. some sort of stack/aggregate consisting of PDI and PDI•-. Chapter 5 surveyed the development and mechanistic investigations of the very first synthetic approach to cleave C-OCF3 bonds (here: an aryl trifluoromethyl ether), which resulted in a controlled liberation and in situ conversion of fluorophosgene for the synthesis of various substrates such as carboantes, carbamates and urea derivatives. The mechanistic studies combined ex situ and in situ NMR illumination studies, transient spectroscopy and radical trapping experiments. Here, transient spectroscopy suggested the formation of a charge-transfer (CT) dimer complex of the photocatalyst as the initial photoexcitation step. The photo-NMR measurements identified a carbonylfluoride compound as key intermediate solidifying the mechanistic proposal of a fluorophosgene promoted reaction. In addition, NMR could unravel an unproductive, second reactive pathway in the dark, which can be almost completely suppressed by the addition of water. With the combined mechanistic studies, an elaborate mechanistic proposal could be made, which covers all aspects of the reaction ranging from the initial photoexcitation, to intermediate detection and the assignment of numerous relevant products and side products. The last chapter of this thesis focused on the execution of ligand based NMR techniques in the field of drug discovery. Here, especially saturation transfer difference (STD) was applied to a system of Histamine-H2 receptor (H2R) antagonists in the presence of H2Rs, which were overexpressed in a parental membrane system. These investigations showed that ligand based NMR methods are very unreliable if non-specific interactions besides the ligand-receptor interactions are present. Therefore, albeit numerous efforts were made, it was not possible to distinguish specific and non-specific binding of H2R antagonists in the presence of H2R, which was incorporated in a membrane system. Nevertheless, sample preparation and composition was tremendously improved by changing out the buffer system and optimization of the sample formulation. In addition, a proposal could be devised, which describes applicable systems for membrane preparations. In summary, this thesis first presented an elaborate review, which surveys the evolution of NMR in combination. Second, the development of a new triple combination consisting of NMR spectroscopy, UV/Vis spectroscopy and in situ illumination (UVNMR-illumination) is described and its applicability as a mechanistic investigation tool in photochemistry is shown for two test systems in the fields of photoswitches and photocatalysis. Third, extensive UVNMR-illumination, in situ NMR illumination and UV/Vis spectroscopic examinations were conducted to unravel the mechanism of a seemingly ConPET driven reaction, in which a novel mechanistic proposal could be made. In addition, in a cooperative project, a photoreaction, which does not rely on radical reactivity, was developed and the underlying mechanism was investigated in detail yielding information about the initial photoexcitation as well as the assignment of key intermediates and the detection of an unproductive dark cycle. The last chapter focused on ligand based NMR methods in drug discovery. Here, valuable information concerning sample preparation and composition could be gathered. Furthermore, it was shown that non-specific interactions are a major issue for ligand based NMR methods; hence, they are only applicable for a minuscule amount of ligands (uncharged ligands with few functionalization) for the investigation transmembrane receptors, which are incorporated in a parental membrane system

ANLN and TLE2 in Muscle Invasive Bladder Cancer: A Functional and Clinical Evaluation Based on In Silico and In Vitro Data

Anilin actin binding protein (ANLN) and transducing-like enhancer protein 2 (TLE2) are associated with cancer patient survival and progression. The impact of their gene expression on progression-free survival (PFS) of patients with muscle invasive bladder cancer (MIBC) treated with radical cystectomy (RC) and subtype association has not yet been investigated. qRT-PCR was used to measure the transcript levels of ANLN and TLE2 in the Mannheim cohort, and validated in silico by The Cancer Genome Atlas (TCGA) cohort. Uni- and multivariate Cox regression analyses identified predictors for disease-specific survival (DSS) and overall survival (OS). In the Mannheim cohort, tumors with high ANLN expression were associated with lower OS and DSS, while high TLE2 expression was associated with a favorable OS. The TCGA cohort confirmed that high ANLN and low TLE2 expression was associated with shorter OS and disease-free survival (DFS). In both cohorts, multivariate analyses showed ANLN and TLE2 expression as independent outcome predictors. Furthermore, ANLN was more highly expressed in cell lines and patients with the basal subtype, while TLE2 expression was higher in cell lines and patients with the luminal subtype. ANLN and TLE2 are promising biomarkers for individualized bladder cancer therapy including cancer subclassification and informed MIBC prognosis

Prognostic significance of EGFR, AREG and EREG amplification and gene expression in muscle invasive bladder cancer

IntroductionMuscle invasive bladder cancer (MIBC) remains a prevalent cancer with limited therapeutic options, obviating the need for innovative therapies. The epidermal growth factor receptor (EGFR) is a linchpin in tumor progression and presents a potential therapeutic target in MIBC. Additionally, the EGFR ligands AREG and EREG have shown associations with response to anti-EGFR therapy and improved progression-free survival in colorectal carcinoma.Materials and methodsWe investigated the prognostic significance of EGFR, AREG, and EREG in MIBC. Gene expression and copy number analyses were performed via qRT-PCR on tissue samples from 100 patients with MIBC who underwent radical cystectomy at the University Hospital Mannheim (MA; median age 72, interquartile range [IQR] 64–78 years, 25% female). Results were validated in 361 patients from the 2017 TCGA MIBC cohort (median age 69, IQR 60–77 years, 27% female), in the Chungbuk and MDACC cohort. Gene expressions were correlated with clinicopathologic parameters using the Mann-Whitney test, Kruskal-Wallis- test and Spearman correlation. For overall survival (OS), cancer-specific survival (CSS) and disease-free survival (DFS) gene expression was analyzed with Kaplan-Meier and Cox-proportional hazard models.ResultsSignificant gene expression differences in EGFR, AREG, and EREG could be detected in all cohorts. In the TCGA cohort, EGFR expression was significantly elevated in patients with EGFR amplification and KRAS wildtype. High AREG expression independently predicted longer OS (HR = 0.35, CI 0.19 - 0.63, p = 0.0004) and CSS (HR = 0.42, CI 0.18 – 0.95, p = 0.0378) in the MA cohort. In the TCGA cohort, high EGFR, AREG, and EREG expression correlated with shorter OS (AREG: HR = 1.57, CI 1.12 – 2.20, p = 0.0090) and DFS (EGFR: HR = 1.91, CI 1.31 – 2.8, p = 0.0008). EGFR amplification was also associated with reduced DFS.DiscussionHigh EGFR and EREG indicate worse survival in patients with MIBC. The prognostic role of AREG should further be investigated in large, prospective series. Divergent survival outcomes between the four cohorts should be interpreted cautiously, considering differences in analysis methods and demographics. Further in vitro investigations are necessary to elucidate the functional mechanisms underlying the associations observed in this study

Integrative Modeling of Quantitative Plasma Lipoprotein, Metabolic, and Amino Acid Data Reveals a Multiorgan Pathological Signature of SARS-CoV-2 Infection.

The metabolic effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on human blood plasma were characterized using multiplatform metabolic phenotyping with nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS). Quantitative measurements of lipoprotein subfractions, α-1-acid glycoprotein, glucose, and biogenic amines were made on samples from symptomatic coronavirus disease 19 (COVID-19) patients who had tested positive for the SARS-CoV-2 virus (n = 17) and from age- and gender-matched controls (n = 25). Data were analyzed using an orthogonal-projections to latent structures (OPLS) method and used to construct an exceptionally strong (AUROC = 1) hybrid NMR-MS model that enabled detailed metabolic discrimination between the groups and their biochemical relationships. Key discriminant metabolites included markers of inflammation including elevated α-1-acid glycoprotein and an increased kynurenine/tryptophan ratio. There was also an abnormal lipoprotein, glucose, and amino acid signature consistent with diabetes and coronary artery disease (low total and HDL Apolipoprotein A1, low HDL triglycerides, high LDL and VLDL triglycerides), plus multiple highly significant amino acid markers of liver dysfunction (including the elevated glutamine/glutamate and Fischer's ratios) that present themselves as part of a distinct SARS-CoV-2 infection pattern. A multivariate training-test set model was validated using independent samples from additional SARS-CoV-2 positive patients and controls. The predictive model showed a sensitivity of 100% for SARS-CoV-2 positivity. The breadth of the disturbed pathways indicates a systemic signature of SARS-CoV-2 positivity that includes elements of liver dysfunction, dyslipidemia, diabetes, and coronary heart disease risk that are consistent with recent reports that COVID-19 is a systemic disease affecting multiple organs and systems. Metabolights study reference: MTBLS2014

mRNA-Expression of KRT5 and KRT20 Defines Distinct Prognostic Subgroups of Muscle-Invasive Urothelial Bladder Cancer Correlating with Histological Variants

Recently, muscle-invasive bladder cancer (MIBC) has been subclassified by gene expression profiling, with a substantial impact on therapy response and patient outcome. We tested whether these complex molecular subtypes of MIBC can be determined by mRNA detection of keratin 5 (KRT5) and keratin 20 (KRT20). Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was applied to quantify gene expression of KRT5 and KRT20 using TaqMan®-based assays in 122 curatively treated MIBC patients (median age 68.0 years). Furthermore, in silico analysis of the MD Anderson Cancer Center (MDACC) cohort (GSE48277 + GSE47993) was performed. High expression of KRT5 and low expression of KRT20 were associated with significantly improved recurrence-free survival (RFS) and disease-specific survival disease specific survival (DSS: 5-year DSS for KRT5 high: 58%; 5-year DSS for KRT20 high: 29%). KRT5 and KRT20 were associated with rates of lymphovascular invasion and lymphonodal metastasis. The combination of KRT5 and KRT20 allowed identification of patients with a very poor prognosis (KRT20+/KRT5−, 5-year DSS 0%, p < 0.0001). In silico analysis of the independent MDACC cohorts revealed congruent results (5-year DSS for KRT20 low vs. high: 84% vs. 40%, p = 0.042). High KRT20-expressing tumors as well as KRT20+/KRT− tumors were significantly enriched with aggressive urothelial carcinoma variants (micropapillary, plasmacytoid, nested)

RANTES/CCL5 and Risk for Coronary Events: Results from the MONICA/KORA Augsburg Case-Cohort, Athero-Express and CARDIoGRAM Studies

BACKGROUND: The chemokine RANTES (regulated on activation, normal T-cell expressed and secreted)/CCL5 is involved in the pathogenesis of cardiovascular disease in mice, whereas less is known in humans. We hypothesised that its relevance for atherosclerosis should be reflected by associations between CCL5 gene variants, RANTES serum concentrations and protein levels in atherosclerotic plaques and risk for coronary events. METHODS AND FINDINGS: We conducted a case-cohort study within the population-based MONICA/KORA Augsburg studies. Baseline RANTES serum levels were measured in 363 individuals with incident coronary events and 1,908 non-cases (mean follow-up: 10.2±4.8 years). Cox proportional hazard models adjusting for age, sex, body mass index, metabolic factors and lifestyle factors revealed no significant association between RANTES and incident coronary events (HR [95% CI] for increasing RANTES tertiles 1.0, 1.03 [0.75-1.42] and 1.11 [0.81-1.54]). None of six CCL5 single nucleotide polymorphisms and no common haplotype showed significant associations with coronary events. Also in the CARDIoGRAM study (&gt;22,000 cases, &gt;60,000 controls), none of these CCL5 SNPs was significantly associated with coronary artery disease. In the prospective Athero-Express biobank study, RANTES plaque levels were measured in 606 atherosclerotic lesions from patients who underwent carotid endarterectomy. RANTES content in atherosclerotic plaques was positively associated with macrophage infiltration and inversely associated with plaque calcification. However, there was no significant association between RANTES content in plaques and risk for coronary events (mean follow-up 2.8±0.8 years). CONCLUSIONS: High RANTES plaque levels were associated with an unstable plaque phenotype. However, the absence of associations between (i) RANTES serum levels, (ii) CCL5 genotypes and (iii) RANTES content in carotid plaques and either coronary artery disease or incident coronary events in our cohorts suggests that RANTES may not be a novel coronary risk biomarker. However, the potential relevance of RANTES levels in platelet-poor plasma needs to be investigated in further studies

Addressing climate change with behavioral science:A global intervention tournament in 63 countries

Effectively reducing climate change requires marked, global behavior change. However, it is unclear which strategies are most likely to motivate people to change their climate beliefs and behaviors. Here, we tested 11 expert-crowdsourced interventions on four climate mitigation outcomes: beliefs, policy support, information sharing intention, and an effortful tree-planting behavioral task. Across 59,440 participants from 63 countries, the interventions' effectiveness was small, largely limited to nonclimate skeptics, and differed across outcomes: Beliefs were strengthened mostly by decreasing psychological distance (by 2.3%), policy support by writing a letter to a future-generation member (2.6%), information sharing by negative emotion induction (12.1%), and no intervention increased the more effortful behavior-several interventions even reduced tree planting. Last, the effects of each intervention differed depending on people's initial climate beliefs. These findings suggest that the impact of behavioral climate interventions varies across audiences and target behaviors.</p

Addressing climate change with behavioral science: a global intervention tournament in 63 countries

Effectively reducing climate change requires marked, global behavior change. However, it is unclear which strategies are most likely to motivate people to change their climate beliefs and behaviors. Here, we tested 11 expert-crowdsourced interventions on four climate mitigation outcomes: beliefs, policy support, information sharing intention, and an effortful tree-planting behavioral task. Across 59,440 participants from 63 countries, the interventions’ effectiveness was small, largely limited to nonclimate skeptics, and differed across outcomes: Beliefs were strengthened mostly by decreasing psychological distance (by 2.3%), policy support by writing a letter to a future-generation member (2.6%), information sharing by negative emotion induction (12.1%), and no intervention increased the more effortful behavior—several interventions even reduced tree planting. Last, the effects of each intervention differed depending on people’s initial climate beliefs. These findings suggest that the impact of behavioral climate interventions varies across audiences and target behaviors

Addressing climate change with behavioral science:A global intervention tournament in 63 countries

Effectively reducing climate change requires marked, global behavior change. However, it is unclear which strategies are most likely to motivate people to change their climate beliefs and behaviors. Here, we tested 11 expert-crowdsourced interventions on four climate mitigation outcomes: beliefs, policy support, information sharing intention, and an effortful tree-planting behavioral task. Across 59,440 participants from 63 countries, the interventions' effectiveness was small, largely limited to nonclimate skeptics, and differed across outcomes: Beliefs were strengthened mostly by decreasing psychological distance (by 2.3%), policy support by writing a letter to a future-generation member (2.6%), information sharing by negative emotion induction (12.1%), and no intervention increased the more effortful behavior-several interventions even reduced tree planting. Last, the effects of each intervention differed depending on people's initial climate beliefs. These findings suggest that the impact of behavioral climate interventions varies across audiences and target behaviors.</p