Method Development for the Detection and Identification of Pathophysiologically Relevant Electrophiles in Pollen

Wang, Jue Theresa. Method Development for the Detection and Identification of Pathophysiologically Relevant Electrophiles in Pollen. 2021, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: https://edoc.unibas.ch/82560/

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Pollen allergy is a complex, multicausal disease with rising prevalence across the world. Currently, pollen allergy is treated mainly by symptom management and allergen-specific immunotherapy. In order to develop new treatment options, a greater understanding of the underlying mechanism of the disease is needed. Based on disease hypotheses like the hapten theory and the danger model, electrophilic small molecules in pollen could play a part in the sensitisation process and the exacerbative nature of the disease. The research on small molecules in pollen is lagging behind in contrast to their protein counterparts, which is why the aim of this project was to develop a method to enable the detection and identification of electrophiles in pollen extracts.
Method development in this work explored i) in situ detection and identification methods with liquid chromatography-mass spectrometry and ii) nucleophilic labelling with an affinity tag in order to facilitate a subsequent purification step, before investigating the potential of using a solid-supported nucleophilic probe. Finally, a probe was developed, consisting of a polystyrene solid support, a hyperacid-sensitive linker and a disulfide-protected cysteine that could act as a nucleophile to capture the electrophilic target molecules upon deprotection. The advantages of the probe are the following: i) the nucleophilic cysteine could be selectively deprotected, and a method was developed to quantify the released cysteine (8.65 ± 2.65 %) and therefore the amount of reactive sites on the resin; ii) the solid nature of the probe enabled a set- up in cartridges intended for solid-phase extraction, which allowed consecutive washes and reagent additions; iii) the hyperacid sensitive linker enabled the release of formed cysteine adducts after reaction; and finally, iv) due to the design of the probe, only mono-addition of cysteine was observed, except in cases where adducts could decompose. The probe was tested on model compounds, a model extract that was spiked with model compound and lastly, on diverse pollen extracts (Ambrosia psilostachya, Ambrosia artemisiifolia, Phleum pratense, Betula pendula, Urtica dioica, Corylus avellana). Both model compound and model extract experiments were successful; adduct formation was observed and the adducts were successfully isolated and characterised by nuclear magnetic resonance. However, due to the low abundance of electrophilic compounds in the extract, it unfortunately was not possible to isolate and characterise any compounds from pollen extracts, aside from two compounds isolated from a larger pollen extract experiment with Ambrosia psilostachya. The two isolated compounds were shown to be coumaroyl spermidine- like structures, however, their exact structure could not be determined.
Advisors:Hamburger, Matthias and Ricklin, Daniel and de Tommasi, Nunziatina
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Ehemalige Einheiten Pharmazie > Pharmazeutische Biologie (Hamburger)
UniBasel Contributors:Wang, Jue Theresa and Hamburger, Matthias and Ricklin, Daniel
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14112
Thesis status:Complete
Number of Pages:XIII, 189
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss141129
edoc DOI:
Last Modified:25 Jun 2021 04:30
Deposited On:24 Jun 2021 10:20

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