Protein PCS NMR spectroscopy under physiological conditions: development of a new high yield cyclization method for rigidified DOTA-based tags with sulfhydryl-reactive activators forming a reductively stable linkage

Müntener, Thomas. Protein PCS NMR spectroscopy under physiological conditions: development of a new high yield cyclization method for rigidified DOTA-based tags with sulfhydryl-reactive activators forming a reductively stable linkage. 2018, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_12978

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This thesis focuses on the development of a new synthetic strategy towards twelve-membered tetraaza macrocycles and the synthesis of new sulfhydryl reactive linker moieties for the application in pseudocontact shift (PCS) NMR spectroscopy.
The first part addresses the development of a new robust synthetic procedure for the selective synthesis of twelve-membered tetraaza macrocycles using natural amino acids and derivatives as cheap chiral building blocks. Cyclization of linear tetrapeptides was attempted but due to the preferred trans configuration of the peptide bond only small amounts of the cyclic tetrapeptide were obtained. Tetraalanine was reduced to tetraalaninol and the cyclization of tetraalaninol in solution was attempted using a preorganized bisaminal approach. This approach was unsuccessful and no product could be isolated. A more flexible mixed amine / amide tetramer was synthesized and successfully cyclized under modest dilution conditions forming a C2 symmetric twelve-membered bislactam. This reaction was highly efficient and afforded various bislactams based on alanine, alanine / valine and alanine / serine in good yields. From these bislactams various valuable intermediates for further tag synthesis are accessible as well as the final tetraaza macrocycles M4-cyclen, M2P2-cyclen and M3O1-cyclen. The newly developed approach allows the synthesis of a variety of differently substituted twelvemembered macrocycles.
The second part primarily focuses on the development of new sulfhydrylreactive linker moieties for site-selective protein tagging. The widely used formation of a disulphide linkage has the inherent problem of being unstable under reductive conditions as they are found in living cells, limiting the range of application to buffered in-vitro applications. Pyridine phenyl sulfone based tags were synthesized and in-cell NMR experiments were conducted with the B1 domain of the streptococcal protein G (GB1). The structure of the protein was successfully calculated with the Rosetta approach using only pseudocontact shifts and residual dipolar couplings (RDCs) obtained from in-cell experiments in oocytes. We proved for the first time that accurate structures of proteins inside cells can be generated solely from experimental PCSs and RDCs within the Rosetta package. The conjugation speed was dramatically increased for the fluorine substituted pyridine phenyl sulfone analogue decreasing the reaction time from 24 h at 40 °C to six hours at 20-25 °C. Further enhancement was achieved using a pyridine thiazol allowing efficient tagging in less than one hour at 20-25 °C and pH 7.0.
The last part deals with various different applications of M8-DOTA in order to solve structural or biological problems. Gd-M8-DOTA was selectively coupled to the Pittsburgh compound B (PiB) for in-vivo magnetic resonance imaging experiments. PiB strongly interacts with the amyloid plaques found in Alzheimer’s disease and thus PiB-Gd-M8-DOTA allows detecting amyloid plaques by MRI.
A second collaboration project addresses the problems found in the assignment of a hexa-polyproline. Due to the repetitive nature of this peptide the chemical shift dispersion was small. Increasing the chemical shift range found in hexa-polyproline was the goal of the attempted conjugation of M8-DOTA to the hexa-polyproline. We showed that M8-DOTA can not be coupled to the N-terminal aniline moiety and a less sterically demanding amine linker is required.
Prospective experiments were carried out on Eu-M8-DOTA and its closely related derivatives, Eu-M4-DOTA and Eu-DOTA, in order to check for possible applications in Förster resonance energy transfer (FRET) spectroscopy. All complexes were not luminescence under UV irradiation and a UV harvesting molecule was required. Eu-azaxanthon-M7-DOTA showed excellent luminescence properties required for potential applications in FRET spectroscopy. Preliminary experiments with Tb-M7PyThiazol-DOTA showed similar properties with a red shifted UV absorption maximum (from 250 nm to 300 nm) indicating great potential for FRET spectroscopy.
Advisors:Housecroft, Catherine E. and Häussinger, Daniel and Wenger, Oliver S.
Faculties and Departments:05 Faculty of Science > Departement Chemie > Former Organization Units Chemistry > Anorganische Chemie (Housecroft)
UniBasel Contributors:Müntener, Thomas and Häussinger, Daniel
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12978
Thesis status:Complete
Number of Pages:1 Online-Ressource (xvi, 285 Seiten)
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edoc DOI:
Last Modified:01 Jul 2020 12:49
Deposited On:25 Mar 2019 15:46

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