Efficient Scalable Synthesis of Shld and Study of Shld-Analogues in vitro and in vivo

Research output: Book/ReportPh.D. thesisResearch

Frederik Præstholm Jørgensen

The work presented in this thesis is related to the synthesis of ligands for the point-mutated protein FKBP12(F36V). FKBP12(F36V) has been used to construct so-called destabilized domains (DDs), which are unstable proteins that are constitutively degraded in vivo. Upon addition of a ligand for the binding domain of the DD, the protein becomes stabilized and will no longer be degraded, resulting in accumulation of the protein in vivo. Expression of DD as a fusion protein attached to a protein of interest (POI), allows spatiotemporal control of the given POI level by addition of ligand. The ligand/DD-POI system can thereby be applied to control various biological functions in both mammalian and plant cells. The most commonly used stabilizing ligand for DD is Shld. The synthesis of Shld is poorly described in literature, and the synthesis of related analogues seemed impractical for a large-scale synthesis of Shld, needed for the DD-POI system to be integrated in agriculture. Chapter 2 of this thesis will describe the efforts made towards optimizing the synthesis of Shld. Shld was synthesized in 9 steps from commercially available and inexpensive building blocks through a convergent synthesis in an overall yield of 40%. Key features of the synthetic procedure involved avoiding protection/deprotection steps, employing organo-catalytic reagents and utilizing a one-pot enantioselective alkylation. Chapter 3 describes development of new Shld-analogues designed to occupy the binding site of FKBP12(F36V) more tightly, giving a stronger binding affinity. 12 Shld-analogues were synthesized for this purpose but none of them exhibited superior binding affinity nor did they facilitate accumulation of the fusion protein RDDKeGFP when treated to transgenic Arabidopsis. However, the obtained results did indicate the importance of an intramolecular arene-arene interaction preorganizing the ligand for binding events as well as stabilizing the binding conformation. Chapter 4 will describe the efforts towards developing plant cell permeable Shld-analogues needed to control protein levels in plants by external application of ligand. 23 Shld-analogues were synthesized and their binding affinities as well as their effect in transgenic Arabidopsis were established. Several of the synthesized analogues displayed both binding affinities, and effect in transgenic Arabidopsis, comparable to that of Shld. No definite conclusions regarding an optimal structure of a plant cell permeable ligand could be drawn from the obtained results, though size and polarity seemed to heavily influence the uptake in plants. Five selected analogues were also tested on transgenic rice. These preliminary studies revealed that two of the selected analogues displayed superior permeability in rice compared to Shld.
Original languageEnglish
PublisherDepartment of Chemistry, Faculty of Science, University of Copenhagen
Publication statusPublished - 2019

ID: 215238191