Chemical Synthesis to Understand and Influence Biological Systems: Spin Labels, Drug Development, and Biosynthetic Investigations

• Author / Creator

  Vuong, Wayne

• Spin labels are a useful tool towards the study of protein conformation and enable the mapping of interactions between specific positions within these systems. Synthetic routes towards three different chiral spin-labelled amino acids have been developed. Compared to commercially available spin labelled amino acids, these compounds better replicate the structures found in canonical amino acids. One of the routes involves direct alkylation of an electrophile onto a chiral glycinate, thereby generating the desired natural stereochemistry at the α-position of the resultant amino acid. The other two routes involve a racemic bond formation reaction between the side chain electrophile and an appropriately protected glycinate nucleophile. This was then followed up with deracemization via dynamic kinetic resolution. While enantioselectivity was not perfect, we demonstrated that two of the three structures produced can be further enantiopurified using D-amino acid oxidase, affording a product with high stereochemical purity as verified through Marfey’s assay. Overall, these novel structures aim to be amenable towards incorporation into peptides or proteins through solid phase peptide synthesis or metabolic incorporation via amber codon suppression.
  A feline drug, GC376, that originally targets the feline coronavirus FCoV was demonstrated to hold potential as a COVID-19 therapeutic. It was shown to possess nanomolar
  activity against the main protease of SARS-CoV-2 as well as the original SARS-CoV, exhibiting inhibition in enzymatic assays as well as reducing viral titers in mammalian cells. This inhibitor works by binding to and inhibiting the activity of a catalytic Cys145 in the active site of the main protease, thereby halting its activity as was verified through X-ray crystal structures as well as 13C-labelled NMR studies. The bisulfite adduct prodrug forms of these compounds showed greater activity, likely owing to improved solubility in aqueous media.
  With the demonstrated activity of GC376 against SARS-CoV-2, improved inhibitors based on this structure were developed. The development of these new inhibitors were concurrently guided by enzymatic assays, mammalian cell assays, and crystallography. This culminated in an SAR study demonstrating that polar groups in the P3 position of the inhibitor or the presence of a nearby chiral methyl group can promote an alternative mode of binding wherein the P3 position (Figure 3.3) preferentially docks in the deeper S4 position of the enzyme (Figure 3.14a), thereby resulting in improved protease inhibition. Studies on the stereochemical characteristics of GC373 and GC376 were also conducted, demonstrating that:

  1. Epimerization of the α-position readily occurs in aqueous media.
  2. GC373 exists as a single enantiomer in aprotic solvents but as a mixture of two diastereomers in aqueous media.
  3. GC376 exists as a mixture of four diastereomers, and the associated diastereomeric ratios of these species were determined. Additionally, it was demonstrated that these bisulfite adducts undergo aggregation and appear to form micelles at sufficiently high concentrations, potentiating delivery of these compounds as micellar solutions. Finally, cation replacement of GC376 was demonstrated to significantly increase solubility; up to 90% when a sodium ion is replaced with a choline. An improved synthetic route to access a building block used to incorporate a γ-lactam ring in numerous antivirals was developed. The improved synthesis of this γ-lactam glutamine analog is scalable, inexpensive, and more than doubles yields when compared to the original synthesis developed by Pfizer. Furthermore, purification is simple, requiring only a quick silica plug in a majority of reactions. In a tangential direction, work was directed towards overcoming metabolic issues with P450 oxidation. Based on reports of oxidation occurring on the γ-lactam ring of nirmaltrevir in Paxlovid, it was anticipated that similar reactions would occur with GC376 as well. Attempts were made to replace the metabolically susceptible CH2 in the ring with an oxygen, with work in this area still ongoing. Efforts were also directed towards understanding the biosynthetic programming of polyketide synthases. To test the theory that regulation is a function of reaction kinetics, a series of polyketide substrates and product standards were synthesized. Work is currently ongoing with expression of the polyketide domains required to do kinetic assays.

• Subjects / Keywords

  • organic chemistry
  • chemistry
  • drug development
  • therapeutic
  • amino acids

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-qz3k-vs12

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.