Investigating biological checkpoints through organic chemistry: Synthesis of the phomopsin sidechain and ATR probes

Biological checkpoints are mechanisms whereby the initiation of later cell cycle events is delayed until earlier events are properly completed. Checkpoints regulate many aspects of the cell cycle. The work described in this thesis is focused on using organic chemistry to generate probes for multiple checkpoints: the mitotic checkpoints, and DNA damage checkpoints and the replication checkpoint.; Phomopsin A is a natural product that inhibits mitosis and has been shown to depolymerize microtubules. Use of this molecule, and synthetic variants of it, will allow study of the interaction of microtubules with proteins required to regulate and execute mitosis, i.e. components of the checkpoints involved in mitosis. Using organic chemistry, work towards the synthesis of the phomopsins is described. This thesis focuses on the phomopsin tripeptide sidechain. The development of a novel methodology, the sulfamidite approach, for the synthesis of α,β-dehydroamino acids is presented. This approach is then applied specifically to (E)-dehydroisoleucine for the synthesis of the phomopsin sidechain.; The focus then changes from probing mitotic checkpoints to understanding the role of the protein ATR (ATM and R&barbelow;ad-3 related) in DNA damage checkpoints and the replication checkpoint. Bifunctional molecules linking wortmannin and biotin were synthesized to identify ATR-associated proteins. Wortmannin is a natural product that binds ATR. By linking it to biotin, streptavidin beads can be used to isolate the bifunctional molecule, the covalently attached ATR, and any ATR associated proteins.; To further study ATR's role, damaged DNA, in the form of cyclobutane-pyrimidine dimers, was generated. The dimer was also incorporated into a 20-base oligonucleotide for use as a probe. Finally, the oligonucleotide was annealed to single stranded DNA and polymerized to generate a double-stranded DNA plasmid with a single pyrimidine dimer. This dimer containing DNA can be added to cycling Xenopus extracts to study the effects on cell cycle checkpoints.; Use of all of these molecules: phomopsin, the biotin-wortmannin conjugates, and the thymidine dimer-containing compounds will hopefully provide insights into biologically complex questions. The biological studies are underway, but even without all of the final results, this work shows the power of organic synthesis within biological systems.