Synthesis of Isotopically Labeled Co-Enzyme to Probe the Active Site of Tryptophan synthase/ New Synthetic Approach to Tetrahydrocannabinol Analogs

Identifying enzyme mechanisms at proton level resolution is the ultimate goal of enzymology. Traditional enzyme mechanistic studies infer protonation states from x-ray crystal structure and optical spectroscopy. This thesis reports work towards the first synergistic combination of x-ray crystallography, computational chemistry, synthetic organic chemistry and solid-state NMR to fully elucidate, at proton level resolution, the full three-dimensional structure of the catalytic site for Tryptophan synthase during active catalysis. Specifically, this thesis describes solutions to the synthetic challenges of introducing site-specific isotopic labels inside the cofactor Pyridoxal-5’-Phosphate (PLP) and highlights a synthetic route that is consistently more cost-effective and higher yielding than previous efforts. The second project presented focuses on efforts towards the synthesis of cannabinoids, cannabidiol (CBD) and tetrahydrocannabinol (THC). Presently, cannabinoids have emerged as compounds of interest for a variety of pharmacologic indications. Although stereochemically simple compounds, economical syntheses of enantiopure cannabinoids remain elusive. Strategies to address facile syntheses of THC and CBD, as well as their analogs, will be presented

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity.

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5'-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography-the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry-to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate Cα and positive charge at C4' of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5′-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallographythe synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistryto interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate C^α and positive charge at C4′ of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites

Protonation States of the Tryptophan Synthase Internal Aldimine Active Site from Solid-State NMR Spectroscopy: Direct Observation of the Protonated Schiff Base Linkage to Pyridoxal-5′-Phosphate

The acid–base chemistry that drives catalysis in pyridoxal-5′-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP’s role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ε-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first ¹⁵N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional ¹³C, ¹⁵N, and ³¹P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established