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I. Development of an NMR-Based Assay for Human Serine Racemase II. Toward a New Variant of the in Situ Enzymatic Screening Method for Catalyst Discovery

Stephany Marie Ramos De Dios, University of Nebraska - Lincoln


Human serine racemase (hSR) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme involved in the racemization and β-elimination of L-serine to give D-serine and pyruvate, respectively. D-Serine is the principal co-agonist of the NMDA receptor, binding at the ‘glycine site’ with 100-fold higher potency than glycine itself. Importantly, this D-amino acid is involved in long-term potentiation (LTP) and synaptic plasticity. Moveover, regulation of D-serine concentration is crucial since elevated levels of D-Ser have been associated with neuronal damage pursuant to ischemic stroke, Alzheimer’s disease, and ALS, whereas reduced levels have been associated with schizophrenia. Pyruvate produced from hSR has been associated with colorectal cancer, suggesting that both reactions catalyzed by hSR are biological important. There is a currently great interest in understanding hSR mechanism and function and in developing hSR activity modulators as a chemical biology tools. Chapter 1 of this dissertation presents a new NMR-based assay that provides information on both catalytic activities of hSR, in parallel, taking advantages of 13C/2H- isotopic crosstalk. This assay was used to screen a library of 1020 small molecules across a variety of chemotypes designed to have ‘drug-like’ parameters, such as possessing ‘privileged’ scaffolds and/or obeying Lipinski’s rules. Interestingly, this work reveals mechanistic insight for hSR, specifically that the rate of H/D exchange is ≥ 13-fold faster than racemization, indicating si-face protonation far outcompetes re-face protonation (racemization) for the putative carbanionic intermediate. The assay was also able to identify an indolo-chroman-2,4-dione inhibitor family that compares favorably with best known hSR inhibitors to date. The second part of this dissertation features efforts to develop a new variant of the In Situ Enzymatic Screening (ISES) method for reporting in real time on relative rates for transformations of interest. Proof of principle of ‘phosphate-ISES’ is demonstrated herein with a platform reaction that targets -(1’-fluoro)vinylglycine via transition metal-mediated intramolecular allylic amination upon an allylic dialkyl phosphate substrate.

Subject Area


Recommended Citation

Ramos De Dios, Stephany Marie, "I. Development of an NMR-Based Assay for Human Serine Racemase II. Toward a New Variant of the in Situ Enzymatic Screening Method for Catalyst Discovery" (2022). ETD collection for University of Nebraska - Lincoln. AAI29169243.