Author

Sam Sung

ORCID Identifier(s)

0009-0009-0842-0898

Graduation Semester and Year

2023

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Daniel W Armstrong

Abstract

Amino acids are essential building blocks in all life on Earth. It was first mistakenly believed that only L-amino acids were of biological relevance in higher organisms, and D-amino acids were laboratory artifacts or biologically irrelevant. It is well accepted today that various D-amino acids exist in different organisms, including humans, and some even play critical roles in biological pathways and processes. In addition, aberrant levels of certain D-amino acids have been reported in patients with varying diseases. It has been proposed that the aberrant variations in the levels of D-amino acids in biological fluids may serve as biomarkers for disease in humans. However, due to the lack of a comprehensive and robust analytical platform a full understanding of D-amino acids remains elusive and most of D-amino acids and their biological roles are not well investigated. An analytical challenge is the analysis of D-amino acids in biological samples, because dominant L-amino acid signals and a mix of other endogenous compounds can interference with detection and quantitation of the trace analytes of interest. Additionally, current methods for the comprehensive analysis of D-amino acids are time consuming. Therefore, the main goals of this dissertation are to a) enhance the sensitivity of current analytical methods and platforms for D-amino acid analysis in biological samples, and b) provide information and methodologies that future studies can use to investigate the roles of D-amino acids in biological systems. A comparison between using UV, MS single quad, multiple reaction monitoring (MRM) detection modes was investigated to determine limits of detection (LOD) for the analysis of AQC-amino acids. This achiral method showed improved detection sensitivity as low as pg and sub-pg levels when using high sensitivity detection modes such as MRM and fluorimetry. A separate study reported the first comprehensive analysis of the intracellular and extracellular profiles of L- and D-amino acid in human breast cancer cells (MCF-7) and non-tumorigenic epithelial breast cells (MCF10A) using HPLC-MS/MS. This study further advanced our knowledge of how D-amino acids can interact with the NMDA receptors in cancer cells. For the differentiation of peptides containing D-amino acids, a novel enzymatic technique using carboxypeptidase Y, coupled with selective retention of D-amino acids and D-amino acid containing peptides on teicoplanin type stationary phases was developed. This enzyme hydrolytically cleaves L-amino acids at the peptide bond from the C-terminus of peptides, but its catalytic efficiency decreases dramatically when confronted with a C-terminal D-amino acid. The strong retention of D-amino acids and D-amino acid containing peptides on teicoplanin stationary phases was then exploited to analyze intact fragments of the carboxypeptidase hydrolysis by LC-MS/MS with little to no interference from more abundant L-amino acid peptides. A fully comprehensive, highly sensitive and selective 2D-HPLC-MS/MS chiral separation method was developed along with a complementary GC-MS/MS method for the study of L- and D-homoserine lactone homologues in the extracellular media of gram-negative bacteria.

Keywords

D-amino acids, D-amino acid containing peptides, AQC-derivatization, carboxypeptidase Y, teicoplanin aglycone, homoserine lactones, HPLC, LC-MS/MS, chiral separations.

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

Included in

Chemistry Commons

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