Graduation Semester and Year

Summer 2024

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Daniel Armstrong

Second Advisor

Purnedu Dasgupta

Third Advisor

Kevin Schug

Fourth Advisor

Carl Lovely

Abstract

This dissertation covers the development of methods to analyze chiral small molecules and d-amino acid containing peptides. The first section covers the development of liquid chromatographic chiral screening approaches for newly synthesized chiral molecules. The next part of the dissertation covers methodologies for the analysis of d-amino acid containing peptides. Lastly the bacterial degradation of three commonly used pesticides by bacteria harvested from Colorado potato beetles (Leptinotarsa decemliniata) is presented.

Twelve new azole compounds and eleven racemic ethanolamine derivatives were synthesized. These twenty-three compounds were evaluated using eight chiral columns, four macrocylic glycopeptides, a cyclodextrin derivative column and a cyclofructan derivative column all of which were bound to superficially porous silica gel particles. The remaining two columns were a cellulose derivative and amylose derivative stationary phase immobilized on fully porous silica gel particles. The enantiomers of the twenty-three compounds could be separated using the three different mobile phase elution modes: normal phase, polar organic and reversed phase. The most effective column for the azole compounds was the MaltoShell column which was able to separate eleven of the twelve compounds. The most effective column for the ethanolamines was the cyclodextrin based column which was able to separate 3 compounds with vastly different structures. Possible mechanisms for chiral recognition are discussed based on the structures of the separated compounds

Carboxypeptidases enzymatically cleave the peptide bond of carboxy-terminal amino acids residues. Carboxypeptidases A and Y have difficulty hydrolyzing the peptide bond of dipeptides and some other amino acid sequences. Herein it is conclusively demonstrated that 6-aminoquinoline-N-hydroxysuccimidyl carbamate (AQC), as an N-blocking group, greatly enhances substrate hydrolysis with carboxypeptidase. AQC addition to the N-terminus of amino acids and peptides also improves chromatographic peak shapes and sensitivities via mass spectrometric detection. These enzymes were used for amino acid sequence determination prior to the advent of modern proteomics. However, most modern proteomic methods assume that all peptides are comprised of l-amino acids and therefore cannot distinguish l-from d-amino acids within the peptide sequence. The majority of existing methods that allow for chiral differentiation either require synthetic standards or incur racemization or degradation in the analysis process. Kinetic studies were used to determine the difference in the rate of hydrolysis between l-amino acid exclusive peptides and d-amino acid containing peptides. It was shown that the l-amino acid exclusive peptides studied are hydrolyzed at a rate two orders of magnitude faster than d-amino acid containing peptides. This difference can be used to completely digest the l-amino acid exclusive peptide while leaving the C terminal d-amino acid intact. The d-amino acid containing peptides can then be preconcentrated and more easily identified. This appears to facilitate scouting for d-amino acid containing peptides.

The Colorado potato beetle (Leptinotarsa decemliniata) is a devastating agricultural pest that has played a major role in the development of the chemical pesticide industry. While the innate resistance mechanisms of the CPB have received much attention, we are only beginning to understand microbial contributions to resistance in this important agricultural pest. Towards this end, we measured the capacity of the CPB’s gut microbiota to degrade three common insecticides: fenitrothion, malathion, and imidacloprid. After 48 hours of in vitro exposure to pesticides, no bacterial isolates degraded imidacloprid, six isolates weakly degraded fenitrothion, and three bacterial isolates (identified as Acinetobacter calcoaceticus, Pseudomonas protegens, and Lactococcus lactis) removed most or all of the malathion. Interestingly, two of these three isolates preferentially degraded different enantiomers of malathion. These findings suggest that in addition to diverse mechanisms of innate pesticide resistance, the Colorado potato beetle may also benefit from an exogenous resistance mechanism: pesticide degradation by its gut microbiota. This work paves the way for further investigation of microbial contributions resistance in this common and voracious crop pest.

Keywords

Chiral separations, Azoles, Kinetics, D-amino acid containing peptides, Carboxypeptidase Y, Pesticide

Disciplines

Analytical Chemistry | Biochemistry | Environmental Microbiology and Microbial Ecology

License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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Available for download on Wednesday, February 12, 2025

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