Dissertations, Theses, and Capstone Projects

Date of Degree

9-2026

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy

Program

Biochemistry

Advisor

Mateusz Marianski

Committee Members

Daron Freedberg

David Mootoo

Emilio Gallicchio

Jianbo Liu

Subject Categories

Biochemistry | Computational Chemistry

Keywords

Glycans, Density Functional Theory, Molecular Dynamics, Stereochemistry

Abstract

Carbohydrates (glycans) are among the most structurally diverse biomolecules and participate in a wide range of biological processes, including molecular recognition, cell adhesion, immune response, host-pathogen interactions, and cellular signaling. Their biological functions are governed by a complex interplay between sequence, stereochemistry, conformational flexibility, and environmental factors. However, the structural heterogeneity and dynamic nature of glycans present significant challenges for both experimental characterization and theoretical modeling. In particular, the relationships between glycan structure, conformational dynamics, and the resulting potential energy surfaces remain incomplete.

This dissertation investigates how external factors modulate the potential energy surfaces of carbohydrates through a combination of density functional theory, ab initio calculations, molecular dynamics simulations, and the interpretation of complementary experimental measurements. Particular emphasis is placed on understanding the structural origins of glycan flexibility and the mechanisms of gas-phase rearrangement reactions that complicate mass spectrometric analyses.

The first part of this dissertation investigates gas-phase carbohydrate rearrangement reactions in mass spectrometry (MS). MS spectra show non-native peaks that originate from a fucose migration along the glycan sequence which can lead to incorrect structure assignment of a glycan. Using a combination of high-level electronic structure calculations, spectroscopy guided structural analysis, we determined the fucose migration product Lewis x and blood group H2 antigens. Furthermore, ab initio molecular dynamics simulations revealed a detailed mechanistic insights into this rearranment. The simulations show that proton transfer events play a central role in initiating rearrangement pathways by activating glycosidic bonds and transiently altering nucleophilic sites within the glycan framework. Multiple competing pathways involving proton shuttling, bond dissociation, non-covalent intermediates, and subsequent reformation of glycosidic linkages were identified. These findings provide atomistic explanations for experimentally observed rearrangement products and establish a mechanistic framework for understanding migration reactions in protonated glycans.

Similar analysis was then performed on the protonated ions of Lewis a and BG-H1 antigens resulted in the assignment of the experimental spectra to the structures of the respective parent ions. By comparing these two pairs of antigens, we determine that the internal rearrangement reaction is triggered by the proximity of the protonated amide group to the glycosidic bond and the mobility of the proton: the Lewis a and BG-H1 structures lacks the mobile proton to initiate the migration, whereas a loosely bound proton in Lewis x and BG-H2 was able to protonate the fucose glycosidic bond. These findings show that the migration of fucose moiety is a sequence-dependent phenomenon and most likely occurs following in-source activation of intact ions during the nanoESI process. Later, we examined the migration of sulfate groups in glycosaminoglycan molecules.

Through combined experimental and computational investigations, the structures of rearrangement products and plausible migration pathways were characterized, providing new insight into the origins of sulfate scrambling observed during tandem mass spectrometry experiments. The results demonstrate that even subtle changes in protonation state, substituent identity, or local molecular environment can substantially alter the topology of the underlying potential energy surface and redirect reaction pathways.

Lastly, we focused on the conformational behavior of Lewis antigens in solution. Extensive microsecond-scale molecular dynamics simulations were performed on Lewis trisaccharides and tetrasaccharides to investigate the interplay between glycosidic torsions, GlcNAc ring-puckering dynamics, substituent effects, and anomeric configuration. The simulations reveal that conformational flexibility cannot be described solely by glycosidic rotations but is strongly coupled to pyranose ring-puckering transitions. Lewis a antigens were found to be substantially more flexible than their Lewis x counterparts, while the influence of anomeric configuration depended on oligosaccharide size. Comparison of complete antigens with their constituent disaccharide fragments demonstrated that neighboring substituents significantly reshape local conformational preferences and restrict accessible torsional space. Furthermore, a comparative analysis of the GLYCAM06j and CHARMM36m force fields showed that both reproduce the same qualitative flexibility trends, although GLYCAM06j predicts broader conformational ensembles and more frequent ring-puckering transitions.

Collectively, this dissertation demonstrates that glycan behavior is governed by a delicate balance between conformational flexibility, intramolecular interactions, and environmental perturbations. By elucidating how structural features and external factors reshape carbohydrate potential energy surfaces, this work advances the molecular-level understanding of glycan dynamics and mechanisms while providing new foundations for the interpretation of glycomics experiments and the development of predictive computational models.

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