Date of Award

2022

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Administrative Home Department

Department of Chemistry

Advisor 1

Shiyue Fang

Committee Member 1

Tarun Dam

Committee Member 2

Marina Tanasova

Committee Member 3

Rebecca Ong

Abstract

Synthetic oligodeoxynucleotides (ODN) have various applications in many areas such as, synthetic biology, chemical biology, antisense drug development, and data storage. As a result, there is a high demand for synthetic ODNs. Many advances have been made with ODN synthesis the purification still remains a bottleneck. The non-chromatographic purification method is developed to address this problem. Similarly, polyethylene glycols (PEGs) have been used in many areas including pharmaceutical applications, surface science, and nanomedicine due to its unique properties. However, monodispersed PEG synthesis is expensive with existing methods. A novel automated solid phase synthesis method is developed to obtain monodispersed PEGs. The first two chapters are an introduction to synthetic oligodeoxynucleotide purification. These chapters describe oligonucleotides and applications, chemical synthesis of oligodeoxynucleotides, impurities in synthetic oligonucleotides, and current purification methods. Even though many chromatographic purification methods are available for synthetic ODN purification, there is no method that is capable of high throughput purification, large scale ODN purification, and long ODN purification. To address this problem, we developed two non-chromatographic purification methods which are based on polymerization, to purify synthetic ODNs. One is catching failure sequences by polymerization, and the second method is catching full-length sequences by polymerization. The second method is less expensive for large-scale and long ODN purification compared to the first method. It is demonstrated that this new non-chromatographic method is suitable for high throughput, large scale, and long ODN purification. The final chapters describe applications of polyethylene glycol and current PEG synthesis methods. For many applications monodispersed PEGs are preferred. However, PEGs synthesized using current synthesis methods are polydispersed and excessive purification is required to obtain monodispersed PEGs. Therefore, it is expensive to obtain monodispersed PEGs using these existing methods. An automated solid-phase stepwise synthesis method is developed for monodispersed PEG synthesis. This solid-phase xxii synthesis method is cost effective because the purification is not required to obtain monodispersed PEGs using this method. Monodispersed PEGs with eight, twelve, and sixteen ethylene glycol units and their derivatives were synthesized using automated stepwise addition of tetra ethylene glycol monomer without chromatographic purification. The monomer consists of a 4,4′ dimethoxy trityl group at one end and the tosyl group at the other hydroxyl group. Wang resin was used as the solid support for the automated synthesis. The automated synthesis cycle consists of three steps, deprotonation, coupling, and detritylation. PEGs were cleaved off from the solid support and analyzed with ESI-MS. After PEG12 synthesis, the coupling step with DMTr monomer had some complications and therefore a new monomer with a smaller protecting group was designed. Using this new monomer monodispersed PEGs with ten, fifteen, and twenty ethylene glycol units were synthesized. The synthesis was done using automated solid-phase stepwise addition of penta ethylene glycol units without chromatographic purification. The penta ethylene glycol monomer consists of a 2-phenethyl group at one end, and the tosyl group at the other hydroxyl group was used for the synthesis. The first step is deprotection and deprotonation, and the second step is coupling. These two steps were performed alternatively until the PEG with the desired length was obtained. After the synthesis, PEGs were cleaved from the solid support and analyzed using ESI-MS.

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