Synthesis and characterisation of a new class of alkene polymers bearing nucleotide functionality

Author: Michael Wilson

Wilson, Michael, 2020 Synthesis and characterisation of a new class of alkene polymers bearing nucleotide functionality, Flinders University, College of Science and Engineering

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DNA is a fundamental compound in biology and, from a polymer science perspective, exceeds most synthetic polymers in terms of size and composition. This thesis outlines a synthetic method for an alkene polymer bearing nucleotide functionality and its subsequent properties.

Alkene polymers are built on the radical polymerisation of alkenes leading to long chain alkyl backbones and are known to be stable for long periods. Combining the selectivity and control of DNA with the rapid synthesis and stability of living radical techniques will allow applications of DNA nanotechnologies in environments where DNA is currently unsuitable.

The background and information relevant to a thorough understanding of this thesis is presented in Chapter 1. This includes discussion of the nature of DNA and variants of its structure, methods for its synthesis, and analysis of the subsequent products. Further, information essential to understanding the polymerisation methods utilised in this work such as the radical polymerisation and the reversible addition-fragmentation chain-transfer method are also discussed. Existing literature discussing the convergence of these two fields is also covered, with the specific methods and materials as they relate to this work presented in the following Chapter 2.

Chapter 3 outlines the initial method developed for the synthesis of a nucleotide functionalised polymer using the phosphoramidite method. This method has been reported previously for the synthesis of DNA and other nucleic acids (broadly poly(ribose-phosphates)). The phosphoramidite method was altered to generate a nucleotide-alkene monomer using a protected cytosine phosphoramidite. This was possible through careful selection of the alkene moiety (hydroxyethyl methacrylate (HEMA)) and the optimisation of experimental parameters. This method was found to successfully generate the polymer as confirmed with proton and 31phosphorus nuclear magnetic resonance (NMR) spectroscopy.

The synthesised HEMA-cytosine monophosphate bioconjugate monomer was polymerised using reversible addition-fragmentation chain-transfer (RAFT) polymerisation. The resulting polymers were limited in their applications as interconversion of the monomer appeared to form a diene that caused the polymer to be cross-linked, resulting in the formation of an insoluble gel. This gel was found to retain the nucleobase functionality and demonstrated the interaction of the nucleotide-alkene with solvents consistent with a hybrid polymer system.

Chapter 4 builds upon Chapter 3, presenting an improved method for the synthesis of a nucleotide-alkene bioconjugate by changing the alkene source from HEMA to 2-hydroxypropyl methacrylate (HPMA) and the nucleotide source to guanosine monophosphate. The HPMA was chosen as the presence of an additional methyl group explicitly prevented the formation of the diene impurity in the monomer. This improved method was shown to result in the HPMA-protected guanosine monophosphate through proton and 31phosphorus NMR spectroscopy along with electrospray ionisation mass spectrometry.

The compatibility of the synthesised HPMA-guanosine monophosphate with RAFT polymerisation was then demonstrated through both in-situ and ex-situ techniques. Samples measured in-situ utilised NMR spectroscopy, with ex-situ measurements using size exclusion chromatography. The poly(HPMA-guanosine monophosphate) formed colloidal suspensions during isolation which lead to further analysis in the following chapter.

Chapter 5 investigated the formation of particles from the poly(HPMA-guanosine monophosphate). It was shown that the polymer was able to replicate the formation of G-quartets normally formed in guanine-rich DNA systems. Through the use of fluorescent dye binding, circular dichroism and fluorescence microscopy, the general structure of these particles was determined to be similar to vesicles or micelles with surfaces covered in active guanine moieties. The aggregation of the particles could be adjusted by the addition of potassium chloride to stabilise the formation of G-quartets between particles.

Finally, Chapter 6 demonstrated the successful synthesis of poly(HPMA-thymine monophosphate) by utilising the synthetic method developed in Chapter 4, thereby demonstrating the flexibility of the synthesis for creating a library of nucleotide-alkene bioconjugate compatible with RAFT polymerisation.

Following this successful synthesis of poly(HPMA-thymine monophosphate), its interactions with single stranded DNA were investigated with the aid of computer modelling and the use of nucleic acid binding dyes. This led to the determination that the poly(HPMA-thymine monophosphate) does bind to a complementary single stranded DNA sequence, but that the structure formed was conformationally distinct to classical double stranded DNA.

Keywords: DNA, Polymer, bioconjugate, DNA analogue, RAFT polymerisation, RAFT polymerization, RAFT, living radical, g-quadruplex, Ascalaph

Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2020
School: College of Science and Engineering
Supervisor: Joe Shapter