UC Irvine

Nucleic acids are among the fundamental building blocks of life, empowering numerous biological functions including genetic information storage, protein production, and cellular signaling. Beside their innate functions in the biology realm, nucleic acids also display vast potential in pharmaceutical, therapeutic, and biotechnology development. However, the scalable synthesis of the monomers of such importance-including nucleosides, phosphoramidites, and triphosphates-remained a major challenge over many decades, especially nucleoside triphosphates. The very properties that grant nucleoside triphosphates dominant roles in life also made them challenging to chemically synthesize and purify on large scales. With the recent emergence of xenobiotic nucleic acids (XNA) and their related biotechnologies such as XNA polymerases, a scalable, time-efficient, and high-yielding triphosphate synthesis method that over encompass more substrate is urgently warranted.In this thesis, we first dive into the developmental history of chemical synthesis of nucleoside triphosphates. Next, we applied the synthesis methods that were pioneered by early chemists on the synthesis of nucleoside triphosphate for threose nucleic acid (TNA) unnatural bps in aim of expanding the TNA genetic alphabet. We also re-evaluated extinction coefficients (ECs) that were crucial to the quantification of nucleic acids monomers and polymers. Finally, a vastly improved triphosphate synthesis based on previous methods was introduced and subsequently applied to some of the ongoing synthesis of base-modified TNA nucleosides.Chapter 1 begins with a review of the history and development of the chemical synthesis of nucleoside triphosphates. We backtrack to the first ever reported chemical synthesis of adenosine triphosphate and examine all the major methods that emerged over the past 70 years. Their advantages are compared and limitations scrutinized for the inspiration and development of improved method that will be discussed in greater detail in Chapter 4.Chapter 2 describes the synthesis of TNA isoG and 5MeisoC phosphoramidites and triphosphates in an effort to expand the TNA genetic alphabet. The study shows the synthesis of unnatural nucleoside triphosphates via our previous method, which contained severe drawbacks despite its superior scalability compared to conventional methods involving HPLC purification. We also tested the ability of DNA-dependent TNA polymerase 10-92 to recognize and incorporate the new triphosphate and discovered the still existing isoG tautomerization issue repeatedly reported in the literature. It was also surprising to see the potential successful incorporation of 5MeisoC across an isoG containing DNA template. Although the incorporation likely induced chain termination, its potential successful incorporation opened up new ideas for future polymerase engineering efforts.Chapter 3 describes the determination of the accurate EC of TNA nucleosides. In the past, the quantifications of TNA related monomers and polymers such as triphosphates and oligonucleotides were conducted under the assumption of their identical UV-absorbance properties with their DNA and RNA counterparts, largely ignoring other factors such as hypochromic effects and sugar backbone differences, compromising the absolute accuracy of measurements. After acquiring and examining the EC of all four standard base TNA nucleosides and two base-modified tUs, we indeed validated the necessity of acquiring EC of any newly emerged XNAs instead of borrowing the ancient number sets from their DNA/RNA parents based on the trend observed.Chapter 4 describes the development of a scalable one-pot multistep synthesis of TNA and DNA nucleosides triphosphates. The method took inspiration from phosphoramidite chemistry and leveraged the advantage of an in-house synthesized pyrophosphate donor that displayed minimal hydroscopic properties. It resulted in a one-pot transformation of nucleoside phosphoramidites to fully protected triphosphates that could be purified via conventional silica gel chromatography and that is free from the complicating byproducts generated by previous methods. The purified fully protected triphosphates could be sequentially subjected to ammonolysis and a final NaClO4 precipitation to give the free triphosphate in high yield and purities. The method has proven to be applicable to not only standard base TNAs but also base-modified tUs and DNAs, further elaborating its generalizability. It was also applied to the synthesis of triphosphates for base-modified tCs in Chapter 5. Furthermore, the utilization of phosphoramidite chemistry, which has been employed and optimized for decades, vastly expanded the substrate repertoire of the new method to encompass modified DNA/RNAs and XNAs that bear a variety of different functional groups that were incompatible with previous methods including pyrroles and thiols.Chapter 5 describes the effort to improve the previous synthesis of base-modified tCs and the application of the new triphosphate synthesis method. Although it still came with a limited scope of substrate, the new synthesis greatly alleviated the issue of extensive reagent usage and prolonged synthesis period characteristic of the previous method. It also warranted future investigation on the cytidine base modification chemistry for a further improved synthesis to help expanding the arsenal of base-modified cytidines and other nucleosides for applications in therapeutics, diagnostics, and biotechnologies.