Scaled synthesis of nucleoside phosphates

Abstract

The research within this thesis is primarily concerned with the synthesis of phosphoanhydrides. Taking advantage of promising synthetic approaches developed in recent years in nucleoside phosphorylation, the thesis focuses on optimizing and increasing the ‘usability’ and ‘scalability’ of existing routes both in batch and in semi continuous flow systems. Specifically, the P(III)-P(V) mixed anhydride method developed by Jessen and co-workers (1) was the starting point of this work. Jessen’s “one pot-three steps” synthesis of phosphoanhydrides1 relies upon the bis-(fluorenylmethyl)-diisopropylamine phosphoramidite reagent. (2-3)
Batch optimization and scale-up of phosphitylating reagent preparation was followed by translation of the process into a continuous flow method. The tetrahydrofuran-based batch approach for the preparation of bis-(fluorenylmethyl)-diisopropylamine phosphoramidite was completed within one hour using a simple, extractive purification process, avoiding chromatography. The approach delivered 86% yield, based on theoretical mass recovery, where the isolated material was shown to be 97% pure based on 31P NMR analysis. Despite the batch process proving successful using tetrahydrofuran, a dichloromethane: tetrahydrofuran-based procedure was required for translating the process to flow. Although the mass recovery and the purity levels of bis-(fluorenylmethyl)-diisopropylamine phosphoramidite obtained in the batch reactions were marginally higher, the experiments performed in flow were effective, delivering 81% yield, based on theoretical mass recovery, and 87% purity based on 31P NMR analysis. The flow approach was also demonstrated to be scalable, delivering 25 g of compound.
Subsequently, the synthesis of ADP from AMP using bis-(fluorenylmethyl)-diisopropylamine phosphoramidite in flow systems was explored. To better inform the flow platform, extensive NMR studies were performed to obtain quantitative, high sensitivity 31P NMR spectra during the phosphoramidite coupling, oxidation and deprodetcion steps for the synthesis of phosphoanhydrides in DMF. NMR parameters were improved by measuring the T1 relaxation and the Ernst angle to ensure a qualitatively satisfactory analysis. Although it was not possible to gain detailed reaction kinetics data, the quantitative 31P NMR methods that were developed proved to be a valuable starting point for further analytical studies and were qualitatively informative for the flow studies. Combining flow chemistry with our 31P NMR work, a workflow approach was developed to deliver a robust and reliable synthesis of nucleotides. The method was upscaled from a 0.27 mL chip to a 2.5 mL reaction coil, delivering 1 g of ADP product, with a 65% yield and a purity >99% by HPLC. The scope of our method was explored through its application to the syntheses of UDP and CDP, and these experiments gave promising starting points for future work.
In summary, starting from the Jessen’s “one pot-three steps” phosphoanhydrides synthesis, an improved the batch synthesis of bis-(fluorenylmethyl)-diisopropylamine phosphoramidite was developed and translated to a continuous flow system. Furthermore, a reliable, robust and scalable workflow method for the synthesis of ADP in a continuous flow system was developed, supported by specific 31P NMR analytical methods.

(1) Jessen, H. J.; Ahmed, N.; Hofer, A. Phosphate esters and anhydrides--recent strategies targeting nature's favoured modifications. Organic & Biomolecular Chemistry 2014, 12 (22), 3526-3530. DOI: 10.1039/c4ob00478g.
(2) Bialy, L.; Waldmann, H. Total synthesis and biological evaluation of the protein phosphatase 2A inhibitor cytostatin and analogues. Chemistry 2004, 10 (11), 2759-2780. DOI: 10.1002/chem.200305543 (acccessed 2020/05/18).
(3) Caron, J.; Lepeltier, E.; Reddy, L. H.; Lepetre-Mouelhi, S.; Wack, S.; Bourgaux, C.; Couvreur, P.; Desmaele, D. Squalenoyl Gemcitabine Monophosphate: Synthesis, Characterisation of Nanoassemblies and Biological Evaluation. European Journal of Organic Chemistry 2011, 2011 (14), 2615-2628. DOI: 10.1002/ejoc.201100036 (acccessed 2020/05/19).