Biocatalytic Synthesis of Antiviral Nucleosides, Cyclic Dinucleotides, and Oligonucleotide Therapies

Abstract

Nucleosides, nucleotides, and oligonucleotides modulate diverse cellular processes ranging from protein production to cell signaling. It is therefore unsurprising that synthetic analogues of nucleosides and their derivatives have emerged as a versatile class of drug molecules for the treatment of a wide range of disease areas. Despite their great therapeutic potential, the dense arrangements of functional groups and stereogenic centers present in nucleic acid analogues pose a considerable synthetic challenge, especially in the context of large-scale manufacturing. Commonly employed synthetic methods rely on extensive protecting group manipulations, which compromise step-economy and result in high process mass intensities. Biocatalytic approaches have the potential to address these limitations, enabling the development of more streamlined, selective, and sustainable synthetic routes. Here we review recent achievements in the biocatalytic manufacturing of nucleosides and cyclic dinucleotides along with progress in developing enzymatic strategies to produce oligonucleotide therapies. We also highlight opportunities for innovations that are needed to facilitate widespread adoption of these biocatalytic methods across the pharmaceutical industry.

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides

“…While most efforts to improve the efficiency of de novo DNA synthesis are centered around TdTmediated, template independent oligonucleotide production, template-dependent approaches are also emerging. (Hoff et al, 2020;Van Giesen et al, 2022;Hoose et al, 2023) A main advantage of templatedependent synthesis is the plethora of polymerases that have been engineered to display very lax substrate requirements and which might be capable of incorporating blocked nucleotides. On the other hand, template-dependent synthesis leads to the formation of dsDNA rather than ssDNA products but this can be circumvented by immobilizing products on solid-support or to an extent by using universal templates.…”

“…In addition, the sustainability (Andrews et al, 2021) as well as the scalability (Molina and Sanghvi, 2019) of phosphorous(III)-based oligonucleotide synthesis are limited which negatively impacts scalable manufacturing. (Van Giesen et al, 2022) Hence, various enzymatic methods are currently developed to alleviate the shortcomings of solid-phase synthesis of nucleic acids. In this context, controlled enzymatic synthesis represents a promising approach where temporarily blocked nucleoside triphosphates are incorporated sequentially into DNA mainly by templateindependent polymerases such as the terminal deoxynucleotidyl transferase (TdT).…”

“…In this context, controlled enzymatic synthesis represents a promising approach where temporarily blocked nucleoside triphosphates are incorporated sequentially into DNA mainly by templateindependent polymerases such as the terminal deoxynucleotidyl transferase (TdT). (Jensen and Davis, 2018;Lee et al, 2019b;Sarac and Hollenstein, 2019;Doricchi et al, 2022a;Lu et al, 2022;Van Giesen et al, 2022;Wang et al, 2022;Ashley et al, 2023;Hoose et al, 2023) The blocking groups can be affixed either at the 3'-hydroxyl moiety to prevent further nucleophilic attack on the -phosphorous of incoming nucleoside triphosphates (Bollum, 1962;Mackey and Gilham, 1971;Chen et al, 2010;Hutter et al, 2010;Gardner et al, 2012;Chen et al, 2013;Mathews et al, 2016;Jang et al, 2019) or on the nucleobase which then act as inhibitors of polymerases. (Bowers et al, 2009;Palluk et al, 2018) While robust protocols have been established for DNA, (Palluk et al, 2018;Lee et al, 2019b;Jung et al, 2022;Venter et al, 2022;Wang et al, 2022) changing the sugar chemistry to ribose (RNA) or to more complex modification patterns deviating from natural systems (xenonucleic acids, XNAs (Chaput and Herdewijn, 2019;Chaput et al, 2020)) raises yet unmet challenges.…”

The Evolving Nature of Biocatalysis in Pharmaceutical Research and Development

“…This perspective specifically aims to focus on recent key examples of implementation of biocatalysis for scalable syntheses of pharmaceutical intermediates. Several excellent reviews have been published on industrial biocatalysis covering various aspects of enzymatic transformations for drug development. ,,− As such, this perspective does not aim to be comprehensive but rather highlight the current state-of-the-art of preparative enzymatic synthesis in an industrial setting. Therefore, the use of biocatalysis for small-scale pharmaceutical metabolite synthesis, lead diversification, ,,, bioconjugation, and selective protein modification − will not be focused upon in this article.…”

Einseitige Borat‐Veresterung während enzymatischer Nukleosid‐phosphorolyse: Scheinbare Gleichgewichtsverschiebungen und kinetische Auswirkungen**

“…Eine solche Reaktion wird typischerweise als (Trans-)Glykosylierung bezeichnet. [9] Obwohl solche (Trans-)Glykosylierungsprozesse als Synthesewerkzeuge gut etabliert sind, [10][11][12][13][14][15][16][17][18][19][20][21] leiden sie inhärent unter thermodynamischen Limitationen, da die Endausbeute dieser Reaktionen ausschließlich von den substratabhängigen thermodynamischen Eigenschaften der jeweiligen (umgekehrten) Phosphorolyseschritte sowie von den angewandten Reaktionsbedingungen bestimmt wird. [9,22,23] Obwohl einige Fortschritte erzielt wurden, um die strenge thermodynamische Kontrolle in diesen Systemen abzuschwächen oder auszunutzen (beispielsweise durch Fällung eines (Neben-)Produkts, [24] die enzymatische Weiterverarbeitung des Produkts [25] oder die Anwendung von einem Überschussreagenz, das zurückgewonnen werden kann [26] ), und die irreversible Phosphorolyse von 6-Oxopurinen gut etabliert ist, [10,27,28] existiert aktuell keine generelle Methode zur direkten Manipulation von Glykosylierungsgleichgewichten.…”