Anke Kurreck

Nucleoside analogues (NAs) are among the most successful small-molecule classes in the fight against cancer and viral infections. Their importance has recently been further underlined by the approval of sofosbuvir for the treatment of hepatitis C virus infections and by developments related to the corona pandemic. In view of the enormous interest from the academic world and the pharmaceutical industry, a variety of chemical and enzymatic synthesis routes have been developed since the first approval of an NA. The selective chemical synthesis of β-anomeric NAs through chemical N -glycosylation remains a challenging and, sometimes, a prohibitively expensive process, thus limiting the available chemical space. In response, full enzymatic cascades for the synthesis of NAs have been developed since the discovery of suitable biocatalysts. Despite significant success, particularly due to advances in enzyme engineering, constraints persist due to a limited substrate scope of the available biocatalysts. This mini-review aims to address both the challenges and potential of chemical and biocatalytic approaches in NA synthesis. It also illustrates how a combined strategy could substantially enhance synthesis efficiency as demonstrated by a few existing examples. Thus the authors hope to encourage scientists from both domains to join their efforts to drive innovative solutions in this field.

Nucleoside-5′-triphosphates (5′-NTPs) are essential building blocks of nucleic acids in nature and play an important role in molecular biology, diagnostics, and mRNA therapeutic synthesis. Chemical synthesis has long been the standard method for producing modified 5′-NTPs. However, chemical routes face limitations, including low regio- and stereoselectivity, along with the need for protection/deprotection cycles, resulting in low yields, high costs, and lengthy processes. In contrast, enzymatic synthesis methods offer significant advantages, such as improved regio- and stereoselectivity and the use of mild reaction conditions, which often leads to higher product yields in “one-pot” reactions. Despite the extensive review of chemical synthesis routes for 5′-NTPs, there has not yet been any comprehensive analysis of enzymatic approaches. Initially, this review provides a brief overview of the enzymes involved in nucleotide metabolism, introducing valuable biocatalysts for 5’-NTP synthesis. Furthermore, the available enzymatic methods for efficient 5′-NTP synthesis using purified enzymes and starting from either nucleobases or nucleosides are examined, highlighting their respective advantages and disadvantages. Special attention is also given to the importance of ATP regeneration systems for 5′-NTP synthesis. We aim to demonstrate the remarkable potential of enzymatic in vitro cascade reactions, promoting their broader application in both basic research and industry.

We present a sustainable biocatalytic process for the one-pot synthesis of Rib1P. By coupling a guanine deaminase to the nucleoside phosphorylase-catalysed Guo cleavage, the reaction was shifted towards almost complete Guo cleavage.

Hepatitis E virus (HEV) poses a significant global health threat, with an estimated 20 million infections occurring annually. Despite being a self-limiting illness, in most cases, HEV infection can lead to severe outcomes, particularly in pregnant women and individuals with pre-existing liver disease. In the absence of specific antiviral treatments, the exploration of RNAi interference (RNAi) as a targeted strategy provides valuable insights for urgently needed therapeutic interventions against Hepatitis E. We designed small interfering RNAs (siRNAs) against HEV, which target the helicase domain and the open reading frame 3 (ORF3). These target regions will reduce the risk of viral escape through mutations, as they belong to the most conserved regions in the HEV genome. The siRNAs targeting the ORF3 efficiently inhibited viral replication in A549 cells after HEV infection. Importantly, the siRNA was also highly effective at inhibiting HEV in the persistently infected A549 cell line, which provides a suitable model for chronic infection in patients. Furthermore, we showed that a 5′ triphosphate modification on the siRNA sense strand activates the RIG-I receptor, a cytoplasmic pattern recognition receptor that recognizes viral RNA. Upon activation, RIG-I triggers a signaling cascade, effectively suppressing HEV replication. This dual-action strategy, combining the activation of the adaptive immune response and the inherent RNAi pathway, inhibits HEV replication successfully and may lead to the development of new therapies.

Nucleoside and nucleotide analogues have proven to be transformative in the treatment of viral infections and cancer. One branch of structural modification to deliver new nucleoside analogue classes explores replacement of canonical ribose oxygen with a sulfur atom. Whilst biological activity of such analogues has been shown in some cases, widespread exploration of this compound class is hitherto hampered by the lack of a straightforward and universal nucleobase diversification strategy. Herein, we present a synergistic platform enabling both biocatalytic nucleobase diversification from 4′‐thiouridine in a one‐pot process, and chemical functionalization to access new entities. This methodology delivers entry across pyrimidine and purine 4′‐thionucleosides, paving a way for wider synthetic and biological exploration. We exemplify our approach by enzymatic synthesis of 5‐iodo‐4′‐thiouridine on multi‐milligram scale and from here switch to complete chemical synthesis of a novel nucleoside analogue probe, 5‐ethynyl‐4′‐thiouridine. Finally, we demonstrate the utility of this probe to monitor RNA synthesis in proliferating HeLa cells, validating its capability as a new metabolic RNA labelling tool.

Nucleoside and nucleotide analogues have proven to be transformative in the treatment of viral infections and cancer. One branch of structural modification to deliver new nucleoside analogue classes explores replacement of canonical ribose oxygen with a sulfur atom. Whilst biological activity of such analogues has been shown in some cases, widespread exploration of this compound class is hitherto hampered by the lack of a straightforward and universal nucleobase diversification strategy. Herein, we present a synergistic platform enabling both biocatalytic nucleobase diversification from 4′‐thiouridine in a one‐pot process, and chemical functionalization to access new entities. This methodology delivers entry across pyrimidine and purine 4′‐thionucleosides, paving a way for wider synthetic and biological exploration. We exemplify our approach by enzymatic synthesis of 5‐iodo‐4′‐thiouridine on multi‐milligram scale and from here switch to complete chemical synthesis of a novel nucleoside analogue probe, 5‐ethynyl‐4′‐thiouridine. Finally, we demonstrate the utility of this probe to monitor RNA synthesis in proliferating HeLa cells, validating its capability as a new metabolic RNA labelling tool.

Modern, highly evolved nucleoside-processing enzymes are known to exhibit perfect regioselectivity over the glycosylation of purine nucleobases at N9. We herein report an exception to this paradigm. Wild-type nucleoside phosphorylases also furnish N7-xanthosine, a “non-native” ribosylation regioisomer of xanthosine. This unusual nucleoside possesses several atypical physicochemical properties such as redshifted absorption spectra, a high equilibrium constant of phosphorolysis and low acidity. Ultimately, the biosynthesis of this previously unknown natural product illustrates how even highly evolved, essential enzymes from primary metabolism are imperfect catalysts.

Nucleoside and nucleotide analogues have proven transformative in the treatment of viral infections and cancer. One branch of structural modification to deliver new nucleoside analogue classes explores replacement of canonical ribose oxygen with a sulfur atom. Whilst biological activity of such analogues has been shown in some cases, widespread exploration of this class of analogue is hitherto hampered by the lack of a straightforward and universal nucleobase diversification strategy. Herein, we present a synergistic platform enabling both biocatalytic nucleobase diversification from 4'-thiouridine in a one-pot process, and chemical functionalization to access new functional entities. This methodology delivers entry across pyrimidine and purine 4'-thionucleosides, paving a way for wider synthetic and biological exploration. We exemplify our approach by enzymatic synthesis of 5-iodo-4'-thiouridine on multi-milligram scale and from here switch to the chemical synthesis of a novel nucleoside analogue probe, 5-ethynyl-4'-thiouridine. Finally, we demonstrate the utility of this probe to monitor RNA synthesis de novo in proliferating HeLa cells, validating its capability as a new metabolic RNA labelling tool.

This publication introduces a thermal segment microwell plate control device, integrating smart sensor technology and standardized well-plate geometry for automated biolab setups. It is tested in a high-throughput enzymatic activity assay.

This Highlight offers a perspective on recent strategies for synthesising purine nucleoside antibiotics, an untapped source of natural products for developing novel antimicrobial agents.