Five‐Component Cascade Synthesis of Nucleotide Analogues in an Engineered Self‐Immobilized Enzyme Aggregate

Nucleoside analogues are important agents in the battle against some of the most serious human illnesses including cancer, parasitic infections and viral diseases. In combating viral infections such as HIV, herpes simplex virus, and hepatitis C, nucleoside analogues are particularly indispensable and target reverse transcriptases, polymerases or other enzymes involved in DNA/RNA synthesis, replication and repair. Most nucleoside analogue drugs are prodrugs that are activated by intracellular 5’-phosphorylation in the target organism. The study of existing and new potential nucleoside therapeutics, which includes structure–activity relationships, kinetics and metabolism of their activated forms, often entails labourintensive chemical synthesis of these metabolites. Chemical synthesis of nucleotide analogues is most commonly performed by 5’-phosphorylation of the corresponding nucleoside analogue, and often requires multiple protective group manipulations to control the regiochemistry of ribose phosphorylation. Moreover, these nucleoside precursors themselves are the products of multistep pathways that must address several potential synthetic challenges—selective activation of the anomeric position for nucleobase addition, control of stereochemistry (or resolution of diasteromeric products) and purification of highly polar, water soluble products. Intermediates in these pathways are often unstable, particularly the anomerically activated ribosides. Each nucleoside analogue presents its own distinct synthetic challenges, which require the development of a variety of nongeneralizable strategies. Recently we provided a biochemical alternative to chemical synthesis of nucleotide analogues by using an engineered hypoxanthine phosphoribosyl transferase (8B3PRT). This transferase, which has markedly relaxed specificity for purine base analogues, facilitated the addition of many base analogues to phosphoribosyl pyrophosphate (PRPP), and created an assortment of monophosphorylated nucleotides. Significant limitations of this approach were the instability and difficulty in handling recombinant enzymes and the expense and instability of the substrate. PRPP decomposes rapidly in solution and at current fine chemical prices costs over 1.2 million dollars per mole. To address these issues, we report the development of a self-immobilized, multiple-enzyme system, a covalently linked enzyme aggregate or CLEA (Figure 1 A), 12] which contains both a purine nucleotide analogue pathway and an auxiliary ATP recycling system. In this pathway (Figure 1 B), ribokinase phosphorylates the 5’ hydroxyl of d-ribose; this results in dribose-5-phosphate, which is subsequently anomerically pyrophosphorylated by phosphoribosyl pyrophosphate synthetase, generating PRPP and consuming two equivalents of ATP. The transferase 8B3PRT then catalyzes the addition of purine nucleobases to the activated sugar, and this potentially provides a variety of nucleotide analogues. ATP hydrolysis products ADP and AMP are recycled to ATP by including adenylate kinase Figure 1. A) General process for generating self-immobilized aggregates. B) Ribose to nucleotide analogues pathway that uses the engineered 8B3PRT combined with ATP regeneration enzymes—all crosslinked in a single aggregate: ribokinase (RK), phosphoribosyl pyrophosphate synthetase (PPS), engineered hypoxanthine phosphoribosyl transferase 8B3PRT (8B3), adenylate kinase (AK), pyruvate kinase (PK). C) HPLC trace of cascade CLEA reaction to form inosine monophosphate.