# Enzymatic Transglycosylation Features in Synthesis of 8-Aza-7-Deazapurine Fleximer Nucleosides by Recombinant E. coli PNP: Synthesis and Structure Determination of Minor Products

## TL;DR

Researchers used recombinant E. coli PNP to synthesize modified nucleosides, discovering unexpected minor products and exploring their formation mechanisms.

## Contribution

The study reveals new enzymatic transglycosylation pathways and the formation of bis-riboside products using a fleximer base.

## Key findings

- Enzymatic transglycosylation produced unexpected minor products like N1-pyridinium-N1-pyrazole bis-ribose derivatives.
- Molecular modeling suggests a unique enzyme-substrate arrangement promotes secondary glycosylation.
- Thermodynamic parameters of intermediates influence product formation.

## Abstract

Enzymatic transglycosylation of the fleximer base 4-(4-aminopyridine-3-yl)-1H-pyrazole using recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of “non-typical” minor products of the reaction. In addition to “typical” N1-pyrazole nucleosides, a 4-imino-pyridinium riboside and a N1-pyridinium-N1-pyrazole bis-ribose derivative were formed. N1-Pyrazole 2′-deoxyribonucleosides and a N1-pyridinium-N1-pyrazole bis-2′-deoxyriboside were formed. But 4-imino-pyridinium deoxyriboside was not formed in the reaction mixture. The role of thermodynamic parameters of key intermediates in the formation of reaction products was elucidated. To determine the mechanism of binding and activation of heterocyclic substrates in the E. coli PNP active site, molecular modeling of the fleximer base and reaction products in the enzyme active site was carried out. As for N1-pyridinium riboside, there are two possible locations for it in the PNP active site. The presence of a relatively large space in the area of amino acid residues Phe159, Val178, and Asp204 allows the ribose residue to fit into that space, and the heterocyclic base can occupy a position that is suitable for subsequent glycosylation. Perhaps it is this “upside down” arrangement that promotes secondary glycosylation and the formation of minor bis-riboside products.