A Ribonucleotide ↔ Phosphoramidate Reaction Network Optimized by Computer-Aided Design

A growing number of out-of-equilibrium systems have been
created
and investigated in chemical laboratories over the past decade. One
way to achieve this is to create a reaction cycle, in which the forward
reaction is driven by a chemical fuel and the backward reaction follows
a different pathway. Such dissipative reaction networks are still
relatively rare, however, and most non-enzymatic examples are based
on the carbodiimide-driven generation of carboxylic acid anhydrides.
In this work, we describe a dissipative reaction network that comprises
the chemically fueled formation of phosphoramidates from natural ribonucleotides
(e.g., GMP or AMP) and phosphoramidate hydrolysis as a mild backward
reaction. Because the individual reactions are subject to a multitude
of interconnected parameters, the software-assisted tool “Design
of Experiments” (DoE) was a great asset for optimizing and
understanding the network. One notable insight was the stark effect
of the nucleophilic catalyst 1-ethylimidazole (EtIm) on the hydrolysis
rate, which is reminiscent of the action of the histidine group in
phosphoramidase enzymes (e.g., HINT1). We were also able to use the
reaction cycle to generate transient self-assemblies, which were characterized
by dynamic light scattering (DLS), confocal microscopy (CLSM), and
cryogenic transmission electron microscopy (cryo-TEM). Because these
compartments are based on prebiotically plausible building blocks,
our findings may have relevance for origin-of-life scenarios.