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

Abstract

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 carbodiimidedriven 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.

Repurposing a catalytic cycle for transient self-assembly

“…A common approach for constructing new chemical reaction cycles is to combine multiple reactions (Figure 1A, Left). The individual reactions can be either catalytic [25][26][27][28][29][30][31][32][33][34] or non-catalytic, [35][36][37][38][39][40][41][42][43] and can incorporate enzymatic reactions. [44][45][46][47][48][49] With biomolecules such as nucleic acids and enzymes, high specificity can be achieved and multiple reactions proceed concurrently under the same conditions.…”

“…Even if feasible combinations are found, the resulting reaction cycles often suffer from problems such as fast background fuel decomposition and formation of side products. 19,22 In the field of dissipative self-assembly, only limited classes of fully synthetic reaction cycles, such as carbodiimide hydration cycles, 31,34,38 a redox reaction between dithionite and oxygen gas 43 and hydrolysis of acid anhydride, 30 have been used to convert monomers from the non-assembling state to the assembling state with good conversion efficiency.…”

“…We note that some of the reported reaction cycles utilize catalytic reactions too. [25][26][27][28][29][30][31][32][33][34][44][45][46][47][48][49] However, in these systems, a catalytic reaction is combined with other reactions (catalytic or non-catalytic) to construct a full reaction cycle (Figure 1A, Left), while in our approach, a single catalytic reaction cycle itself is used as a chemical reaction cycle (Figure 1A, Right). We also note that virtually all the chemically fueled out-of-equilibrium systems reported to date can be regarded as driven by catalysis, 22,23,54 in that these systems provide an alternative pathway for the fuel-to-waste conversion.…”

Steric Control over the Threading of Pyrophosphonates with One or Two Cyanostar Macrocycles during Pseudorotaxane Formation

“…By contrast, the supramolecular chemistry of organic pyrophosphates is underexplored [48,58,59] and, to the best of our knowledge, the recognition of organic pyrophosphonates is unexplored. As dianions with tunable organic substituents, these compounds would extend the scope of anion recognition towards designer guest molecules, [60–62] which could play a role in carbodiimide‐driven chemical reaction cycles [63–69] …”

“…As dianions with tunable organic substituents, these compounds would extend the scope of anion recognition towards designer guest molecules, [60][61][62] which could play a role in carbodiimide-driven chemical reaction cycles. [63][64][65][66][67][68][69] A promising candidate for the binding of such large anions is the cyanostar (CS) macrocycle (Figure 1A) [45] that has a positively polarized central cavity in which it can bind anions through multiple CH hydrogen bonds. Cyanostar has been reported to bind organophosphates and organophosphonates in co-assemblies, where two (or more) macrocycles π-stack in a dimeric fashion with multiple equilibria.…”