Influence of Phosphate Activation Chemistry on the Selection of the Primordial Genetic Alphabet

RNA copying under mild conditions compatible with protocell integrity requires the input of chemical energy to drive the synthesis of activated nucleotides such as phosphorimidazolides. Recently, two potentially prebiotic classes of phosphate-activating agents have been explored, one based on isonitrile-aldehyde chemistry, the other on imine diimidazole (IDI)-N-cyanoimidazole (NCI) chemistry. Because such highly electrophilic activating agents may lead to undesirable nucleotide modifications, we have examined the reaction of both types of activating agents with the canonical ribonucleotides A, U, C, and G, and the potentially primordial nucleotides 2-thio-C (s²C), 2-thio-U (s²U), and inosine (I). We find that the isonitrile-aldehyde system shows minimal hydroxyl modification but does modify the nucleobases of U, G, s²U, and I. Except for guanosine, these modifications are readily reversible. In contrast, IDI-NCI systems acylate ribonucleotide hydroxyls while modifying nucleobases only transiently; mildly acidic pH suppresses undesired modifications. Both classes of activating agents modify 2-thiopyrimidines on the sulfur, with the isonitrile-aldehyde reaction promoting desulfurization and thus conversion to the canonical pyrimidines. To evaluate compatibility with model protocells, we tested the effects of activation chemistry on fatty acid vesicles and found that protocell integrity was preserved at moderate reagent concentrations. Our findings show that the potentially primordial s²U, s²C, and I nucleotides are more sensitive to modification than the canonical U, C, and G nucleotides, potentially contributing to the chemical selection of the early genetic alphabet.