Thermodynamic Constraints on Glycerol-Based Proto-Nucleotides: Phosphate versus Arsenate in Early Backbone Evolution

The emergence of the first nucleic acids required the prebiotic formation of nucleosides and nucleotides under chemically challenging conditions. Because assembly of the canonical ribose–phosphate framework is disfavored in water, simpler ancestral backbones may have preceded RNA and DNA. Here, semiempirical prescreening and density functional theory (DFT) calculations were used to evaluate the thermodynamics of glycerol-based nucleosides and nucleotides as candidate proto-nucleic-acid building blocks. Two assembly routes were examined: a classic pathway, in which glycerol first condenses with a recognition unit and then with an ionized linker, and an alternative pathway, in which glycerol first condenses with the ionized linker and only then with the recognition unit. Although Gibbs free energy is a state function, the two pathways access different regions of the potential energy surface and converge to distinct local minima, leading to pathway-dependent differences in the computed thermodynamic quantities. Glycerol-derived nucleosides are predicted to form favorably in both vacuum and implicit aqueous solution when combined with either canonical or the studied non-canonical bases. Glycerol-based nucleotides are, likewise, thermodynamically accessible by both pathways, although the classic route is consistently more favorable than the alternative route. Among the most stabilized products are derivatives containing adenine, uracil, and C-glycosylated barbituric acid. Replacement of phosphate by arsenate produces only modest energetic and structural changes, indicating that arsenate-containing analogues cannot be excluded on thermodynamic grounds alone. However, the consistently longer As–OC3 distances relative to P–OC3 suggest weaker backbone bond and therefore a plausible hydrolytic disadvantage for arsenate. These results support the view that glycerol-based backbones could have participated in early proto-nucleic-acid chemistry and suggest that phosphate may have been selected not because arsenate analogues were thermodynamically inaccessible, but because phosphate-containing backbones were more kinetically and hydrolytically persistent.