Boron-assisted abiotic polypeptide synthesis

The emergence of proteins and their interactions with RNA were pivotal in the origin and early evolution of life. Understanding the abiotic synthesis of peptides, particularly in environments conducive to both peptide and RNA formation, is crucial to reconstructing the prebiotic world. Previous research has shown that peptide synthesis is favored under highly alkaline conditions, but these conditions are detrimental to RNA stability. This incompatibility has presented a challenge in understanding the co-evolution of these essential biopolymers. The role of boron, known to facilitate abiotic RNA synthesis in prebiotically plausible conditions, in peptide formation has remained unclear. This study aims to investigate the influence of boric acid on amino acid polymerization to determine whether it can promote peptide synthesis under conditions compatible with RNA stability, potentially bridging the gap in understanding the origins of these key biomolecules and their interactions.

Extensive research has explored prebiotic peptide synthesis in various geological settings, including volcanic and hydrothermal fields, and under different conditions of pH, mineral presence, and salinity. Highly alkaline conditions have been identified as favorable for peptide synthesis, but the resulting peptides are typically short unless chemically activated. However, the highly alkaline environment is incompatible with the stability of RNA. Previous studies have highlighted the importance of borate in RNA synthesis, as borate binds and stabilizes ribose, facilitating regioselective phosphorylation and ribonucleotide formation. The potential interaction between borate and peptide formation has been discussed, but its effect remains to be elucidated. This research builds upon these previous findings, focusing specifically on the effect of boric acid on the abiotic polymerization of amino acids under conditions that are conducive to RNA stability.

The researchers conducted simple thermal evaporation experiments of glycine (Gly) solutions containing boric acid at various pH levels (2, 3, 6, 8, and 10) and temperatures (90°C and 130°C). The experiments involved heating 600 µL of a 0.5 mol L⁻¹ Gly solution with varying amounts of boric acid for 200 hours in 1.5 mL glass vials. The molar ratio of Gly to boric acid was varied (0:1, 0.1:1, and 1:1). The resulting products were analyzed using liquid chromatography-mass spectrometry (LC-MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) to identify and quantify the resulting Gly peptides. LC-MS/MS was used to quantify short Gly oligomers using commercially available standards. FTICR-MS was utilized to identify longer peptides, given the low solubility of long oligomers. The effects of wet-dry cycles were also investigated. Additional experiments were conducted with alanine (Ala) to assess the generality of the boron-assisted synthesis. The formation of borate esters between Gly and boron species was investigated using negative electrospray ionization mass spectrometry (ESI-MS) and ¹¹B-nuclear magnetic resonance (NMR) spectroscopy to characterize the interaction between glycine and boron species at different pH values. FT-IR analysis was used to further support the formation of esters. All experiments were meticulously controlled using baked glass vials and ultrapure water to minimize contamination.

The study found that boric acid significantly enhanced the polymerization of glycine, yielding peptides up to Gly39 in length. The yield of short Gly peptides (Gly2-5 and diketopiperazine [DKP]) and the length of detectable peptides were dependent on the amount of boric acid and the pH of the solution. The presence of boric acid led to substantially higher yields compared to control experiments without boric acid. Near-neutral pH conditions (pH 6-8) at 130°C proved optimal for the formation of long Gly oligomers in the presence of boric acid. In contrast, in the absence of boric acid, higher yields and lengths of oligomers were observed under acidic and alkaline conditions. The catalytic effect of boric acid was confirmed at a lower temperature (90°C) but was less effective at 130°C under acidic conditions due to the formation of black by-products. Wet-dry cycles did not significantly impact the yields or lengths of the peptides. The boron-assisted peptide synthesis was also confirmed for alanine, although longer reaction times were required for polyalanine formation. Negative ESI-MS and ¹¹B-NMR analyses confirmed the formation of borate esters (Gly-B and Gly-BA) between Gly and boron species in near-neutral and acidic/alkaline solutions, respectively. The boron species act as catalysts, continuously promoting peptide synthesis as they are released in the same form after peptide bond formation. Under neutral conditions with boron, the reaction converted 50% of Gly to peptides with limited formation of by-products, unlike the reactions under highly acidic and alkaline conditions without boron, which showed significantly higher by-product formation.

The findings demonstrate that boric acid facilitates abiotic polypeptide synthesis under near-neutral pH conditions, which are compatible with RNA stability, unlike the previously reported highly alkaline conditions. This suggests that the same prebiotic environments might have supported the synthesis of both RNA and peptides, potentially facilitating their interactions. The length of the synthesized peptides (up to 39 amino acids) falls within the range of small proteins with diverse biological functions, supporting the plausibility of biologically relevant peptides forming in such environments. The observed catalytic effect of boric acid on both Gly and Ala suggests its potential role in the polymerization of multiple amino acids. The co-occurrence of boron species in evaporitic basins with other organic compounds and amino acids, under near-neutral conditions, could have provided a favorable environment for the formation of primordial functional polymers composed of peptides and RNA. The potential for RNA-peptide interactions is supported by the existence of amino acid riboswitches involved in translation, and the stabilizing effects of some peptides on RNA stability.

This study provides compelling evidence for boron-assisted abiotic polypeptide synthesis under prebiotically plausible conditions compatible with RNA stability. The findings suggest that evaporitic basins on early Earth, enriched in boric acid and other organic molecules, may have provided an ideal environment for the formation and interaction of primordial peptides and RNAs, potentially playing a key role in the origin of life. Further research should investigate the polymerization of a wider range of amino acids and the specific mechanisms of boron catalysis in peptide synthesis.

The study primarily focused on glycine and alanine. While these are common amino acids, further research is needed to determine the efficacy of boron-assisted synthesis with other amino acids. The high temperatures used in the experiment might not perfectly reflect the temperature range of all potential prebiotic environments. Although wet-dry cycles were considered, more detailed investigation of the influence of environmental fluctuations on the reaction is warranted.