Boron-assisted abiotic polypeptide synthesis

RNAs store genetic information and catalyze reactions, and their interactions with proteins underpin core biological processes. Understanding how such interactions arose is central to origin-of-life research, particularly at the boundary between an RNA world and modern DNA-protein systems. While amino acids could have been supplied prebiotically by terrestrial synthesis and extraterrestrial delivery, peptide formation without biological machinery typically requires highly alkaline or acidic conditions and often yields short oligomers, conditions unfavorable for RNA stability. Prior work suggests borate/boric acid are crucial for stabilizing ribose and enabling nucleotide phosphorylation, making boron-rich environments promising for RNA formation. However, how boron affects amino acid polymerization has remained unclear. This study tests the hypothesis that boric acid catalyzes peptide bond formation at acidic to near-neutral pH, enabling polypeptide synthesis under conditions compatible with RNA stability.

Prebiotic peptide formation has been explored in volcanic, hydrothermal, seafloor, and tidal flat settings, with effects of minerals, salts, ions, and pH examined. High pH favors peptide formation but usually yields limited-length oligomers (e.g., Gly20) unless amino acids are chemically activated; alternative multi-step chemistries (e.g., aminonitrile coupling) have limitations in plausibility and length (e.g., Gly17). In contrast, borate minerals stabilize ribose among aldopentoses and boric acid aids regioselective phosphorylation of ribose and nucleosides, fixing ribose in the furanose form and enabling ribonucleotide formation, placing boron-rich environments at the center of plausible RNA synthesis scenarios. Although borate’s potential role in prebiotic peptide formation has been suggested, concrete evidence was lacking. In organic synthesis, borate/boric acid form esters with carboxylates and catalyze amide formation in anhydrous systems, hinting at a catalytic role in aqueous prebiotic contexts.

Thermal evaporation experiments were conducted in baked glass vials (1.5 mL; 32 mm height; 6 mm top i.d.; 9.5 mm bottom i.d.) placed in an aluminum block within an electric furnace. Standard starting solutions contained 600 µL glycine (Gly) at 0.5 mol L−1 with 0.5 mmol boric acid; pH was adjusted with NaOH or HCl (unadjusted pH: 6.5 without boric acid; 5.3 with boric acid). Reactions were typically run at 130 °C up to 200 h across pH 2–10, and selected acidic runs at 90 °C. Wet-dry cycling was tested by adding 600 µL water and vortexing at 100 h and 200 h. After heating, products were dissolved in 300 µL water for analysis. Short oligomers (Gly2–Gly5) and DKP were quantified by UPLC-MS/MS (Shimadzu LCMS-8040) using a VC-50 2D column (2.0 × 150 mm, 5 µm) at 50 °C with a linear acetonitrile gradient (70% to 37% over 17 min; 0.25 mL min−1). Positive-mode ESI conditions: nebulizer 2.5 L min−1; drying gas 10 L min−1; desolvation 250 °C; heat block 400 °C. Commercial standards for Gly2–Gly5 and DKP were used for quantification. For samples forming long oligomers/polymers, residual insoluble material after initial water extraction (300 µL) was re-extracted with 10–100 µL water or water/methanol and analyzed by MALDI FTICR-MS (solariX 9.4T) or MALDI-TOF (REFLEX III) with 2,5-dihydroxybenzoic acid matrix. Formation of boron esters with Gly (Gly-B and Gly-BA) in starting solutions at varying pH was monitored by negative-mode ESI-MS via direct infusion (instrument settings as above). 11B-NMR (JNM-ECA800, 800 MHz) collected 8–32 scans with 2 s relaxation delay; chemical shifts referenced to boric acid (pH 7, 16.067 ppm); samples dissolved in D2O. FT-IR (JASCO FT/IR-6300, 4 cm−1 resolution, 100 scans) probed dried mixtures of Gly with boric acid.

• Boric acid enabled abiotic polymerization of glycine up to at least Gly39 under simple thermal evaporation at near-neutral pH (6–8) and 130 °C for 200 h; without boric acid, products were limited to Gly2 and Gly13 under the same pH and temperature. • Total yield of short oligomers (Gly2–Gly5 and diketopiperazine) at pH 6 with boron was higher by four orders of magnitude compared to boron-free controls (boron-free was ~0.00025 of the boron-assisted yield). • In the presence of boron, short oligomer yields were comparable across pH, but long oligomers formed most substantially at near-neutral pH (6–8). In the absence of boron, both yields and lengths were higher under acidic or alkaline conditions than near-neutral. • Catalytic effects of boron species were also observed at 90 °C under acidic conditions; at 130 °C and low pH, formation of black by-products suppressed peptide formation. Wet-dry cycling did not significantly change yields or lengths. • Alanine showed enhanced oligomerization in the presence of boric acid, suggesting generality, though longer durations appear necessary to reach long polyalanine. • Reaction mass balance: without boron under highly acidic/alkaline conditions, 70–90% of initial Gly was converted/consumed with substantial by-products; with boron at neutral pH, ~50% conversion and ~50% consumption of initial Gly occurred, indicating improved selectivity with fewer by-products. • Spectroscopic evidence indicates formation of borate esters with glycine (Gly-B and Gly-BA) in starting solutions across pH, with 11B-NMR showing typical bulk speciation (boric acid dominant < pH 9; borate > pH 9), but ester speciation deviating from bulk behavior. FT-IR shifts (to ~1227 cm−1) are consistent with ester formation. • Proposed mechanism: boron esters of carboxylate catalyze amide bond formation; boron species are released after peptide bond formation, enabling catalytic turnover.

The findings demonstrate that boric acid catalyzes peptide bond formation at acidic to near-neutral pH, conditions that are much more compatible with RNA stability than the highly alkaline or acidic regimes typically invoked for prebiotic peptide synthesis. Given evidence for boron-rich evaporitic settings and near-neutral ocean pH in the early Archean (and possibly Hadean), evaporitic basins could have simultaneously supported RNA formation and peptide polymerization. The ability to reach peptide lengths up to 39 residues approaches the regime of small functional proteins, aligning with hypotheses that short peptides aided ribozyme function. The coexistence of catalytically effective boron species for both ribonucleotide formation and peptide polymerization suggests a unified prebiotic environment where proto-peptides and RNAs co-emerged and interacted, potentially facilitating the transition to RNA-dependent protein synthesis.

Boric acid markedly enhances abiotic peptide synthesis from amino acids at acidic to near-neutral pH via formation of boron esters that catalyze amide bond formation, enabling glycine oligomerization up to at least 39-mers under simple evaporative heating. This identifies boron-rich evaporitic environments as plausible sites for concurrent RNA and peptide formation on early Earth. Future work should clarify the relative catalytic roles of Gly-B versus Gly-BA, extend testing to diverse proteinogenic amino acids and mixtures, quantify kinetics across broader temperature–pH regimes closer to natural settings, and assess functional interactions between abiotically formed peptides and RNA systems.

Detection of long oligomers was limited by low solubility, restricting routine LC-MS quantification to short peptides (Gly2–Gly6) and requiring specialized MS for less soluble higher oligomers. At 130 °C under strongly acidic conditions, extensive by-product (black material) formation suppressed observable peptide synthesis. Only glycine (and preliminary alanine) were systematically tested; generality to other amino acids and complex mixtures remains to be established. The specific catalytic contributions of Gly-B versus Gly-BA esters are unresolved. Experimental temperatures are higher than many natural evaporitic settings, though catalysis was also evident at 90 °C.