Ester-Mediated Amide Bond Formation Driven by Wet-Dry Cycles: A Possible Path to Polypeptides on the Prebiotic Earth

Abstract

Although it is generally accepted that amino acids were present on the prebiotic Earth, the mechanism by which α-amino acids were condensed into polypeptides before the emergence of enzymes remains unsolved. Here, we demonstrate a prebiotically plausible mechanism for peptide (amide) bond formation that is enabled by α-hydroxy acids, which were likely present along with amino acids on the early Earth. Together, α-hydroxy acids and α-amino acids form depsipeptides-oligomers with a combination of ester and amide linkages-in model prebiotic reactions that are driven by wet-cool/dry-hot cycles. Through a combination of ester-amide bond exchange and ester bond hydrolysis, depsipeptides are enriched with amino acids over time. These results support a long-standing hypothesis that peptides might have arisen from ester-based precursors.

Keywords: chemical evolution; day-night cycle; depsipeptides; origins-of-life; proto-peptides.

scheme 1

Proposed reaction scheme for lactic acid (LA)-mediated peptide bond formation and depsipeptide elongation by ester–amide exchange (also see SI: Mechanism). Here, a free amino acid (G, A, etc.) with a neutral -NH₂ terminus (in equilibrium with -NH₃⁺) reacts with a lactic acid dimer to form amide-linked depsipeptides. Elongation leads to longer oligomers.

Figure 1

Demonstration of depsipeptide formation from lactic acid (LA) and glycine (G) through ester-amide exchange. a) Negative electrospray mass spectrum of LA after 4 wet–dry cycles (wet phase 65 °C, 5.5 h; dry phase 85 °C, 18 h). Polyesters are observed with n≥10. b) Mass spectrum of a 1:1 mol LA/G mixture after 4 cycles. The most intense signals correspond to mixed depsipeptides. LA-G depsipeptides are observed up to n=10 (inset).

Figure 2

Amino acid/peptide bond enrichment in depsipeptides following environmental cycling. a) Mass spectral region showing LA-A pentamers after 1, 4, 8, and 12 cycles. LA residue mass=72.0211 Da; A residue mass=71.0371 Da. As the number of cycles increases, the pentamer series is enriched in A residues, as evidenced by a mass decrease of 0.9840 Da per exchanged residue. Also see Figure S5 and S6. b) Isotopically-corrected MS data for n=2 through n=8 oligomers shows the relative amounts of A incorporated as a function of environmental cycles. The theoretical limit of A in each oligomer, shown in black, corresponds to n−1 residues as the N-terminal lactamide is not thought to exchange. c) Emergence of amide bonds as demonstrated by IR spectroscopy. Spectra are of (orange) LA + glycine (G) monomers before cycling, (yellow) LA-G mixture after 1 cycle, (light green) LA-G mixture after 8 cycles, and (green) LA-G mixture after 16 cycles, where LA was re-added after the 8th cycle. The band in top spectrum at 1710 cm⁻¹ corresponds to free carboxylic acids of monomers. Upon cycling, reduction of the carboxylic acid band is observed, and the formation of ester bonds is observed (shoulder at 1730 cm⁻¹). Eventually, amide bond bands are observed (Amide I, 1640 cm⁻¹; Amide II, 1530 cm⁻¹), similar to those of a polyglycine spectrum (black, Ref. 13). Full IR spectra are provided in Figure S7–S9.

Figure 3

a) Amide bond yield as a function of wet–dry cycles, determined by ¹H NMR spectroscopy. After 8 cycles, approximately 10 % of G monomer has been incorporated into depsipeptides. Amide bond yields increase significantly when LA is re-added after cycle 8, resulting in approximately 40 % of G being incorporated into depsipeptides. b–d) Sequencing of depsipeptides by tandem MS reveals internal amino acid incorporation. b) Sequencing of 2LA+4G, theoretical precursor ion [M−H]⁻=389.13 Da, formed after 8 cycles. CID collision energy=20 eV. The primary sequence is LA-G-G-G-G-LA. c) Sequencing of 2LA+3A, theoretical precursor ion [M−H]⁻=374.16 Da, formed after 12 cycles. CID collision energy=15 eV. The primary sequence is LA-A-A-A-LA. d) Sequencing of 2 LA+2G+1A, theoretical precursor ion [M−H]⁻=346.13 Da, formed after 4 cycles. CID collision energy=20 eV. LA-A-G-G-LA, LA-G-A-G-LA, and LA-G-G-A-LA sequences are observed.

Figure 4

Depsipeptide formation and enrichment within an alanine-lactic acid sample that had an initial pH of 7. a,b) Mass spectra of n=4 alanine-lactic acid depsipeptides formed after 1 cycle and after 8 cycles. Before the first cycle, the solution was titrated to pH 7 by the addition of triethylamine. Because the sample became more acidic as solvent and base were evaporated (i.e., pH 3.5), triethylamine was added before each subsequent cycle to bring the sample back to pH 7. After 8 cycles, we observe enrichment of the 2LA+2A depsipeptide and reduction of the 4LA oligoester. c) Tandem MS sequencing of 2LA+2A. Theoretical precursor ion [M−H]⁻=303.12 Da, formed after 8 cycles. CID collision energy=12 eV. The primary sequence is LA-A-A-LA.