Thioesters provide a plausible prebiotic path to proto-peptides

Abstract

It is widely assumed that the condensation of building blocks into oligomers and polymers was important in the origins of life. High activation energies, unfavorable thermodynamics and side reactions are bottlenecks for abiotic peptide formation. All abiotic reactions reported thus far for peptide bond formation via thioester intermediates have relied on high energy molecules, which usually suffer from short half-life in aqueous conditions and therefore require constant replenishment. Here we report plausible prebiotic reactions of mercaptoacids with amino acids that result in the formation of thiodepsipeptides, which contain both peptide and thioester bonds. Thiodepsipeptide formation was achieved under a wide range of pH and temperature by simply drying and heating mercaptoacids with amino acids. Our results offer a robust one-pot prebiotically-plausible pathway for proto-peptide formation. These results support the hypothesis that thiodepsipeptides and thiol-terminated peptides formed readily on prebiotic Earth and were possible contributors to early chemical evolution.

Fig. 1. Proposed acyl substitution reactions for peptide bond formation (amidation) through a thioester-amide exchange.

Under conditions of low water activity, mercaptoacids condense to form thioesters, which can be exchanged for amide bonds in the presence of amino acids. Structures are drawn in their predominant form under acidic dry-down reactions (pH ~3.0).

Fig. 2. Analytical data confirms the formation of thiodepsipeptides in dry-down reactions of tg and Ala.

a Negative-ion mode ESI-MS spectrum of thioglycolic acid (tg) and alanine (Ala) after being dried at 65 °C for 7 days, indicating a variety of thiodepsipeptides. All labeled species correspond to [M-H]⁻ ions. Species that are labeled with H₂O correspond to species that have an additional H₂O unit, which could be the result of non-covalent adducts. b Fourier transform infrared spectroscopy (FTIR) shows shifts in the C=O band and in the amide regions upon dry-down of tg and Ala, supportive of thiodepsipeptide formation. c HPLC chromatograms of products resulting from drying tg alone or tg with Ala at 65 °C for 7 days. Comparison between the two dry-down reaction products via hydrophobicity-based separation on a C18 column allows the identification of various oligomers that contain Ala. c(tgA) is the cyclic form of tgA, i.e., thiazinedione.

Fig. 3. Amide bonds form upon dry-down of amino acid and a mercaptoacid as indicated by MS/MS Higher-energy C-trap dissociation (HCD) analysis.

tg and Ala were dried at 65 °C for 7 days; the resulting products were analyzed by negative-ion mode MS/MS. Tandem MS analysis was conducted using m/z 162.0 as the precursor ion. Negative-ion mode MS/MS analysis of the [tg+Ala-H₂O] molecule showed a base peak at m/z 88.0385, being the y₁ ion of the sequence tgA. This fragmentation pattern is supportive of the formation of amide bonds in these dry-down reactions.

Fig. 4. Dry-down reactions of tg and Ala produce oligomers containing amide bonds.

¹H NMR spectra of: a A fresh, unreacted mixture of thioglycolic acid (tg) with alanine (Ala). b The authentic standard of Ala acylated with tg at the α-amine (tgA) demonstrating the down-field shift of the α-resonance upon amidation of Ala. c Sample of tg dried with Ala in a 5:1 molar ratio at 65 °C for 7 days. The starting tg material contains some thioester-linked dimer (tg)₂. c(tgA) is the cyclic form of tgA, i.e., thiazinedione.

Fig. 5. Thiodepsipeptides form under a range of pH conditions and temperatures.

a Chromatograms of products resulting from thioglycolic acid (tg) and alanine (Ala), in a 5:1 molar ratio, being dried at 65 °C for 1 week in various initial reactions pH. Products are observed throughout the whole range of pH conditions tested. b Chromatograms illustrating the temperature dependence of thiodepsipeptide formation under unbuffered, acidic conditions (pH ~3). Products are detected when mixtures of tg and Ala are dried and maintained at temperatures as low as 25 °C. (tg)₂ refers to a thioester-containing homodimer of tg. c(tgA) is the cyclic form of tgA, i.e., thiazinedione.