Aqueous microdroplets enable abiotic synthesis and chain extension of unique peptide isomers from free amino acids

Abstract

Amide bond formation, the essential condensation reaction underlying peptide synthesis, is hindered in aqueous systems by the thermodynamic constraints associated with dehydration. This represents a key difficulty for the widely held view that prebiotic chemical evolution leading to the formation of the first biomolecules occurred in an oceanic environment. Recent evidence for the acceleration of chemical reactions at droplet interfaces led us to explore aqueous amino acid droplet chemistry. We report the formation of dipeptide isomer ions from free glycine or L-alanine at the air-water interface of aqueous microdroplets emanating from a single spray source (with or without applied potential) during their flight toward the inlet of a mass spectrometer. The proposed isomeric dipeptide ion is an oxazolidinone that takes fully covalent and ion-neutral complex forms. This structure is consistent with observed fragmentation patterns and its conversion to authentic dipeptide ions upon gentle collisions and for its formation from authentic dipeptides at ultra-low concentrations. It also rationalizes the results of droplet fusion experiments that show that the dipeptide isomer facilitates additional amide bond formation events, yielding authentic tri- through hexapeptides. We propose that the interface of aqueous microdroplets serves as a drying surface that shifts the equilibrium between free amino acids in favor of dehydration via stabilization of the dipeptide isomers. These findings offer a possible solution to the water paradox of biopolymer synthesis in prebiotic chemistry.

Keywords: air–water interface; dipeptide; mass spectrometry; origin of life; prebiotic chemistry.

Fig. 1.

Reactions, experimental setups, and spray parameter effects associated with the spontaneous generation of putative dipeptides from free amino acids in aqueous microdroplets. A and B show condensation reactions yielding GlyGly and AlaAla from free Gly and Ala, respectively. C and D illustrate the experimental arrangements including high voltage (HV) utilized to generate microdroplets in nESI (C) and ESSI (D). E through H show the full-scan nESI spectra for aqueous solutions (5 mM) of Gly (E and G) and Ala (F and H) in both the positive (E and F) and negative ion (G and H) modes. The observed presumptive dipeptide peaks are labeled in all cases. I and J summarize the effects of the spray distance and voltage, respectively, on the positive ion conversion ratio to GlyGly. Conversion ratios were calculated using the average of 50 scans in triplicate. Error bars indicate SDs.

Fig. 2.

Comparison of nESI-MS/MS spectra obtained for the standard (Top Row, A–D) and droplet-synthesized (Bottom Row, E–H) dipeptides. Results are shown for both protonated (A and E) and deprotonated (B and F) GlyGly, as well as protonated (C and G) and deprotonated (D and H) AlaAla. Droplet-synthesized spectra (Bottom Row) were obtained by spraying pure amino acid solutions in water, whereas standard spectra (Top Row) were acquired from aqueous solutions of commercial dipeptide standards. Annotated in blue are the unique peaks in the spectra of the standard compounds, while the unique reaction product fragments are in pink.

Fig. 3.

Differences on the MS/MS spectra of the isomeric dipeptide species before (Bottom) and after (Top) collisional heating (30 s at CE < fragmentation threshold). Results for both the protonated (A) and deprotonated (B) GlyGly isomer, as well as the protonated (C) and deprotonated (D) AlaAla isomer, are shown. Annotated in blue are the unique peaks present in the spectra of the isomeric compound prior to heating, which disappear partially or completely after collisional heating. Annotated in pink are the unique standard dipeptide fragments which appear in the isomer spectra after collisional heating.

Fig. 4.

Two-dimensional plots of m/z vs. drift time of aqueous solutions with different proportions of Gly and GlyGly (concentration of Gly increases from Top to Bottom). Results for both the positive (Left) and the negative (Right) ion mode are included. The solutions analyzed were as follows: 100% GlyGly (A and F), 1:125 GlyGly-Gly (B and G), 1:375 GlyGly-Gly (C and H), 1:500 GlyGly-Gly (D and I), and 100% Gly (E and J). Average drift times of the protonated isomeric and standard species were determined as 2.92 ± 0.02 ms and 3.31 ± 0.01 ms, respectively. For the deprotonated species, the average drift times were 1.54 ± 0.01 ms (isomer) and 1.71 ± 0.02 ms (authentic). Note that IMS conditions are substantially different for the two polarities as they were optimized separately to maximize resolution in drift time separation.

Fig. 5.

Droplet-fusion experiment (A), proposed isomer chain extension reaction from structure 1 (B), and resulting full-scan mass spectra (C–F). When aqueous solutions of Gly are sprayed from both nESI emitters (C), di-, tri-, and tetrapeptides are observed in the mass spectrum. Similarly, if both emitters are used to spray GlyGly (E), only tetra and hexapeptides are obtained, as would be expected. When spraying Gly from one nESI emitter and GlyGly from the other (D), a range of oligomers up to hexapeptide species is detected. Finally, when spraying mixtures of Gly and Ala from both emitters, all possible homo- and heterostructures are represented in the m/z values observed up to tripeptides (F). The generated peptide species are highlighted in all cases. During droplet fusion, the unique microdroplet-synthesized dipeptide isomers (either oxazolidinone as shown or as an ion-neutral complex) serve as chain extension species that undergo further condensation reactions with other amino acids or peptides to yield authentic higher-order peptides (B).

Scheme 1.

Proposed mechanism for the microdroplet-mediated formation of [GlyGly+H]⁺ dipeptide isomers that lead to authentic [GlyGly+H]⁺ from free Gly. Protonation of free Gly on the carbonyl oxygen leads to condensation with a neutral Gly molecule followed by cyclization to oxazolidinone 1. This species has two ion-neutral complex forms where the neutral is either water (2) or methylamine (3).