Oxazolone mediated peptide chain extension and homochirality in aqueous microdroplets

Abstract

Peptide formation from amino acids is thermodynamically unfavorable but a recent study provided evidence that the reaction occurs at the air/solution interfaces of aqueous microdroplets. Here, we show that i) the suggested amino acid complex in microdroplets undergoes dehydration to form oxazolone; ii) addition of water to oxazolone forms the dipeptide; and iii) reaction of oxazolone with other amino acids forms tripeptides. Furthermore, the chirality of the reacting amino acids is preserved in the oxazolone product, and strong chiral selectivity is observed when converting the oxazolone to tripeptide. This last fact ensures that optically impure amino acids will undergo chain extension to generate pure homochiral peptides. Peptide formation in bulk by wet-dry cycling shares a common pathway with the microdroplet reaction, both involving the oxazolone intermediate.

Keywords: microdroplet chemistry; origin of homochirality; origin of life; peptide formation; prebiotic chemistry.

Scheme 1.

Proposed mechanism for peptide formation mediated by oxazolidinone/oxazolone intermediates. All reactions, except as noted, occur in microdroplets. Free amino acids condense to form oxazolidinone 1, which undergoes isomerization, resulting in the generation of dipeptide (investigated in ref. 31). Through simple dehydration, oxazolidinone 1 converts to oxazolone 2, the key intermediate involved in peptide formation. It reacts with water or with a third amino acid to form dipeptide and tripeptide, respectively. The resulting di- or tripeptide undergoes a similar process, involving oxazolidinone/oxazolone formation, to further extend the peptide chain. The strong preference for L-amino acid during chain extension enforces the L-homochirality of peptides.

Scheme 2.

Bulk synthesis of intermediates of peptide chain extension, oxazolones. A and B illustrate the synthetic routes to oxazolone 5 and enol isomer, oxazole 7, respectively.

Fig. 1.

Dipeptide formation through oxazolone hydration in microdroplets. (A) Illustration of microdroplet production by nESI; online microdroplet reaction and follow-up MS detection. Conversion from oxazolone to dipeptide by reaction with H₂O occurs in microdroplets. (B) Mass spectra in the positive mode, showing the conversion from 5a and 5b to 8a and 8b respectively, in water microdroplets in ambient air. (C) Mass spectra in the positive mode, showing the online conversion from 5b to 8b in ACN/DCM (v/v = 1:1) microdroplets in ambient air and with added water vapor. (D) Comparison of MS/MS spectra obtained for the standard dipeptide (Top row) and the product generated in microdroplets (Bottom row) in both positive and negative modes; the fragments annotated with blue columns represent the diagnostic peaks of authentic dipeptides.

Fig. 2.

Peptide chain extension reaction from the oxazolone intermediate. (A) Conversion from oxazolone to tripeptide by reacting with glycine in microdroplets. (B) Mass spectrum in the positive mode, showing the conversion from 5a to both dipeptide and tripeptide in water microdroplets containing glycine. (C) Comparison of MS/MS spectra obtained for the standard tripeptide (Top row) to the product generated in microdroplets (Bottom row) in both positive and negative modes; the fragments annotated in blue columns represent the diagnostic peaks of the authentic tripeptide.

Fig. 3.

Chiral preference during the process of peptide chain extension. (A) Chiral oxazolone-directed diastereoselective tripeptide formation occurs due to preferential L-Alanine reaction. (B) MS/MS spectra of generated tripeptide or isomers when 5b was mixed with D-Ala (Left) and L-Ala (Right). The spectrum from the reference tripeptide, H-Gly-Val-Ala-OH is displayed in the Middle panel for comparison. Note that its diastereomer, H-Gly-Val-D-Ala-OH presents an identical MS² spectrum (SI Appendix, Fig. S10). (C) Reaction between 5b and racemic alanine with D-Ala labeled by deuterium, with the results of MS/MS spectra of the ions at m/z 246 derived from L-isomer (Left) and m/z 249 derived from D-isomer (Right). (D) Reaction between 5b and racemic alanine with L-Ala labeled by deuterium, with the results of MS/MS spectra of the ions at m/z 246 derived from D-isomer (Left) and m/z 249 derived from L-isomer (Right).

Fig. 4.

Wet-dry cycling to synthesize Gly₂. (A) Illustration showing the bulk cycling process, including a hot phase and a cool phase. Glycine solution (15 μL, 10 mM) was used as starting material. After one cycle, the residue from the aqueous amino acid mixture was redissolved in 0.1% acetic acid aqueous solution for nESI-MS/MS analysis. (B) MS/MS spectra of Gly₂ and its isomer at m/z 133 generated by wet–dry cycles with the hot phase at RT for 24 h (Left), 40 °C for 2 h (Middle), and 75 °C for 2 h (Right). The diagnostic fragments from the Gly₂ and its oxazolidinone isomer (1) are annotated in blue and red, respectively. Peaks annotated in purple are shared fragments.