Qualitative Monitoring of Proto-Peptide Condensation by Differential FTIR Spectroscopy

Abstract

Condensation processes such as wet-dry cycling are thought to have played significant roles in the emergence of proto-peptides. Here, we describe a simple and low-cost method, differential Fourier transform infrared (FTIR) spectroscopy, for qualitative analysis of peptide condensation products in model primordial reactions. We optimize differential FTIR for depsipeptides and apply this method to investigate their polymerization in the presence of extraterrestrial dust simulants.

Figure 1

(a) Diagram of a wet–dry cycle. Monomer solutions are heated (open system), removing water and forming polymers. Water is then reintroduced and incubated with sample (closed system; temperature and length of time adjustable) to hydrolyze unstable polymers in solution. Cycles are then repeated as desired. FTIR analysis is performed after the hot/dry step, as material is in a solid and/or gel state and easy to transfer to an ATR target. (b) Monomers and polymers studied in this work; chiral centers are marked with asterisks. Hydroxy acids are abbreviated with lower case letters and amino acids are abbreviated with upper case letters. Depsipeptides are copolymers of hydroxy acids and amino acids.

Figure 2

(a) Overview of differential FTIR spectroscopy for proto-biopolymer analysis. Monomer control spectra are subtracted from cycled sample spectra to generate difference spectra. (b) Difference FTIR spectra for lactic acid + glycine (a + G) depsipeptides after 1, 4, 8, and 12 wet–dry cycles in the main fingerprint region. Marked signals correspond to ester (1748 cm^–1) and amide/peptide (1652 and 1539 cm^–1) backbone linkages in depsipeptides. Assignments of labeled signals are provided in Table 1. No signal normalization was used.

Figure 3

Key ester/amide changes with wet–dry cycling and comparison to MALDI-TOF MS, an orthogonal technique. (a) Differential FTIR ester and amide signals from Figure 2b. Amide I (1652 cm^–1) and amide II (1539 cm^–1) bands increase with cycling, whereas the ester C=O band (1748 cm^–1) increases to 4 cycles and then decreases. These data are consistent with amide bond formation via activated ester intermediates. (b) Comparison to MALDI-TOF MS, an orthogonal technique, for the same a + G depsipeptide samples. The median glycine content in all depsipeptides increases from 1 cycle to 12 cycles, consistent with differential FTIR spectroscopy.

Figure 4

Normalization examples for glycolic acid and valine (g + V) depsipeptides. (a) Raw FTIR spectra without normalization and resulting difference spectra. Inconsistent amounts of sample placed on ATR target resulted in baseline drift across the fingerprint region, including in the key ester/amide I region for depsipeptides (blue box). (b) FTIR spectra after normalization at 2880 cm^–1 (valine methine C–H str.) and resulting difference spectra. The ester/amide I region is improved, but few negative signals are observed (a.k.a. depletion with cycling). This is likely due to overlapping O–H str. at the normalization value and varying amounts of water between control and cycled samples. (c) FTIR spectra after normalization at 1456 cm^–1 (assigned to asymmetric CH₃ bend) and resulting difference spectra. Ester/amide I region is clean, and both positive and negative changes are observed with cycling.

Figure 5

Differential FTIR spectra of lactic acid and alanine (a + A) depsipeptides subjected to wet–dry cycling in the absence or presence of model dust simulants. (a) FTIR spectra without model dust simulant added. Ester formation (light red) is pronounced at 1 cycle and conversion to amide/peptide (light blue) is observed at 4 cycles. With the addition of (b) 1.0 mg of LHS-1D lunar dust simulant or (c) 1.0 mg of JEZ-1 Martian dust simulant to samples, spectra show reduced esterification, yet amide/peptide formation persists.