Impact of D2O on peptidization of l-hydroxyproline

Abstract

This is our fourth consecutive study carried out in an order to collect experimental evidence regarding the impact of heavy water (D2O) on the spontaneous oscillatory peptidization of proteinogenic α-amino acids and this time its subject matter is l-hydroxyproline (l-Hyp). Our three earlier studies have been focused on the two sulfur-containing α-amino acids, i.e., l-cysteine (l-Cys) and l-methionine (l-Met), and on one structurally related α-amino acid, i.e., l-proline (l-Pro). It seemed interesting to assess the effect induced by D2O on l-Hyp and to compare it with the effects valid for l-Cys, l-Met, and l-Pro. As analytical techniques, we used high-performance liquid chromatography with the evaporative light-scattering detection (HPLC-ELSD), mass spectrometry (MS), and scanning electron microscopy (SEM). The obtained results make it clear that the impact of heavy water on dynamics of spontaneous peptidization of l-Hyp differs from that exerted on three other α-amino acids discussed (although in all four cases, heavy water significantly hampers spontaneous oscillatory peptidization). With l-Hyp, an increasing proportion of D2O in the reaction mixture results in decreasing yields of both, the soluble lower molecular weight peptides and the insoluble higher molecular weight peptides. With the two sulfur-containing compounds (l-Cys and l-Met), an increasing proportion of D2O in the reaction mixture resulted in growing yields of the soluble lower molecular weight peptides at an expense of decreasing yields of the insoluble higher molecular weight peptides. With l-Pro, still different pattern was observed, namely that the hampering effect of D2O on peptidization was not monotonously dependent on the concentration of D2O in the system, but it was the strongest pronounced for 10% (v/v) D2O in the employed binary methanol–water solvent (with the investigated proportions of D2O in this solvent changing from 0 to 30%). We hope that firm quantitative results presented in this study (and also in the three earlier studies from this cycle) can prove an inspiration for future researchers interested in getting a deeper insight into the role of D2O in life processes, and more specifically in the kinetic and mechanistic aspects thereof. Perhaps it might be noteworthy to add that out of four α-amino acids investigated so far, which can be divided into two groups of the endogenous (l-Cys and l-Pro) and exogenous (l-Met and l-Hyp) species, the endogenous species undergo spontaneous peptidization following the circadian rhythm, whereas the exogenous ones (including l-Hyp) do not.