From our earlier investigations, it comes out that proteinogenic amino acids can undergo spontaneous oscillatory reactions of chiral inversion and peptidization. l-Serine (l-Ser) is an important proteinogenic amino acid with many vital functions in human and mammalian organisms, e.g., it is responsible for good condition of the nervous cell membranes. It undergoes spontaneous oscillatory processes of chiral inversion and peptidization, and the goal of this study was to compare the dynamics of its peptidization with that of d-Ser and dl-Ser (racemate). The main analytical technique used in our experiment was TLC-densitometry, and the auxiliary chromatographic techniques were HPLC–evaporative light scattering detector and LC–MS. The results obtained witness to the differences in peptidization dynamics of the two Ser enantiomers (l and d) and of the racemic mixture thereof (dl). It was shown that dl-Ser characterizes with the higher, and l- and d-Ser with the lower peptidization yields.

Introduction

The research on spontaneous oscillatory chiral conversion of the low-molecular-weight carboxylic acids was initiated a decade ago with an article demonstrating this phenomenon upon an example of S(+)-ibuprofen (1), and later it was confirmed as a general phenomenon with a selection of other compounds from the groups of profen drugs, hydroxyl acids and amino acids (e.g., (24)). Then, it was found out that spontaneous oscillatory chiral conversion of the aforementioned compounds is accompanied by spontaneous oscillatory condensation of the same compounds and the two processes are running in the parallel (57). Studies (17) furnish general information on possible molecular mechanisms of these two oscillatory processes, which involve a non-chiral enol/enolate ion intermediary step and can be illustrated by the scheme given in Figure 1A (for the sake of this study, showing an example of l-Ser).

Figure 1.

(A) The parallel processes of chiral conversion and peptidization of l-Ser; and (B) chemical structures of l-Ser and d-Ser.

Figure 1.

(A) The parallel processes of chiral conversion and peptidization of l-Ser; and (B) chemical structures of l-Ser and d-Ser.

So far, spontaneous peptidization of amino acids in abiotic media has not attracted sufficient interest from the side of the amino acid and protein researchers and an evident proof of this lack of interest is, e.g., a poor choice of the HPLC columns available on the market and dedicated to the enantioseparation of native, underivatized amino acids. In the past, spontaneous peptidization of amino acids has been sporadically investigated, and mostly by those interested in abiogenesis and the development of a prebiotic universe under the environmental conditions stimulating the origin of biological life. In model experiments, the employed working conditions used to be quite drastic, in order to reproduce the terrestrial or extraterrestrial environments from before the millennia. Among the best-known experiments are those performed by Fox and Harada, and dealing with thermal copolymerization of amino acids to the products resembling proteins and therefore denoted as proteinoids (8, 9).

Our interest in spontaneous oscillatory processes running in abiotic liquid systems and involving amino acids resulted in the investigation of chiral conversion (10, 11) and peptidization (e.g., (1215)), using mainly TLC-densitometry (1113) and HPLC (1416) with different detectors [diode array detector (DAD), evaporative light scattering (ELSD) and MS] as analytical tools. In some of these investigations, we focused on peptidization of binary amino acid mixtures and a possibility of the heteropeptide formation (e.g., (14, 16, 17)). Selection of the binary amino acid mixtures (e.g., l-Pro-l-Phe, l-Hyp-l-Phe, and l-Pro-l-Hyp) with different peptidization dynamics of each counterpart was deliberate, in order to trace its impact on an overall dynamics of co-peptidization. In the study of Sajewicz et al. (16), a theoretical model was developed extensively discussing four logical cases of peptidization dynamics with two different amino acids present in the solution. Case 1 does not assume any intermolecular interactions between the two counterparts, which results in formation of homopeptides only. In that case, peptidization dynamics of the two amino acids resembles those of pure amino acids in monocomponent solutions. Cases 2–4 anticipate different cooperative effects with (i) one amino acid governing an overall peptidization dynamics, (ii) another amino acid governing an overall peptidization dynamics and (iii) peptidization dynamics in the mixture different from those valid for the single peptidization counterparts.

Amino acid enantiomers are two different yet structurally very similar compounds. The aim of this study was to compare peptidization dynamics of two enantiomers in an abiotic solution versus their peptidization dynamics in a binary mixture. Moreover, we were searching for an experimental evidence of heteropeptide formation in an enantiomer mixture. Our choice was l- and d-Ser, because of the importance of the proteinogenic l-Ser as a building block of the mammalian and human proteins (Figure 1B). Our interest in Ser was instigated by a growing amount of information on an effective chiral conversion of l-Ser to d-Ser in various different brain regions of mammals under a catalytic influence of serine racemase (e.g., (1820)). Although the role of this chiral conversion in the metabolic pathway is not yet fully understood, this finding undermines an established concept of l-homochirality of the mammalian and human proteins. Moreover, certain scientific reports point out to a possible role of d-Ser played in neuropsychiatric and neurodegenerative conditions (e.g., (21, 22)). Another interesting feature of l-Ser is its documented ability to relatively easily racemize, which was perceived by geochemists involved in the protein geochronological studies (23, 24).

Experimental

Reagents

In this experiment, we used l-Ser purchased from Reanal (Budapest, Hungary), and d-Ser and dl-Ser purchased from Sigma-Aldrich (St Louis, MO, USA). All amino acids were of analytical purity. Methanol, 1-propanol (Sigma-Aldrich, St. Louis, MO, USA) and 2-propanol (Merck, Darmstadt, Germany) were of HPLC purity. Ninhydrin, zinc (II) nitrate, glacial acetic acid and ammonia (PHH POCh, Gliwice, Poland) were also of analytical purity. Water was de-ionized and double distilled in our laboratory by means of the Elix Advantage model Millipore System (Molsheim, France).

TLC-densitometric comparison of peptidization yields with l-, d- and dl-Ser

Thin-layer chromatographic experiments were performed on the commercial 20 × 20 cm plates pre-coated with microcrystalline cellulose (Merck; cat. no. 1.05716). Concentrations of l-Ser, d-Ser and dl-Ser in 70% aqueous methanol were 1 mg mL−1 (i.e., 9.44 × 10−3 mol L−1). The l-Ser solution in 70% aqueous methanol was used for the microscopic measurements as well.

Chromatographic plates were activated by heating for 30 min at 110°C prior to applying the amino acid samples. Just before the chromatographic analysis, zinc(II) nitrate was added to the six samples, i.e., to the fresh l-, d- and dl-Ser solutions, and the l-, d- and dl-Ser solutions after 11 days of aging (the molar ratio of amino acid-to-zinc(II) nitrate was 1:1). The 1-propanol–0.5% aqueous ammonia (7:3, v/v) mixture was used as the mobile phase. Samples were spotwise applied to the plates in the 5-µL aliquots, 1.5 cm apart and 1 cm above the lower edge of the plate. The chromatograms were developed to the distance of 7 cm, and the development time was ca. 2 h. After the development, the plates were dried and the chromatograms were visualized by spraying the plates with the 0.5% ninhydrin solution in 2-propanol, followed by heating for 5 min at 110°C.

The visualized chromatograms were densitometrically scanned with use of a Desaga (Heidelberg, Germany) model CD 60 densitometer equipped with the Windows-compatible ProQuant software. Concentration profiles of the development lanes were recorded in the reflectance mode at the carefully selected wavelength, λ = 550 nm. The obtained results were additionally recorded with a digital camera. All TLC experiments were performed in triplicate.

HPLC-ELSD comparison of peptidization dynamics with l-, d- and dl-Ser

Monitoring of spontaneous non-linear peptidization dynamics of l-, d- and dl-Ser was performed with use of the achiral HPLC-ELSD. The l-, d- and dl-Ser solutions were prepared in 70% aqueous methanol at the concentration equal to 1 mg mL−1 (i.e., 9.44 × 10−3 mol L−1), then placed in an autosampler and recording of the chromatograms for the l-, d- and dl-Ser sample was carried out in the 8-min intervals for the period of 40 h. The analyses were carried out using the Varian model 920 liquid chromatograph equipped with the Varian 900-LC model autosampler, the gradient pump, the Varian Pro Star 510 model column oven, the Varian 380-LC model ELSD detector (Varian Inc., Palo Alto, CA, USA), the ThermoQuest Hypersil C18 (150 × 4.6 mm i.d.; 5 µm particle size) column and the Galaxie software for data acquisition and processing. Other working conditions are as follows: sample aliquots, 3 μL; mobile phase composition, acetonitrile–1% aqueous CH3COOH (10:90, v/v); and mobile phase flow rate, 0.8 mL min−1 (in the isocratic mode). The column was thermostatted at 35°C.

MS evidence of peptidization of l-, d- and dl-Ser

To prove the presence of the peptides in the investigated amino acid solutions, the mass spectrometric analysis was performed for the samples of l-, d- and dl-Ser in 70% aqueous methanol, aged for 9 months. For this investigation, we used Thermo LCQ Deca XP Plus MS system (Thermo Scientific, Waltham, MA, USA) under the following working conditions: the ESI mode (ESI–MS scan, positive ionization, capillary voltage 50 V, needle voltage 5 kV and needle temperature 250°C). All MS experiments discussed in this study were performed at least in duplicate.

Results

TLC-densitometric comparison of peptidization yields with l-, d- and dl-Ser

Different peptidization dynamics of l-, d- and dl-Ser dissolved in 70% methanol were demonstrated with use of the thin-layer chromatography. In Figure 2A, the densitograms are presented valid for the freshly prepared solutions of l-, d- and dl-Ser, and also for those aged for 11 days. In Figure 2B, the picture of the chromatogram is presented, showing the chromatographic spots of the freshly prepared solutions of l-, d- and dl-Ser, and also of those aged for 11 days. The retardation coefficient (RF) values of the monomeric Ser (l, d and dl) are equal to 0.32 ± 0.02.

Figure 2.

(A) Densitograms obtained for the freshly prepared solution of (a) l-Ser, (b) d-Ser and (c) dl-Ser, and for the solutions thereof stored for the period of 11 days. (B) Picture of the developed chromatogram, where (1, 2): freshly prepared l-Ser solution; (3, 4): l-Ser solution aged for 11 days; (5, 6): freshly prepared d-Ser solution; (7, 8): d-Ser solution aged for 11 days; (9, 10): freshly prepared dl-Ser solution and (11, 12): dl-Ser solution aged for 11 days.

Figure 2.

(A) Densitograms obtained for the freshly prepared solution of (a) l-Ser, (b) d-Ser and (c) dl-Ser, and for the solutions thereof stored for the period of 11 days. (B) Picture of the developed chromatogram, where (1, 2): freshly prepared l-Ser solution; (3, 4): l-Ser solution aged for 11 days; (5, 6): freshly prepared d-Ser solution; (7, 8): d-Ser solution aged for 11 days; (9, 10): freshly prepared dl-Ser solution and (11, 12): dl-Ser solution aged for 11 days.

HPLC-ELSD comparison of peptidization dynamics with l-, d- and dl-Ser

The HPLC-ELSD technique was used to demonstrate the non-linear concentration changes of the investigated amino acids in the course of time, thus revealing an oscillatory nature of peptidization in the solutions of l-, d- and dl-Ser.

In Figure 3, superposition is shown of three chromatograms valid for l-Ser and registered for the fresh sample (after 0.12 h aging) and for those after 4.8 and 39.83 h aging. In Figure 4, we show the time series of the peak heights registered for the l-, d- and dl-Ser samples in the course of 40 h aging.

Figure 3.

Superposition of the selected HPLC-ELSD chromatograms recorded for l-Ser in 70% aqueous methanol after 0.12, 4.83 and 39.83 h storage time.

Figure 3.

Superposition of the selected HPLC-ELSD chromatograms recorded for l-Ser in 70% aqueous methanol after 0.12, 4.83 and 39.83 h storage time.

Figure 4.

Time series of chromatographic peak heights for l-Ser, d-Ser and dl-Ser in 70% aqueous methanol registered with the ELSD detector.

Figure 4.

Time series of chromatographic peak heights for l-Ser, d-Ser and dl-Ser in 70% aqueous methanol registered with the ELSD detector.

MS evidence of peptidization of l-, d- and dl-Ser

The mass spectrometric results prove that amino acids stored in an abiotic environment undergo spontaneous peptidization. In Figures 5A–C, the mass spectra that were recorded for the l-Ser, d-Ser and dl-Ser samples stored for the period of 9 months are given.

Figure 5.

Mass spectra registered with use of LC–MS and implemented with the tabulated interpretation of the selected m/z signals for the aged samples of (A) l-Ser, (B) d- and (C) dl-Ser, respectively.

Figure 5.

Mass spectra registered with use of LC–MS and implemented with the tabulated interpretation of the selected m/z signals for the aged samples of (A) l-Ser, (B) d- and (C) dl-Ser, respectively.

Discussion

TLC-densitometric comparison of peptidization yields with l-, d- and dl-Ser

First, it has to be emphasized that the enantioseparation of amino acids still is a very challenging analytical task. However, TLC-densitometry of amino acids and, more specifically, the enantioresolution thereof can be achieved in a more straightforward and easier manner than with the other chromatographic techniques. There are at least two reasons, which justify this statement. One reason is that for the direct enantioseparation of amino acids (i.e., for that without a preliminary derivatization) only one chiral HPLC column is available on the market, whereas TLC-densitometry offers a possibility of an in-home preparation of a wide selection of dedicated and reasonably well-performing chiral stationary phases and/or a variety of specially devised mobile phases. Another reason is that the enantioresolution of chiral counterparts obtained with aid of TLC-densitometry is preserved on a chromatographic plate, thus enabling further investigations. This conclusion is based on our long-time experience, and it can also be derived from the monograph on chromatographic analysis of amino acids, with a considerable part dedicated to the achievements of the enantioseparation with aid of TLC-densitometry (25).

From our earlier investigations, it comes out that amino acids can undergo spontaneous oscillatory inversion and peptidization (although Ser has never been investigated before). The current comparison of the Ser enantiomers shows that the peptidization dynamics of l-, d- and dl-Ser dissolved in 70% aqueous methanol considerably differ. Investigations with use of TLC revealed that the most dynamic peptidization process takes place with the racemic Ser mixture (dl-Ser). Namely, the band intensity of the monomeric dl-Ser obtained for the fresh sample equaled to 836.45 mAV, and the analogous signal obtained from the aged sample equaled to 332.86 mAV only, showing a concentration drop equal to 503.59 mAV (Figure 2A-C). In the case of d-Ser, the analogous band intensity values were 958.00 and 621.08 mAV, respectively, with the band intensity drop equal to 336.92 mAV, which is a proof of the lower peptidization yields (Figure 2A-B). The least dynamic peptidization was observed with l-Ser. In this case, band intensities of the monomeric l-Ser in the fresh sample and in that after 11 days aging were practically the same and equal to 824.87 mAV. It needs adding that chromatographic spots of the monomeric l-, d- and dl-Ser in the fresh and aged samples (visualized with ninhydrin; Figure 2B) do not show the effect of peptidization as clearly as do the respective densitograms.

HPLC-ELSD comparison of peptidization dynamics with l-, d- and dl-Ser

The superposition of the three chromatograms valid for l-Ser and registered for the fresh sample (after 0.12 h aging) and for those after 4.83 and 39.83 h aging (Figure 3) was made for the sake of comparison to emphasize the general trend of sample aging, which was the concentration drop of the monomeric l-Ser as a result of peptidization. The analogous trend was observed with d-Ser and dl-Ser, too.

In Figure 4, we showed the time series of the peak heights registered for the l-, d- and dl-Ser samples in the course of 40 h aging. From the obtained plots, it is evident that the amino acid concentrations were changing in a non-linear (oscillatory) fashion. Changes in the l-, d- and dl-Ser concentrations are well expressed in terms of the peak intensity changes. For l-, d- and dl-Ser, the respective oscillatory peak intensity ranges are 145.03–201.44, 114.90–165.90 and 123.40–157.10 mV. The oscillation range of the peak maxima valid for l-Ser is somewhat higher than those valid for d-Ser and dl-Ser. This result remains in conformity with those originating from TLC-densitometry as an additional proof that with d- and dl-Ser, higher peptidization yields are observed compared with l-Ser (and hence, the intensity ranges of the respective peak maxima for the monomeric d- and dl-Ser are lower than that valid for l-Ser).

MS evidence of peptidization of l-, d- and dl-Ser

Mass spectra obtained for l-, d- and dl-Ser confirm differentiated peptidization dynamics with these three amino acids. From the TLC results, a conclusion can be drawn that the peptidization rate with dl-Ser is the higher one, whereas peptidization rates with l-Ser and d-Ser are lower. From Figures 5A and B, it can be seen that the mass spectra valid for the aged l- and d-Ser samples after the 9-month storage period are quite similar to one another in this sense that relative intensities of the respective peptides are rather low when compared against the predominant molecular ion at m/z 105.9. In Figure 5C, the mass spectrum valid for the dl-Ser sample is given, with the predominant molecular ion at m/z 105.9, yet with relative intensities of the spontaneously formed peptides considerably higher than those observed for individual enantiomers (Figures 5A and B). Moreover, in Figures 5A–C the tabulated proposals are presented of the possible peptide structures attributed to the selected m/z signals in the respective mass spectra. In certain cases, the numbers of the corresponding peptide bonds (–CO–NH–) are equal to several dozens.

Conclusion

The goal of this study was to demonstrate different peptidization dynamics of l-, d- and dl-Ser, and it was obtained with the use of the TLC-densitometry as the main analytical technique, and HPLC-ELSD and LC–MS as the auxiliary chromatographic techniques. Although each chromatographic technique contributed in its own way to adding to a multidimensional picture of the phenomenon investigated, the experimental evidence furnished by TLC-densitometry can be regarded as the most straightforward and therefore the most comprehensive also. On the basis of the results originating from the three different chromatographic techniques, it was unequivocally established that dl-Ser undergoes a more rapid spontaneous oscillatory peptidization process, whereas peptidization of l- and d-Ser is perceptibly slower. At this stage, the reason for the difference in peptidization rates between l- and d-Ser is not yet clear.

Acknowledgment

A.G. acknowledges the financial support of the DoktoRIS project, co-financed by the European Union within the European Social Fund.

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