Phosphoric acid salts of amino acids as a source of oligopeptides on the early Earth | Communications Chemistry

Communications Chemistry volume 7, Article number: 185 (2024) Cite this article

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Because of their unique proton-conductivity, chains of phosphoric acid molecules are excellent proton-transfer catalysts. Here we demonstrate that this property could have been exploited for the prebiotic synthesis of the first oligopeptide sequences on our planet. Our results suggest that drying highly diluted solutions containing amino acids (like glycine, histidine and arginine) and phosphates in comparable concentrations at elevated temperatures (ca. 80 °C) in an acidic environment could lead to the accumulation of amino acid:phosphoric acid crystalline salts. Subsequent heating of these materials at 100 °C for 1–3 days results in the formation of oligoglycines consisting of up to 24 monomeric units, while arginine and histidine form shorter oligomers (up to trimers) only. Overall, our results suggest that combining the catalytic effect of phosphate chains with the crystalline order present in amino acid:phosphoric acid salts represents a viable solution that could be utilized to generate the first oligopeptide sequences in a mild acidic hydrothermal field scenario. Further, we propose that crystallization could help overcoming cyclic oligomer formation that is a generally known bottleneck of prebiotic polymerization processes preventing further chain growth.

Being a fundamental building block of ribonucleic acids, phosphates have always been considered as one of the mandatory ingredients of the prebiotic brine from which life has emerged on our planet. In addition, partly due to their unique proton-transfer properties1, phosphate derivatives represent excellent catalysts for organic chemical syntheses2.

In the past, various strategies have been proposed for the formation of peptide bonds between amino acid monomers in a prebiotic context3. In order to overcome the thermodynamic barrier of condensation reactions in water, activation by a volcanic gas, carbonyl sulfide, was suggested4. Copper-catalyzed peptide bond formation has also been described in various geological settings5,6, including environments of high-salt concentrations5,7. Liquid SO2 was also suggested to be a highly potent environment for copper- and iron-catalyzed peptide bond formation on the early Earth8,9. Further, it has been demonstrated that drying of glycine on silica or titania surfaces may induce formation of oligoglycines10,11. Silica-catalyzed formation of mixed oligopeptides up to the length of heptamers has also been reported12. Other metal-oxide and silicate minerals13,14 as well as iron(III) oxide nanoparticles15 have also been found to be effective at catalyzing the oligomerization of glycine. Dry-wet cycling has been suggested to positively influence the yield of amino acid polymerization processes16. Recently, a very original approach has been suggested to overcome the thermodynamic barrier of peptide bond formation reactions in water that involves formation of depsipeptides17,18,19,20. These oligomers contain a mixture of amide and ester bonds that upon dry-wet and hot-cold cycling transform into oligopeptides. Carbodiimide-promoted C-terminal elongation of peptides was reported21. A complex reaction network connecting prebiotic synthesis of cysteine with its catalytic role in amino acid polymerization has been proposed22.

Concentrated phosphoric acid is often used as a dehydrating agent and catalyst. For example, alcohols can be converted into alkenes using concentrated phosphoric acid23. Since peptide bond formation between two amino acid monomers is essentially a condensation reaction, it was reasonable to expect that a concentrated phosphoric acid medium may assist peptide bond formation as well.

Catalytic amounts of phosphoric acid have been shown to be efficient in the condensation of solid amino acids24. Nevertheless, the reaction required harsh conditions (160–210 °C), the reaction temperature being only ca. 30 degrees lower than that of the well-known synthesis of peptides by heating of solid amino acids that requires temperatures far above 100 °C25. Peptide bond formation in the presence of added inorganic phosphate26 or polyphosphate27 salts also required temperatures much higher (140 °C)26 or extended reaction times (several hundreds of hours)27. Trimetaphosphate28,29 and diamidophosphate30 assisted peptide bond formation has also been proposed in the literature.

Facile salt formation (co-crystallization) of various amino acids with phosphoric acid is demonstrated by the numerous crystal structures available in the Cambridge Structural Database31. In these crystals the protonated amino acid serves the role of cations and H2PO4− is the charge compensating anion. An inspection of the structure of the 1:1 salt of glycine and phosphoric acid32 suggested that in the crystalline material the amino acid and phosphate ions are arranged in a favorable way for a phosphoric acid catalyzed peptide bond formation between the glycine monomers. Since concentrated phosphoric acid has the highest proton-conductivity among all known materials1, we thought that the assembly of phosphates present in amino acid:phosphoric acid salts could catalyze the proton transfer processes in the course of the peptide bond formation. This motivated us to scrutinize the role of co-crystallization of amino acids with phosphoric acid in the prebiotic production of simple peptides in a drying lagoon scenario. In the current work we illustrate that by selecting conditions that allow for fast proton transfer processes within arrays of phosphoric acid molecules or its conjugate anions the well-established protocol by Fox and Harada24,25,33 can be easily adapted to demonstrate prebiotic polymerization of amino acids at significantly lower (thus prebiotically more plausible) temperatures.

Figure 1 demonstrates the MALDI-TOF mass spectra of polymerization products formed upon a 1 day-long heat-treatment of glycine:H3PO4 1:1, l-arginine:H3PO4 1:1 and l-histidine:H3PO4 1:2 salts at 100 °C. For all three studied amino acids at least dipeptide formation has been confirmed. In case of glycine, formation of hexamers is also apparent. An extended (3 days long) sample treatment yielded 24-mers from glycine, while no apparent oligomer extension was observed for histidine and arginine, as both amino acids formed only dimers. In case of glycine after the 3-days-long heat-treatment the sample had a glassy, rather than crystalline appearance and its color changed to green. MALDI-TOF mass spectra of this sample are shown in Fig. 2. MALDI-TOF mass spectra of the products formed upon extended heat-treatment (3 days, 100 °C) of arginine:H3PO4 1:1 and histidine:H3PO4 1:2 crystals are shown in Fig. S1 of the Supplementary information.

Unlabeled signals correspond to MALDI matrix clusters. MALDI-TOF mass spectra of the commercial untreated amino acids are shown in Fig. S2 of the Supplementary information.

Gn: glycine multimer (n = 2–24). Unlabeled signals correspond to MALDI matrix clusters.

Oligomer formation was also confirmed by an LC-MS analysis of the polymerization products (see Fig. 3). The analysis has shown that heat-treatment of glycine:H3PO4 crystals yields up to 14-mers upon a 3 days long treatment. Using the same technique, the longest oligomers detected from salts of the other two amino acids were trimers only. Nevertheless, the analysis has clearly shown that the amount of oligomeric products increases upon a prolonged heat-treatment of the H3PO4 salts of arginine and histidine. A 1 week-long heat-treatment did not yield any significant improvement of the reaction yields, being in the order of <1% (for MALDI-TOF mass spectra see Fig. S3 in the Supplementary information).

The bars represent relative abundance of the forms derived from the corresponding extracted ion chromatograms. Gn (n = 2–14), Rn (n = 2–3), and Hn (n = 2–3) stand for multimers formed from glycine, arginine and histidine, respectively. Since the amount of unreacted glycine cannot be straightforwardly quantified by LC-MS the data for glycine do not include the monomer fraction. We note that the applied LC-MS detection technique provides straightforward information about the variation of the relative polymerization yields under various reaction conditions, nevertheless, the method cannot be reliably applied to estimate absolute polymerization yields.

Based on peak areas of individual species detected by LC-MS, 0.16 ± 0.03% and 0.52 ± 0.10% of the l-arginine and l-histidine monomers, respectively, have been incorporated in oligopeptides upon heating of the amino acid–phosphoric acid crystals for 3 days.

It is well-know that liquid chromatographic separation of oligoglycine sequences and glycine monomers is notoriously problematic34. Previously, some of us have demonstrated that capillary electrophoresis in combination with mass spectrometry can be successfully used for quantification of oligopeptides formed in prebiotic amino acid polymerization experiments8,9. Applying the same methodology for the quantification of polymers formed in glycine:phosphoric acid 1:1 crystals we have found that about 10.7 ± 0.6% of glycine has been converted into an oligomeric state after a 3-days long heat-treatment at 100 °C. Electropherograms and extracted ion chromatograms used for the quantitative analysis are shown in Fig. 4.

Electropherogram (A) and combined extracted ion chromatograms (B) detected for a sample containing 1:1 glycine:H3PO4 crystals heat-treated at 100 °C for 3 days. Gn (n = 1–6) denote covalent multimers of glycine.

For comparison, we have also tested the oligomerization of an amorphous glycine:phosphoric acid mixture. For this purpose we made an aqueous mixture of NaH2PO4 with glycine in a 1:1 ratio and subsequently acidified the solution with 1 molar equivalent of HCl. After drying the solution, we obtained a material which contained an amorphous phase concentrating phosphoric acid and glycine, in addition to crystalline NaCl and a smaller amount of crystalline diglycine hydrochloride35 (NaCl:(GlyH)Cl·Gly molar ratio of ca. 4.4:1, see Fig. S4 in the Supplementary information). A 3-days long heat-treatment of the dry material at 100 °C resulted in the formation of oligomers of the length of up to 7-mers only (see Fig. 5). In contrast, thermal treatment of the 1:1 glycine:H3PO4 crystalline salts under the same conditions yields remarkably longer oligoG sequences (up to 24-mers, see Fig. 2). We note that polymerization of the amorphous mixtures of phosphoric acid with arginine and histidine proceeds with similar yields as that of the crystalline materials (<1%). Nevertheless, the dimer fraction is dominated by the cyclic diketopiperazine products (see Figs. S5 and S6 in the Supplementary information) that are dead-ends of amino acid polymerization processes.

Gn: glycine n-mer (n = 2–7). Unlabeled signals correspond to MALDI matrix clusters. Time dependence of oligomer relative abundance from an LC-MS analysis (on the bottom) of the same material. Since the amount of unreacted glycine cannot be straightforwardly quantified by LC-MS, the data shown in the bottom graph do not include the monomer fraction.

Under prebiotic conditions concentrated phosphoric acid:amino acid mixtures could easily accumulate upon evaporation of water even from highly diluted (micromolar), mildly acidic solutions at elevated (ca. 80 °C) temperatures. Dissolution of nitrogen oxides formed in atmospheric lightning36 could produce nitric acid for the conversion of phosphate salts into phosphoric acid. If the sufficient (roughly molar) concentration is reached, crystallization of amino acid:phosphoric acid salts is a facile process. Subsequent incubation of the crystalline materials at slightly higher temperatures (100 °C) then yields oligopeptides. Thus, the prebiotic settings we propose (see Fig. 6) are very simple and are compatible with an acidic hydrothermal field37 scenario. Under the same conditions the pure crystalline amino acids do not oligomerize (see Fig. S7 in the Supplementary information): the lowest reported temperature at which polymerization was observed in the absence of added materials by early studies33,38 is ca. 170 °C.

The first step involves concentration of amino acid: phosphoric acid mixtures to reach a roughly molar solution enabling crystallization. Oligopeptides are then produced by heating the crystalline material at 100 °C.

Polymerization of aspartic acid when heated with catalytic amounts of H3PO4 is a well-documented process39. Based on thermogravimetric measurements Wang et al. have found that a diluted mixture of phosphoric acid with aspartic acid can be concentrated to a mixture of concentrated phosphoric acid and amino acids by heating above 80 °C. Upon further heating the material loses water due to amide bond formation between the aspartic acid monomers. Our observations are consistent with this previous study, suggesting that under prebiotic conditions concentrated phosphoric acid:amino acid mixtures could easily accumulate upon evaporation of water at elevated (ca. 80 °C) temperatures. In addition, our results indicate that oligopeptides did form with considerable yields upon incubation of the phosphoric acid:amino acid salts at slightly higher temperatures (100 °C).

We note that polymerization of 1:1 glycine:phosphoric acid and glycine:polyphosphoric acid mixtures was also attempted in preceding studies26. Nevertheless, at the two reported temperatures, i.e., 75 and 100 °C, no oligomerization was observed. One of the possible reasons could be that drying of the materials was performed at high temperatures (140 °C) that interfered with crystallization of the glycine:phosphoric acid 1:1 salt. We have repeated the experiment and have found that above 120 °C the material does not crystallize, essentially forming an amorphous syrup. Using our state-of-the-art MALDI-TOF MS technique we detected oligomer formation in the sample after a 3 days long heat-treatment at 120 °C (see Fig. S8 in the Supplementary information). Nevertheless, the oligomeric products are noticeably shorter than those obtained when heating the crystalline material. Note, that we have also observed a noticeable deterioration of the polymerization yield for the 1:1 amorphous mixture of glycine with phosphoric acid (prepared by acidification of NaH2PO4 at room temperature, see above) as compared to the crystalline salt form material. (Crystalline diglycine hydrochloride, (GlyH)Cl·Gly, also present in this sample as minor component, does not polymerize.)

In our experiments the highest oligomerization yields and the longest oligopeptides were observed for glycine-phosphoric acid crystalline salts, suggesting that the activation mechanism is the most efficient in this case. Early studies by Fox and Harada also note that glycine has a distinctly higher propensity to polymerize as compared to other amino acids. They associate this fact with the absence of side chains on glycine that enables a better positioning of the amino acid molecules for peptide bond formation25.

Especially, the heat-treated 1:1 glycine:phosphoric acid crystalline salts exhibited a prominent polymerization activity, suggesting that the crystal structure may provide with favorable conditions for a phosphoric acid catalyzed peptide bond formation between the glycine monomers. As Fig. 7 illustrates, the crystal structure32 is built by alternating layers of negatively (−1) charged phosphate anions and +1 charged glycinium (NH3+CH2COOH, GlyH+) cations. Inside the glycinium layer the GlyH+ ions are assembled in a way that enables close contact between the amino and carboxylic ends of two adjacent monomers, the distance between the adjacent amino N and carboxylic C atoms being 3.67 Å. The phosphate layer held together by a network of interconnected P–O–H…O–P H-bonds between the phosphate anions may serve as a unique proton-transfer catalyst. We reiterate, that phosphate assemblies are responsible for the fact that phosphoric acid has the highest proton-conductivity among all known materials1. Thus, it could enable (i) deprotonation of the N-terminus of the glycinium cations in the initial step and (ii) elimination of water from the covalent adduct of two glycine units leading to amide bond formation (as shown in Fig. 7). Note, that early studies by Harada and Fox also propose an acid-catalysed mechanism for the formation of peptide bonds in a prebiotic context24,25. Nevertheless, due to the low amount of phosphoric acid used, these previously reported synthetic routes were found to operate at temperatures far above 100 °C.

The stick-and-ball models show a possible catalytic geometry for the peptide bond formation, which includes two amino acids and two phosphates. Distance between the amino N and carboxylic C atoms (highlighted in yellow) in two adjacent glycine molecules is 3.67 Å. On the right: schematic mechanistic model for the phosphoric acid-catalyzed peptide bond formation inside the crystalline material.

We note that, in principle, similar proton-conducting arrays may also form in phosphorous acid salts of amino acids. Phosphorous acid could be even more relevant to a prebiotic early Earth environment than phosphoric acid itself, since it could form via the corrosion of the meteoritic mineral schreibersite in a more reducing atmosphere likely present on our planet 4 billion years ago40,41. Indeed, we have found that crystallization of glycinium phosphite is even more facile than that of the corresponding phosphate salt. Heating of the dry crystalline material did produce oligomers, nevertheless their length is lower than that obtained from glycinium phosphate under the same conditions. Notably, the efficiency of the reaction is lower because the chain grow is impeded by formation of diketopiperazine in the first chain-extension step (see Fig. S9 in the Supplementary information). We reiterate that, in contrast to glycinium phosphite, no diketopiperazine formation was observed when polymerizing the crystalline glycinium phosphate salt. The likely reason for the diketopiperazine formation in glycinium phosphite crystals42 could be that in the crystal the H2PO3– ions form only chains; whereas in the glycinium phosphate crystals32 the H2PO4– ions are arranged into 2D sheets. The H2PO3– chains give sufficient flexibility to the material for cyclization reactions leading to diketopiperazine. It is also to note that in the (GlyH)(H2PO3) crystals no direct inter-glycinium hydrogen bonds are observed (although the GlyH+ cations are very close to each other), whereas they are present in the crystalline (GlyH)(H2PO4) salt. See Fig. S10 in the Supplementary information.

On the contrary, while crystallization of the glycine:HCl 1:1 salt is pretty facile, as we have shown above in connection to the polymerization of the glycine:NaH2PO4:HCl 1:1:1 mixture, crystals of diglycine hydrochloride, i.e., (GlyH)Cl Gly do not promote the polymerization of glycine. Likewise, absolutely no polymers form upon heat-treatment of crystals with a [(GlyH)2(SO4)]·Gly and (GlyH)2(SO4) composition (see Fig. S11 in the Supplementary information).

As an alternative pathway, one may think of the intermediacy of phosphoric acid activated amino acids in the reaction mechanism24. However, computations by Martínez-Bachs and Rimola have shown, that even when using a better phosphorylating agent, like ATP, the activation of the carboxylic group of glycine is an endergonic process43. N-phosphorylamino acids have also been reported to polymerize to short oligopeptides44. Nevertheless, synthesis of N-phosphorylamino acid monomers requires reduced phosphate esters45, that are not available in our reaction mixture. Indeed, neither our MALDI-TOF nor the LC-MS analysis indicated presence of phosphorylated peptides or amino acids in the reaction products. Thus, phosphorylated amino acid intermediates are not likely to be involved in the above-described reaction network.

Since phosphates are important building blocks of nucleic acids, there is no question that they had to be present in the prebiotic pool. One of the most popular proposals for the accumulation of phosphates involves extraterrestrial delivery in the form of the meteoritic mineral schreibersite. The ultimate products of the corrosion of this meteoritic mineral in water are phosphate salts46. It has been proposed that schreibersite could serve as a suitable prebiotic phosphorylating agent at the synthesis of nucleotides47. As we have shown above, co-drying of amino acids and phosphates in a low pH environment enables formation of oligopeptide sequences from amino acid precursors. It has been demonstrated that drying in acidic environments is also compatible with nucleotide polymerization reactions48. This suggests that nucleic acids and peptides, i.e., the two biopolymer classes forming the chemical foundations of terrestrial life, could simultaneously emerge upon drying of the same phosphate-containing acidic prebiotic brine. In addition, as our results demonstrate, in the presence of commeasurable amounts of phosphates and amino acids the polymerization proceeds already at 100°C, especially for crystals of glycinium dihydrogen phosphate. In this latter case, most likely the fortuitous mutual orientation of hydrogen-bonded phosphate and protonated amino-acid ions provides favorable conditions for a facile solid-phase oligomerization process.

In the current work, we propose a simple prebiotic route for the oligomerization of amino acids, catalyzed by phosphoric acid that could accumulate upon drying a phosphate-containing prebiotic pool in an acidic environment at temperatures ≤100 °C. The mechanism exploits the unique proton conductivity of phosphoric acid chains present in concentrated phosphoric acid or in salts/co-crystals of phosphoric acid with amino acids. These ordered phosphoric acid arrays make possible fast proton transfer processes involved in the condensation reaction leading to amide bond formation. The reaction proceeds under mild conditions, at 100 °C, i.e., at markedly lower temperatures than those previously reported24 for synthetic routes that utilize phosphoric acid in catalytic amounts only. Since phosphates are fundamental components of nucleic acids, our results indicate that the first oligopeptides and oligonucleotides are of common origin: both could simultaneously emerge from the same phosphate containing prebiotic brine upon drying in an acidic environment at elevated temperatures, i.e., 80–100 °C. This proposal is in line with the conclusions of a recent study on selection of the amino acid components of early oligopeptides also favoring an acidic environment for the emergence of the first biopolymers on our planet49.

Glycine (CAS 50046, Cat. No. 50046), l-arginine (CAS 74793, Cat. No. W381918), l-histidine (CAS 53319, Cat. No. 71001), formic acid (FA, CAS 64186, Cat. No. 33015), trifluoroacetic acid (TFA, CAS 76051, Cat. No. 302031) and H3PO3 99% (CAS 13598-36-2 Cat. No. 215112) were purchased from Merck. Sodium dihydrogen phosphate (NaH2PO4·2H2O, CAS 13472350) and 0.5 M H2SO4 solution (CAS 7664-93-9, Cat. No. 44490) was purchased from Penta Chemicals. Phosphoric acid 85% (CAS 7664382, Cat. No. 10048-A85) and hydrochloric acid 35% (CAS 7647-01-0, Cat. No. 10033-A35) were purchased from Lach-Ner, Neratovice. Acetonitrile was from Riedel de Haën (CAS 75058, Cat. No. 34967). 2,5-dihydroxybenzoic acid (DHB, CAS 490799, Cat. No. 8201345) was purchased from Bruker. All materials (except for DHB and H3PO3) used were of molecular biology grade purity. Nuclease-free water was used throughout the whole study.

Crystallization of amino acid:phosphoric acid salts was performed in plastic Petri dishes from aqueous solutions according to the procedures given in preceding studies32,50,51. We note that the crystalline salt formation was even more facile when using Petri dishes made of glass. Nevertheless, in order to rule out the possibility that silica could catalyze the reported peptide bond formation, as reported by previous investigations10,11,12, we made all experiments in plastic vessels. Crystallinity and composition of the salts obtained, i.e., (GlyH)(H2PO4), (l-ArgH)(H2PO4)·H2O and (l-HisH)(H2PO4)H3PO4, was verified by powder X-ray diffraction (see Fig. S12 in the Supplementary information). For microscopic images of the crystals obtained see Fig. S13 in the Supplementary information. MALDI-TOF mass spectra of the untreated crystalline (GlyH)(H2PO4), (L-ArgH)(H2PO4)·H2O and (L-HisH)(H2PO4)·H3PO4 salts are shown in Fig. S14 in the Supplementary information. Crystallization of the phosphorous and sulfuric acid salts of glycine was performed in an analogous way as that of the phosphoric acid salt.

The same procedure was used for the preparation of glycine:phosphoric acid amorphous product from NaH2PO4 and HCl, with the difference that an equivalent amount of NaH2PO4 and HCl were used to substitute for phosphoric acid (for diffraction pattern see Fig. S4 in the Supplementary information).

Amorphous syrups made by mixing phosphoric acid with arginine and histidine in 1:1 and 1:2 ratio, respectively, were prepared by collecting the amorphous phase present in the crystallization mixture at 100 °C just right at the beginning of the appearance of the first crystals.

The materials were then transferred into plastic Eppendorf tubes and heat-treated at 100 °C for 1–7 days. All reactions were run in pentaplicates. As negative controls, the untreated starting materials were analyzed to rule out the possibility of presence of oligomeric traces resulting from amino acid synthesis or artificial oligomer formation induced by the applied analytical methods themselves (see Fig. S2 in the Supplementary information).

The samples were dissolved in 1% FA to 5 mg·ml−1 concentration and mixed with the MALDI matrix solution (20 mg·ml−1 DHB in 1% TFA:acetonitrile, 1:1 v/v mixture) in a 1:4 ratio (v/v). The mixture in a volume of 0.6 µl was deposited on a ground steel sample plate. After drying at room temperature, the samples were introduced to an Ultraflextreme spectrometer (Bruker, Bremen, Germany) and analyzed in reflectron positive ion detection mode under FlexControl 1.4 software (Bruker). The data were processed by FlexAnalysis 3.4 software (Bruker).

The samples were further diluted in 0.1% FA to 167 µg·ml−1 concentration and 1 µl of this solution was analyzed by a Ultimate 3000 RSLCnano system (Thermo Fisher Scientific) on-line connected to an Impact II Ultra-High Resolution Qq-Time-Of-Flight mass spectrometer (Bruker) operated under Hystar 3.2 (Bruker) and otofControl 1.8 (Bruker) software, respectively. The peptides were separated by using an Acclaim Pepmap100 C18 column (2 µm particles, 75 μm × 500 mm; Thermo Fisher Scientific, Waltham, MA, USA) by the following gradient program (mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in 80% acetonitrile; flow rate 300 nl/min): the gradient elution started at 2% of mobile phase B and increased from 2% to 60% during the first 25 min and then increased to 95% of mobile phase B for the final 5 min. Equilibration of the analytical column was done before sample injection into sample loop. The data were processed by DataAnalysis 4.2 software (Bruker). Extracted ion chromatograms used for quantification of the peptide products shown in Figs. 3, 5 and S9 (Supplementary information) are presented in Figs. S15–S19 in the Supplementary information.

Separation of the oligopeptide fractions was performed using an Agilent 7100 CE system coupled to a Thermo Scientific Q Exactive Plus mass spectrometer using a home-built sheath-flow ESI interface. ChemStation software was used for data acquisition and instrument control. All analyses were conducted at 25 °C with an aqueous acetic acid solution (6%) as BGE in positive polarity mode +15 kV. For quantification experiments the total length of the silica capillaries used were 40 cm, for the MS connection the total length used was 70 cm. The polyimide coating was removed (2 mm) at the MS end of the capillary. For ESI-MS analysis the application of an external voltage (3.6 kV) to the stainless steel emitter and a sheath liquid flow using the BGE (flow rate 1.8 µL/min) ensured a stable electrospray. Prior to the first use capillaries were conditioned with water (2 min), followed by 0.1 M NaOH (20 min), water (2 min) and BGE (2 min). Between runs the capillaries were flushed with water (2 min), 0.1 M NaOH (7 min) followed by water (2 min) and BGE (2 min). In all cases, samples were injected hydrodynamically by applying a pressure of 50 mbar for 4 s followed by injection of BGE with a pressure of 50mbar for 4 s. Conversion for glycine was estimated from quantification of the G2, G3 and G5 oligopeptide fractions.

PXRD measurements were performed on Malvern Panalytical Aeris diffractometer using CuKα radiation (λ = 1.5418 Å) and hybrid PIXcel detector. Data were collected at room temperature in the 2θ range 5-50°. Microsample PXRD (μ-PXRD) measurements were performed at 100 K on Rigaku XtaLAB Synergy-DW diffractometer using CuKa radiation, HyPix-Arc 150° detector and Oxford Cryosystems Cryostream 800 Plus temperature attachment. Data were collected in the 2θ range 4–52°. The experimental diffraction patterns were compared with the Rietveld-fitted theoretical diffractograms simulated from the single-crystal X-ray data with the use of HighScore Plus52 program. For the analyses, CIF files deposited at the Cambridge Crystallographic Data Center with the following refcodes were used: GLYCPH0132 for (GlyH)(H2PO4), LARGPH0453 for (l-ArgH)(H2PO4)·H2O, HISTPA1654 for (l-HisH)(H2PO4)·H3PO4, DGLYHC0135 for (GlyH)Cl·Gly, ICSD 2894855 for NaCl and QQQFGD0256 for (GlyH)2(SO4). Data for (GlyH)(H2PO3) and [(GlyH)2(SO4)]·Gly were taken from low-temperature (100 K) single-crystal CIF files obtained for the crystals isolated from the samples. The models (ferroelectric phases, in both cases monoclinic P21 space groups) corresponded to the deposited structures (LABSEM05 and TGLYSU19, respectively).

All data supporting the findings of this study are available within this article and the Supplementary Information. HPLC/MS data are available from the authors upon request.

The HighScore Plus program used for X-ray diffraction analysis is available from https://www.malvernpanalytical.com/en/products/category/software/x-ray-diffraction-software/highscore-with-plus-option.

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Financial support from the Czech Science Foundation (GAČR, grant. no. 22-25057S) and from the Ministry of Education, Youth and Sports, Czech Republic (grant No. CZ.02.01.01/00/22_008/0004587) is greatly acknowledged. CIISB, Instruct-CZ Center of Instruct-ERIC EU consortium, funded by MEYS CR infrastructure project LM2023042 and European Regional Development Fund-Project “UP CIISB” (No. CZ.02.1.01/0.0/0.0/18_046/0015974), is gratefully acknowledged for the financial support of the measurements at the CEITEC Proteomics Core Facility. OT thanks the Max‐Planck‐Society (Max‐Planck‐Fellow Research Group Origins of Life) for financial support.

These authors contributed equally: Judit E. Šponer, Rémi Coulon.

Institute of Biophysics of the Czech Academy of Sciences, Královopolská 135, Brno, Czech Republic

Judit E. Šponer, Rémi Coulon & Jiří Šponer

CATRIN—Regional Centre of Advanced Technologies and Materials, Šlechtitelů 27, Olomouc, Czech Republic

Judit E. Šponer, Rémi Coulon, Michal Otyepka & Jiří Šponer

Department of Physical Chemistry, Faculty of Science, Palacký University, 17. listopadu 12, Olomouc, Czech Republic

Rémi Coulon

IT4Innovations, VSB—Technical University of Ostrava, 17. listopadu 2172/15, 708 00, Ostrava, Poruba, Czech Republic

Michal Otyepka

Department Chemie, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, München, Germany

Alexander F. Siegle & Oliver Trapp

University of Wrocław, Faculty of Chemistry, 14 F. Joliot-Curie, Wrocław, Poland

Katarzyna Ślepokura

Central European Institute of Technology, Masaryk University, Campus Bohunice, Kamenice 5, Brno, Czech Republic

Zbyněk Zdráhal & Ondrej Šedo

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Project design, and supervision: J.E.S and O.S.; experimental design and data acquisition: J.E.S., O.S., Z.Z., A.S., O.T., K.S. and R.C.; data analysis: J.E.S., O.S., K.S., and R.C.; funding acquisition: J.E.S., Z.Z., O.T., and M.O.; analysis and writing of the manuscript: J.E.S., J.S., O.S., Z.Z., A.S., O.T., K.S., R.C., and M.O.

Correspondence to Judit E. Šponer or Ondrej Šedo.

The authors declare no competing interests.

Communications Chemistry thanks Matthew Pasek and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Šponer, J.E., Coulon, R., Otyepka, M. et al. Phosphoric acid salts of amino acids as a source of oligopeptides on the early Earth. Commun Chem 7, 185 (2024). https://doi.org/10.1038/s42004-024-01264-6

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Received: 22 January 2024

Accepted: 30 July 2024

Published: 22 August 2024

DOI: https://doi.org/10.1038/s42004-024-01264-6

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