Abstract
Under the RNA first hypothesis for the origin of life, RNA that emerges from prebiotic chemistry performed both catalytic and informational roles. Ribose is the only sugar in RNA; thus, many have sought to understand how ribose might have emerged on a prebiotic Earth. Ribose can be formed from formaldehyde with small amounts of glycolaldehyde by formose-like processes. However, under the strongly alkaline conditions of the reaction, ribose is consumed as it is formed. Here, we show that borate significantly decreases the consumption of the ribose formed in the formose reaction, which results in higher amounts of ribose that remained as the reaction progressed. Given a longer timescale of prebiotic chemical reactions governed by geological processes, borate-rich environments could have contributed to accumulating ribose on prebiotic Earth. Borate could be available on proto-continents and is known to contribute to ribonucleoside synthesis, ribose 5-phosphate synthesis, and nucleoside phosphorylation. Therefore, such environments might have promoted chemical reactions to RNA.
Keywords
Introduction
In modern Terran biology, RNAs hold copies of gene information as sequences of nucleobases for gene expression and can catalyze many biological reactions, including splicing and translation. Thus, RNA is regarded as a central molecule that organizes the primordial life system before transitioning to the system based on RNA–protein and modern DNA–RNA systems (Gilbert, 1986; Benner et al., 1989; Joyce, 1989; Orgel, 2004). Ribose is a central molecule in RNA that connects nucleobases to the sugar-phosphate backbone.
Ribose and other sugars can form from formaldehyde by the formose reaction in which formaldehyde reacts in the presence of calcium hydroxide to form diverse sugars (Breslow, 1959; Shapiro, 1988; Schwartz and de Graaf, 1993). Higher pH, temperature, and glycolaldehyde concentration substantially promote the reaction to form ribose and other aldopentoses (Shigemasa et al., 1977; Ono et al., 2024). However, ribose is a labile and reactive sugar compared with many other sugars (Larralde et al., 1995; Dworkin and Miller, 2000). Therefore, the yield of ribose in the formose reaction is relatively low (Ono et al., 2024). This has led some to conclude that ribose was not a building block of primordial life (Larralde et al., 1995).
However, borate (i.e., dissolved boroxide ion) can stabilize ribose by forming ribose–borate complexes in solutions (Chapelle and Verchere, 1988; Prieur, 2001; Li et al., 2005; Scorei and Cimpoiaşu, 2006; Šponer et al., 2008; Furukawa et al., 2013). An increase in the formation of ribose with borate in a part of the formose reaction between glycolaldehyde (i.e., C2 aldehyde) and glyceraldehyde (i.e., C3 aldehyde) is seen (Ricardo et al., 2004). However, in the formose reaction from formaldehyde (i.e., C1 aldehyde) with a small amount of glycolaldehyde, which are precursors that were likely available on prebiotic Earth, borate-moderated carbohydrate evolution forms branched pentoses as the major pentose product (Kim et al., 2011).
The formose reaction forms many sugars simultaneously, and the abundance of these sugars changes as the reaction progresses. Quantitative analysis of the evolution of the sugar product mixture is required to understand the complex reaction but has been difficult to perform. Recently, complex mixtures of formose sugars were investigated using gas chromatography–mass spectrometry with aldonitrile acetate derivatization and two-dimensional gas chromatography–mass spectrometry with conventional derivatization (Furukawa et al., 2019; Abe et al., 2024; Ono et al., 2024; Vinogradoff et al., 2024). These investigations showed the effects of chemical conditions on the yields of specific sugars (Abe et al., 2024; Ono et al., 2024; Vinogradoff et al., 2024). In the present study, we investigated the effects of borate on the formose reaction by using gas chromatography–mass spectrometry with aldonitrile acetate derivatization.
Materials and Methods
Experimental procedures
Formaldehyde (C1), glycolaldehyde (C2), calcium hydroxide, and boric acid were added to 15 mL of 45°C hot water with continuous stirring in a polytetrafluoroethylene (PTFE) bottle to be 100 mM formaldehyde and 10 mM glycolaldehyde. The pH of the starting solution was adjusted with Ca(OH)2 at 12. The pH was adjusted to ca. 12 (>11) by adding Ca(OH)2 by 3–12 h during the reaction. An aliquot of the solution (1 mL) was collected from the bottle at the scheduled time (after 5, 60, 120, 180, 240, 360, 420, 480, and 540 min and 24, 48, and 72 h), and the collected solution was dried under vacuum after it was acidified to pH 2 with HCl solution.
Product analyses
The dried products were derivatized, as described elsewhere (Furukawa et al., 2019). The derivatized sugars were dissolved in 100 µL of organic solvent (hexane and ethyl acetate) and analyzed with a gas chromatograph–mass spectrometer (GC-MS) (GCMS-QP2010; Shimadzu) with a capillary column (DB-17MS, 60 m, 0.250 mm inner diameter, 0.25 µm film thick; Agilent). The injection was conducted with a 1:10 split mode at 250°C with a sample volume of 1 µL. The total flow and column flow of the He carrier were 11.8 and 0.8 mL/min, respectively. The elution was conducted from 50°C for 2 min, ramped up to 120°C at 15°C/min, held for 5 min, ramped up to 160°C at 5°C/min, 195°C at 3°C/min, held for 15 min, ramped up to 240°C at 3°C/min, and held for 10 min. The source and interface temperatures were 200°C and 250°C, respectively. For the identification and quantification, we analyzed all 20 kinds of reference sugars and 5 kinds of sugar-related compounds to identify their characteristic mass fragments and to construct their calibration lines (Supplementary Table S2 and Supplementary Fig. S4). The calibration lines were constructed for ribose and lyxose with m/z 217, arabinose and xylose with m/z 242, branched aldopentoses with m/z 145, linear ketopentoses with m/z 273, aldohexoses with m/z 145, and ketohexoses with m/z 345. Quantification was conducted with the calibration lines as external standards. At each time of the product analysis, we measured an external standard of ribose to correct the inclination of the calibration lines based on the sensitivity at that time. We have investigated how additives such as calcium and borate affect the efficiency of derivatization and verified that these amounts of additives do not affect the derivatization efficiency with this protocol. The datapoints in Figs. 3, 4, and 5a and Supplementary Fig. S1 (linear aldopentoses and branched aldopentoses, and linear aldohexoses) show the average of the double or triplicate experiments. Error bars show standard deviation of the triplicated experiments. The datapoints for linear ketopentoses are from a single experiment. The datapoints in the experiments with NaOH and the neutral solvent were also from a single experiment.
Gas chromatograph–mass spectrometer (GC-MS) chromatograms of product sugars, sugar acids, and sugar alcohols in borate-free experiments after 24 h. The lines show mass chromatograms of specific m/z indicated in the legend.
Structures of product sugars.
Amounts of product ribose in different borate concentration experiments. Error bars show standard deviation of the triplicated experiments.
Amounts of linear aldopentoses in the borate-free experiment
Amounts of total product branched aldopentoses
Formaldehyde solution (36.0–38.0%; Wako), glycolaldehyde dimer (Sigma-Aldrich), calcium hydroxide (>96%; Wako), and boric acid (>99.5%; Wako) were used for the starting materials of the experiments. All the water was prepared with Milli-Q Integral (>18.2 MΩ.cm, TOC < 5 ppb). For identification and quantification standard,
Results
Product sugars
Incubation of alkaline solutions (pH ∼12) containing 100 mM formaldehyde (C1), 10 mM glycolaldehyde (C2), and Ca2+ (as the hydroxide) with/without borate (0, 10, 40, and 80 mM) at 45°C formed various sugars, including linear aldopentoses (ribose, xylose, arabinose, and lyxose), linear ketopentoses (ribulose and xylulose), branched aldopentoses ((2R,3S)−2,3,4-trihydroxy-2-(hydroxymethyl)butanal and (2S,3S)−2,3,4-trihydroxy-2-(hydroxymethyl)butanal), linear hexoses (allose, mannose/altrose, glucose/gulose, galactose, and fructose), and aldotetroses (threose and erythrose) (Figs. 1 and 2; Supplementary Table S1). We also sought to find other sugars in the product mixtures, including idose, talose, tagatose, sorbose, psicose, and 2-deoxyribose. However, these were below our detection limits. Sugar alcohols of ribitol, arabitol, and xylitol and sugar acids of xylonic acid and ribonic acid were also identified (Fig. 1).
Effects of borate on pentoses
The yields of product ribose typically reached a maximum earlier than 30 min and then decreased gradually. The maximum yield was highest at 0.14 mM in the borate-free experiment, but lower with higher borate concentrations (i.e., 0.042 mM in 10 mM borate and 0.025 in 80 mM borate) (Fig. 3). The decrease in the yields of ribose after the maximum as the reaction proceeded depended significantly on borate concentrations. The rate of decrease was highest in the borate-free experiment and lowest in the borate-rich experiment (80 mM), resulting in approximately six times more ribose in the borate-rich experiment than in the borate-free experiment at 72 h (Fig. 3). Other linear aldopentoses (i.e., arabinose, xylose, and lyxose) were also influenced by borate concentrations, but the effect was smaller than with ribose, resulting in lower amounts remaining compared with ribose after 24 h of experiments (Fig. 4).
Borate also influenced the amounts of other pentoses formed, in particular threo- and erythro-branched aldopentoses. Their yields reached a maximum at 0.04 mM within 1 h and decreased very rapidly, dropping below the detection level (<0.0001 mM) at 24 h in the borate-free experiment (Fig. 5A). In experiments with borate (10–80 mM), their maximum yields were substantially higher, 0.5–0.8 mM. The yields decreased far more gradually than in the borate-free experiment, to 0.015 and 0.081 mM in, respectively, the 10- and 80-mM borate experiments after 72 h. Similar stabilization effects were also seen with ketopentoses, but the effects by borate were smaller than those with threo- and erythro-branched aldopentoses (Fig. 5B).
In borate-free experiments, branched aldopentoses and linear ketopentoses formed earlier than linear aldopentoses, including ribose. Then, they decreased by more than three orders of magnitude in 10–24 h (Fig. 6A). Conversely, in borate-containing experiments, the amounts of branched aldopentoses and linear ketopentoses decreased by less than only one order of magnitude in 72 h (Fig. 6B).
Amounts of total linear aldopentose, total linear ketopentose, and total branched aldopentose in the borate-free
To understand the mechanisms that created the differences in the yields of pentoses under different conditions, we also investigated the evolution in the amounts of tetroses formed. Erythrose formed very quickly, reaching its maximum of 0.26–1 mM at 5 min to 1 h in experiments at all borate concentrations (Fig. 7A). The yields decreased substantially at all borate concentrations, but the decrease was smaller in borate-containing experiments. The yields of threose also reached their maximum (0.2–0.5 mM) very early (5 min to 1 h) (Fig. 7B). The decrease after the maximum was significantly affected by borate. In the borate-free experiment, the yield was decreased to 0.00024 mM at 24 h. However, with borate, the yield decreased to 0.004 and 0.25 mM in 10 and 80 mM borate experiments, respectively. Linear aldohexoses were formed only in the experiment free from borate (Fig. 1). They did not form at detectable amounts in borate-containing experiments.
Amounts of product tetroses.
The C5 sugar alcohols of ribitol, arabitol, and xylitol were seen after 1 h in the experiment with 80 mM borate; the amounts slightly increased afterward. In the borate-free experiment, formation of C5 sugar alcohols was delayed (Supplementary Fig. S1). The C5 aldonic acids (i.e., ribonic acid and xylonic acid) formed later than the C5 sugar alcohol formation (after 24 h) in the borate-free experiments (Supplementary Fig. S2). Their formation was not confirmed in the borate-rich experiment.
Several pathways yield linear aldopentoses, including ribose, by aldol additions that start with formaldehyde (C1) and glycolaldehyde (C2). A simple path is the reaction between glycolaldehyde (C2) and glyceraldehyde (C3), yielding linear aldopentoses and linear ketopentoses (Kim et al., 2011; Robinson et al., 2022; Tabata et al., 2023; Ono et al., 2024) (Fig. 8). Another pathway to yield linear aldopentoses is the reaction between tetroses (C4) and formaldehyde (C1) via linear ketopentoses. The interconversion between linear ketopentoses and linear aldopentoses happens by an isomerization known as the Lobry de Bruyn–van Ekenstein transformation (Speck, 1958) (Fig. 8). Borate is known to promote this isomerization (Furukawa et al., 2013).
Major reaction path of the formose reaction to form pentoses. The red and blue arrows represent aldol condensation and retro-aldol reaction, respectively. The dotted lines represent the reactions that are strongly inhibited by borate.
The reaction between tetroses (C4) and formaldehyde (C1) also yields branched aldopentoses. The branched aldopentoses were then fragmented in a retro-aldol reaction to give glycolaldehyde (C2) and glyceraldehyde (C3) (Fig. 8). This reaction seems to be significant, as indicated by the quick decay of branched aldopentoses followed by the increase of linear aldopentoses (Figs. 5 and 6). Borate substantially increased the formation of the branched aldopentoses by stabilizing these sugars (Figs. 5 and 6). This inhibits the retro-aldol reaction into glycolaldehyde (C2) and glyceraldehyde (C3), which results in limiting the formation of linear aldopentoses from glycolaldehyde (C2) and glyceraldehyde (C3) (dotted blue arrows in Fig. 8).
Furthermore, higher amounts of tetroses formed in borate-rich experiments indicate that tetroses, in particular threose, were stabilized by borate (Fig. 8). This further suggests that the reaction that forms linear ketopentoses between tetroses (C4) and formaldehyde (C1) was substantially suppressed by borate (dotted long red arrow in Fig. 8). Thus, these suppressions would have led to a lower maximum yield of linear aldopentoses, including ribose, in borate-rich experiments.
The drop in the yields of sugars after their maxima indicates the stability of these sugars under these conditions. Although the maximum yields of linear aldopentoses became lower in experiments with higher borate concentrations, borate lowered their rates of decay substantially, which resulted in their having higher residual amounts in experiments with higher borate concentrations after the reaction had progressed (Figs. 3 and 4).
Many sugars, ribose in particular, are known to bind borate (Chapelle and Verchere, 1988; Scorei and Cimpoiaşu, 2006; Šponer et al., 2008; Furukawa et al., 2013; Franco et al., 2019). When the sugar ring is closed in their furanose and/or pyranose forms, the lack of an aldehyde group increases the stability of the sugar, as a free aldehyde is required for base-catalyzed enolization. The absence of linear aldohexoses in borate-rich experiments is consistent with borate stabilizing ring-closed forms. These two effects of borate (i.e., reduction of the maximum yield and improvement of the stability) explain the apparently opposite effects of borate reported in previous studies (Ricardo et al., 2004; Kim et al., 2011; Furukawa et al., 2013).
Alkaline pH is known to promote Cannizzaro and cross-Cannizzaro reactions that form sugar alcohols and sugar acids from corresponding sugars (Swain et al., 1979). Different yields of sugar alcohols and sugar acids formed as the reaction progresses in the present study indicate their formation not by a Cannizzaro reaction but by a cross-Cannizzaro reaction (Supplementary Fig. S1), where formaldehyde is the reducing agent. Borate promotes the synthesis of C5 sugar alcohols (i.e., ribitol, arabitol, and xylitol) but conversely limits the synthesis of C5 aldonic acids (i.e., ribonic acid and xylonic acid) (Supplementary Fig. S1). Borate binds aldopentoses to fix their configuration and reduce their reactivities; the formation of lower amounts of aldonic acids in borate-rich experiments is consistent with this effect. However, the reason for its effects on C5 sugar alcohols is unclear.
Threose is another sugar that has been investigated for prebiotic interest because this sugar works as a component of threose nucleic acid that can form complementary base pairs with DNAs and RNAs (Schöning et al., 2000). Borate substantially increased the yields of both threose and erythrose (Fig. 7), but more for threose. This is consistent with a previous investigation that formed these sugars from glycolaldehyde (Kim et al., 2011). The binding of borate to the 1′- and 2′-diol of threose would stabilize this molecule, fixing its configuration into furanose.
Calcium ion is known to promote the formose reaction and potentially influence its detailed outcome. We investigated the effects of borate in an alkaline sodium-rich solution for comparison. The yields of ribose and other sugars were lower in the alkaline Na+ solutions, but an effect of borate in stabilizing ribose was seen (Supplementary Fig. S2).
Boron is one of the incompatible elements in rock-forming minerals and is thus concentrated in felsic rocks such as in continental crust rather than in mantle and oceanic crust (Marschall, 2018). The development of vast areas of continental crust (as on modern Earth) was not expected on Hadean Earth before the representative oldest traces of life (at ∼3.9 Ga) (Ohtomo et al., 2014; Tashiro et al., 2017). However, the oxygen isotope compositions of 4.4 Ga zircon found in Australia indicate the formation of at least some felsic magma on the early Hadean Earth (Wilde et al., 2001). The formation of small proto-arc that contains felsic rocks has been proposed for early Archean Earth (Nutman et al., 2015). Furthermore, boron-containing minerals have been found in 3.8 billion-year-old metasediments in Isua, Greenland (Appel, 1995; Grew et al., 2015; Mishima et al., 2016). The δ11B value of the B-containing mineral, tourmaline, indicates that this mineral was formed from sediments in a closed basin with a hydrothermal boron charge, which suggests the formation of a boron-rich proto-arc basin (Grew et al., 2015). Formaldehyde and glycolaldehyde were continuously formed from CO2 and H2O by photochemical reactions globally on prebiotic Earth (Pinto et al., 1980; Cleaves, 2008; Harman et al., 2013). Because these molecules are highly soluble in water, they were provided to the oceans continuously, either directly or as bisulfite addition products with SO2. When the aldehyde-containing seawater was captured in a boron-rich basin, the reactions investigated in this experiment could be possible, although the pH of the environment may have been less alkaline than in the present study. This would have slowed the reactions (Furukawa and Kakegawa, 2017).
Ribose could also be formed in a neutral solution in comparable concentrations to the borate-rich alkaline solution in this study (Ono et al., 2024). However, its decay rate is higher than in the borate-rich alkaline solution (Ono et al., 2024). Furthermore, ribose was the least abundant aldopentose in the neutral experiment without borate. In boron-rich neutral solution, boric acid does not provide significant effects on sugar synthesis (Supplementary Fig. S3). Although borate-rich alkaline environments were less common on prebiotic Earth than neutral open oceans, these comparisons suggest that borate-rich alkaline environments were more suitable for ribose accumulation on prebiotic Earth.
In forming RNA abiotically on prebiotic Earth with ribose, ribose must react with other molecules to form ribonucleosides, or ribose needs to react with phosphate to form ribose 5-phosphate. The accumulation of ribose by borate contributes to these reactions. The selective accumulation of ribose over other linear aldopentoses found in this study further underlines the importance of the borate-rich environments. Previous studies have found that borate contributes to the reaction between ribose and small reactive molecules to yield ribonucleosides (Becker et al., 2016, 2019). Another previous study showed that borate contributes to the reaction between ribose and phosphate to yield ribose 5-phosphate (Hirakawa et al., 2022; Takabayashi et al., 2023). Borate is also key to making ribonucleoside triphosphates (Kim et al., 2016). Thus, the accumulation of ribose in borate-rich environments is compatible with the following prebiotic chemical evolution to RNA. Boron-rich evaporative environments might also have provided primordial proteins because boric acid significantly promotes peptide synthesis from amino acids (Sumie et al., 2023). They imply that boron-rich environments around proto-continents offered a suitable platform for the formation and interaction of prebiotic functional polymers that could record information and catalyze reactions.
Authors’ Contributions
Conceptualization: Y.F.; methodology: Y.F. and H.J.K.; experiments: Y.T.; data analysis: Y.T. and Y.F.; writing—original draft preparation: Y.F.; writing—review and editing: Y.T., H.-J.K., S.A.B., and T.K.; visualization: Y.T. and Y.F.; and funding acquisition: Y.F. All authors have read and agreed to the published version of the article.
Footnotes
Author Disclosure Statement
The authors declare no competing interests.
Funding Information
This work was supported by JSPS KAKENHI to Y.F. (22H00165 and 22H00164) and by NSF EAR-2213438.
Data Availability
Data are provided within the article. The datasets used or analyzed during this study are available from the corresponding author upon reasonable request.
Supplemental Material
Associate Editor: David A. Baum
Abbreviations
References
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