A hypothesis on the possible contribution of free hypoxanthine and adenine bases in prebiotic amino acid synthesis

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Experimental conditions were devised, imitating prebiotic conditions in hydrothermal vents, to examine the possible prebiotic role of free purine bases in the direct synthesis of amino acids. Hypoxanthine, the biochemical precursor of adenine and guanine, was able to capture nitrite ions and be reductively transformed into adenine. Transfer of the exocyclic group of adenine into uracil for cytosine formation was also possible, but the reverse transamination was not observed. In combination with previous work proving the feasibility of reductive amination of pyruvate into alanine by cytosine, it is concluded that the pyrimidine hypoxanthine could have evolutionarily appeared as an efficient capturer of inorganic nitrogen species, passing them to uracil, for further amination of α-keto acids into amino acids.


Introduction
It has been proposed that life originated in a hot, acidic, reducing, anaerobic environment, rich in hydrogen gas and nitrogen, sulfur and iron compounds, such as is encountered in hydrothermal vents (1-7). Two different lines of thought are known concerning the prebiotic evolutionary course. According to one of them, amino acids were formed first and were subsequently randomly polymerized to polypeptides (6,8,9). The other proposal is that RNA was formed first by random polymerization of ribonucleotides (10) and the first RNAs are believed to have catalyzed random polypeptide synthesis driven by the need for stability of this primitive ribosome. In a recent third alternative view (11), the amino acids may have started at some evolutionary point to be synthesized in a way dependent on the bases, which could be the origin of the contemporary interdependence of polypeptide-nucleic acid synthesis. This eliminates a significant degree of randomness, since it creates spatial proximity of amino acids to bases, facilitating a subsequent polymerization into polypeptides and RNA, respectively. Since it is believed that RNA is the earliest form of nucleic acid and DNA appeared later, as a more stable depository format of the genetic material (12,13), the RNA bases were examined for any probable catalytic role in amino acid synthesis. Thymine is actually 5΄-methylated uracil and cytosine is uracil aminated at carbon 4.

In a previous work (11), it has been shown that cytosine can reductively aminate the α-keto acid pyruvate into the amino acid alanine. Accordingly, uracil and cytosine could be considered as the two alternative states of the pyrimidine ring, with cytosine donating its 4-amino group for reductive amination of an α-keto acid to the corresponding amino acid, being converted into uracil in the process. Two questions arose from this. The first is what could be the source of amino groups regenerating cytosine from uracil? The second question arising was what could then be the possible role of the purine bases, if the pyrimidines were enough for base-dependent amino acid synthesis to originate? A similar role for hypoxanthine and its 6-aminated state, adenine, could not be proven and a different role for the purine pair had to be sought. An answer to these two questions is attempted in this article.

Results
Reductive amination of pyruvate into alanine by ammonium or by free cytosine
A reaction scheme for the reductive amination of pyruvate into alanine by ammonium or by cytosine in a non-aqueous environment is shown in Figure 1. The feasibility of the reaction with ammonium is shown in Figure 2. Higher temperatures and reducing agents, either hydrogen generated in situ from HCl and zinc or zinc alone favored the reaction. There was no need for the presence of uracil. A hydrophobic medium, xylene in this case, was required. The results show that reductive amination of α-keto acids into amino acids by ammonia directly, without an intervening stage of cytosine formation, might have been possible in prebiotic hydrophobic micro-environments. About 0.1±0.03% of the original pyruvate was transformed into alanine, as calculated by comparison of the integrated mass spectrometry of the alanine peaks to those of the standard alanine.

Microsoft Word - test_figs.docx

Figure 1 | Reactions employed for the synthesis of alanine by reductive amination of pyruvate by ammonium or cytosine. Anhydrous conditions were achieved by carrying out the reactions in xylene. Reduction of the imine intermediate was done using hydrogen generated in situ from zinc powder and 2M HCl. Liberation of the formed alanine from its conjugate to cytosine was effected by sulfonizing the base moiety using 2M sodium bisulfate and leaving the sample at low temperatures for detaching the alanine. For the sake of comparison the sulfonization step was also done on reactions involving ammonium.

 

 

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Figure 2 | Results of reactions for synthesis of the amino acid alanine (ala) from pyruvate (pyr) using ammonium ions. Frames A1 to A4 are ninhydrin-stained paper chromatograms of the reaction products. Reactions carried out at room temperature (RT) were compared to those at 60ºC. Ala: alanine standard. A1. Reactions involving no reduction by Zn + HCl and no NaHSO3 treatment step produced no amino acid product. A2. Reactions involving a NaHSO3 treatment but no Zn + HCl did produce alanine at higher temperatures, since NaHSO3 is a weak reducer, but only traces of alanine at room temperature. A3. Reactions using only HCl and then NaHSO3 treatment. Some alanine was produced at higher temperatures and only traces at RT. The NaHSO3 concentration in the reaction is expected to be lower since this compound reacts with HCl, producing SO2. A4. Optimum conditions for alanine synthesis were employed. Hydrogen was produced in situ from zinc and HCl. Zinc alone also acted as reducer. The simultaneous presence of hypoxanthine (hyp) did not significantly alter the yield.B. LC-MS chromatogram of the reaction products when 60ºC and H2 were used. Upper trace shows the total positive ion current. The lower peak trace shows the presence of alanine, detected as a positive M+1 ion with proton, at m/z=90.

 

The ability of the cytosine exocyclic amino group to be transferred to pyruvate for alanine formation had been shown before (11), but the results of similar experiments with the other bases are also shown in Figure 3. A reaction scheme is provided in Figure 1. Transamination by the adenine or guanine exocyclic amino groups was not possible. Uracil produced no results either, indicating that any nitrogen-containing groups originating from purine degradation do not participate in reductive amination of the keto acid. The yield of transformation of pyruvate into alanine was about 0.3±0.1%.

 

393-Fig3

Figure 3 | In vitro synthesis of alanine via reductive amination of pyruvate by nucleic acid bases. A. Paper chromatogram of the reaction products stained with ninhydrin for amino acid detection. Only cytosine (C) produced alanine. Transamination by adenine (A) was not successful. B. Scan of the LC-MS chromatogram of the cytosine- and uracil-containing reactions for alanine M+H+ (m/z = 90) ion peaks. Alanine was detected only in the cytosine-containing reaction.

 

Migration of the adenine exocyclic amino group to uracil
The reaction scheme used in this and similar transamination experiments is shown in Figure 4 and the results of reactions in which purine-pyrimidine pairs were incubated in xylene are shown in Figure 5. When the reaction contained adenine and a 10-fold molar excess of uracil, the red-brown color characteristic of the presence of adenine did not develop upon addition of NaOCl. Instead a very weak yellow color, characteristic of the presence of cytosine, showed up (Figure 5A), indicating the deamination of most of the adenine into hypoxanthine and the amination of some uracil into cytosine. The products were confirmed by Liquid Chromatography-Mass Spectrometry (LC-MS) analysis (Figure 5B). The cytosine negative ion (m/z = 110) was detected at elution time 4.9 min. Hypoxanthine showed as its positive ion with H+ (m/z = 137) at 4.8 min. The yield of transformation of uracil into cytosine was about 0.7±0.2%, whereas the deamination of adenine into hypoxanthine was almost quantitative. These significant losses of ammonia during transamination could be attributed to the far from perfect reducing conditions in the reaction mixtures and to the expected loss of liberated volatile ammonia at 60ºC, during the imine intermediate step. Nevertheless, the experiment sufficiently proves the feasibility of amino group transfer from adenine to uracil. The reverse reaction, containing cytosine and a 10-fold molar excess of hypoxanthine, did not show significant transfer of the amino group from cytosine to hypoxanthine.

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Figure 4 | Reaction conditions for the transamination of uracil by adenine. The bases were mixed together in xylene and were incubated under mild heating overnight.

 

 

393-Fig5

Figure 5 | Exocyclic amino group transfer between the free purines and pyrimidines.
A. Adenine (A), hypoxanthine (H), cytosine (C) and uracil (U), alone or in combination, were heated overnight at 60ºC in xylene, in the presence of a small amount of zinc powder. The presence of adenine is shown by the developing reddish color using the NaOCl test. In the presence of 10-fold molar excess of uracil the adenine color is so greatly diminished as to be hardly distinguishable from that of hypoxanthine, indicating de-amination of the purine. This transamination was also possible at 45ºC. In the presence of 10-fold molar excess of hypoxanthine the cytosine color hardly changes, the reaction being conducted either in xylene or in water. This indicates little, if any, formation of adenine. B. LC-MS analysis of the adenine-uracil reaction products. The cytosine negative ion was detected as a m/z = 110 peak at elution time 4.9 min. Hypoxanthine was detected as a positive M+H ion of m/z = 137 at elution time 4.8 min.

 

Synthesis of adenine from hypoxanthine and nitrite ions
The possibility was examined that nitrogen species less reduced than ammonia, such as nitrite ions (NO2-), might be utilized as a source of amino groups for amino acid synthesis. The reaction conditions are presented in Figure 6 and the results are shown in Figure 7. Nitrite can be reduced to ammonia by zinc, as indicated by the brownish color development on addition of the Nessler reagent. Free ammonia was not detected when hypoxanthine was also present in the reaction. However, ammonia not produced from nitrite, but added to the reaction together with hypoxanthine, cannot be captured by hypoxanthine to produce adenine. Hypoxanthine was able to become adenine only when it was simultaneously present in the aqueous reaction with sodium nitrite and in the presence of a reducing agent, such as zinc powder. An explanation for the observed results could be that any ammonia synthesized by reduction of the nitrite ion was immediately captured by hypoxanthine. However, color developed even in the absence of Zn. It seems probable that the nitrite ion reacts directly with the hypoxanthine to give a nitroso product that is subsequently reduced into adenine. In LC-MS analysis of the hypoxanthine-NaNO2 reaction products, a negative ion of m/z = 134 was detected at elution time 4.7 min. This fits the adenine negative ion. Its amount is 5±0.8% of the original hypoxanthine. A second peak of the same m/z appears when the reaction is done in xylene. Its identification has not been pursued further, but it fits the hypoxanthine imine with ammonia, which is equivalent in molecular weight to the adenine tautomers (14). Similar reactions between uracil and nitrite reveal only traces of cytosine (Figure 8), at least an order of magnitude less than the amounts of adenine synthesized from hypoxanthine + NO2-. Therefore, the amination of hypoxanthine using nitrite is a far more efficient process than the amination of uracil.

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Figure 6 | The reaction of hypoxanthine with nitrite ions under aqueous conditions. Zinc powder was used as reducer but adenine could be synthesized even in its absence (Figure 7).

 

 

393-Fig7

Figure 7 | Capture of nitrite ions by hypoxanthine. A. Overnight reactions of hypoxanthine with sodium nitrite or ammonia (as NH4Cl) at 60ºC in water. Ammonia was detected using the chromogenic Nessler reagent, only in the nitrite reaction, in the presence of Zn. The adenine color did develop in the NaOCl test, when the reaction of hypoxanthine with NaNO2 was done in aqueous environment, irrespective of the presence of Zn. Sodium nitrite alone did not produce a color with NaOCl. No adenine was detected in reactions of hypoxanthine with ammonia. B. Negative ion peaks adenine of m/z = 134 throughout the LC-MS chromatogram of the hypoxanthine-NaNO2 reaction. When the reaction is done in aqueous medium, a peak appears at retention time 4.7 min, fitting the adenine ion. A second peak of the same m/z appears at 4.8 min when the reaction is done in xylene.

 

 

393-Fig8Figure 8 | LC-MS spectrum of positive ion peaks of m/z=112 among the products of a reaction of uracil with sodium nitrite in the presence of Zn powder. Traces of peaks at retention time 4.3-4.7 min of the LC chromatogram fit the cytosine M+1 (111+H+) positive ion. Yet, the intensity of the peak is an order of magnitude lower than that of adenine obtained from hypoxanthine + nitrite (Figure 7B) under identical reaction and product analysis conditions.

 

 

Discussion
The source of α-keto acids on prebiotic earth has been investigated to a significant extent. Pyruvate is present in hydrothermal vents, synthesized at high temperatures from alkyl thiols and carbon monoxide, under the catalytic action of iron-sulfur centers or other transition metal sulfides (15). But pyruvate and other α-keto acids could also be derived from α-hydroxy acids upon oxidation by sulfur/iron sulfite mixtures (16,17). These prebiotic α-hydroxy acids can form by aldehyde rearrangements, as is the case of lactic acid synthesis from glyceraldehyde, catalyzed by the mineral iron(III) hydroxide oxide, Fe(OH)O (18). Glyceraldehyde is considered an ingredient of the prebiotic atmosphere (19,20). Also, the α-keto acids pyruvic acid, oxaloacetic acid and α-ketoglutaric acid have been identified in carbonaceous meteorites and in laboratory reactions pyruvic acid can produce oxaloacetic acid (21). Experiments show that pyruvate can form by a condensation of a ketene with HCN to give first pyruvonitrile(CH3COCN), a pyruvate precursor, whereas other nitriles lead to various other keto acids (22).

Nucleic acids are easily depurinated (23) and their N-glycosidic bonds are easily cleaved (24) under hot acidic conditions. It is therefore probable that in prebiotic hydrothermal vents or similar environments, free bases were encountered initially perhaps alone and later together with nucleic acids. Free nucleic acid bases can be synthesized from aqueous ammonium cyanide or from a mixture of methane, ammonia and water (7), but also from formamide (25,26). The presence of H2, ammonia and formamide, as well as the low pH and high temperature in hydrothermal vents, are well documented (1-3). The keto acid amination conditions used in this work are similar to the conditions encountered in hydrothermal vents. In water, the bisulfite ion is in chemical equilibrium with SO2. Bisulfite and SO2 are present in magma and volcano emissions (27,28), but have not been reported for submarine hydrothermal vents (2). This is expected because SO2 is rapidly converted into bisulfite ions upon contact with water and the bisulfite itself readily reacts with dissolved oxygen to give sulfate ions. Its existence in hydrothermal microenvironments under oxygen-free prebiotic conditions is a reasonable expectation.

In the reductive amination of α-keto acids into amino acids, the imine synthesis step requires an anhydrous environment, achieved in our experiments by carrying out the reaction in xylene. Xylene is not a prebiotic compound and was used purely to facilitate the heat transfer processes in the reaction mixtures, since the reaction ingredients are poorly soluble in it. In prebiotic times an anhydrous microenvironment around an α-keto acid could be created if several purine or pyrimidine molecules, forced by the repulsive forces of the surrounding water, were aggregated to form a hydrophobic cavity containing the α-keto acid. Such a hydrophobic microenvironment exists even today in the interior of the nucleic acid double helices. As has been proposed before (29), the formation of ribonucleosides and then oligonucleotides would facilitate the bases staying together and the formation of such a hydrophobic cavity, establishing the three-base codon as the minimum required size for the enclosure of α-keto acids and their amination to amino acids.

If pyrimidines were sufficient to affect the amination of α-keto acids into amino acids (11), the question arises of what could be the role of purines and why these bases are constituents of contemporary nucleic acids. As it has been shown in this work, adenine cannot effect the formation of alanine from pyruvate and a role for the hypoxanthine-adenine couple, similar to that of uracil-cytosine, is unlikely. Biosynthetically, hypoxanthine is the common precursor of adenine and guanine. If we accept the view that biochemical pathways are traces of the evolutionary order of appearance of their individual reactions, it is reasonable to suggest that the first purine with a biological role to appear was hypoxanthine.

Reactions of hypoxanthine with ammonium chloride did not synthesize adenine (Figure 7A). Such an amination is not favored thermodynamically, as shown by the equilibrium constant Keq = [inosine][NH3] / [adenosine][H2O] = 38 (30). Amination of hypoxanthine into adenine by nitrite ions has been shown in this article to be possible and more efficient than the amination of uracil into cytosine. The transamination of uracil into cytosine by the produced adenine has also been proven. Hypoxanthine and adenine could then also be seen as alternative interchanging forms of the same molecule, as in the case of the uracil/cytosine couple, mediating the more efficient capture of nitrite and possibly other nitrogen sources for the amination of uracil.

A possible order of prebiotic reactions leading to amino acid synthesis is shown in Figure 9. At the earliest evolutionary stage α-keto acids could be directly aminated into amino acids by free ammonium ions and inorganic reducing power, as has been shown also before (31,32).Theoretically, the process would be more efficient if carried out in a hydrophobic environment, such as inside pyrimidine or other lipophilic compound aggregates, because the imine formation step would be facilitated in this case.

393-Fig9

Figure 9 | A possible prebiotic role of nucleic acid bases in amino acid synthesis. At the earliest evolutionary stage (A), direct reductive amination of α-keto acids into amino acids could be done using ammonia. At stage (B) uracil captures nitrite by reductively transforming it into the exocyclic 6-amino group of cytosine in an aqueous environment and then cytosine uses it for amino acid synthesis under anhydrous conditions. At stage (C), purines act as more efficient nitrite harvesters and reducers, passing the amino group to uracil for cytosine formation.

 

Nitrite anions and other nitrogen sources captured by uracil, although rather inefficiently, could produce cytosine after being reduced by inorganic reducers. Cytosine, as all other nucleic acid bases, is an order of magnitude more soluble in hydrophobic solvents than ammonia (33,34) and better suited for transaminations of keto-acids in a lipophilic environment. Hypoxanthine is actually a modified uracil resulting from the removal of the 2-keto group and attachment of an imidazole ring. This modified base can more efficiently capture and reduce nitrite, even from aqueous reductive media, and can transfer the 6-amino group of the resulting adenine into uracil, regenerating cytosine spent in amino acid synthesis. Ribosylation of the bases and polymerization of the ribonucleosides would produce the first RNAs. The prebiotic cooperative action of the purine-pyrimidine couple in amino acid synthesis could have been imprinted as an evolutionary trace manifested by the proximity and complementarity of the bases in the double stranded regions of the RNA and their proximity in the pyrimidine-base-purine (YNR) format of the ancient codons. It also suggests a spatial proximity of the codons to the coded amino acid, as has been proposed before (29).

Conclusion
Hypoxanthine, the purine precursor of adenine and guanine, can be transformed into adenine by reacting with nitrite ions under aqueous conditions. Adenine can then donate its exocyclic amino group to uracil, generating cytosine, which, in a hydrophobic environment, can reductively aminate pyruvate into alanine. The evolutionary role of purines could therefore be that of an efficient initial harvester of nitrogen species less reduced than ammonia, passing them to pyrimidines as amino groups, to be eventually used for amino acid synthesis from α-keto acids.

Methods
The reactions for synthesis of amino acids via reductive amination of α-keto acids by free nucleic acid bases and the analysis and identification of the products were conducted by an adaptation of the methodology described before (11). One hundred mg of pyrimidine or purine base were mixed with 100 mg of sodium pyruvate and 100 mg anhydrous sodium sulfate in 1 ml xylene. The preparations were left at 60ºC for 24 h to achieve imine formation. Sodium sulfate was included to help remove traces of water present in the reactants as well as water generated during the Schiff base formation. In reactions including them, sodium nitrite or ammonium chloride were included in amounts of 70 mg and 50 mg respectively, but the removal of water using Na2SO4 was omitted to facilitate some dissolution of the salts into the xylene solvent. After the imine formation step, to reduce the imine,10 mg of solid zinc powder and 10 μl 2M HCl were added under the xylene layer and mixed gently with the zinc-pyruvate-base solid mixture to generate in situ hydrogen gas at 60ºC for 1 h. To split the base-amino acid conjugate into free base and amino acid 300 μl freshly made 2M NaHSO3 were added and the reaction was left at 4ºC overnight. Reactions containing NH4Cl were similarly treated with Zn + HCl and NaHSO3, except when the effect of no reducing power was to be tested.

In reactions examining the exchange of exocyclic amino groups between purine-pyrimidine base pairs, 11 mg of pyrimidine or 13 mg of purine were mixed with 10-fold molar excess of the other base in the pair in 1 ml xylene and were left with some zinc powder at 60ºC overnight. The exchange in the hypoxanthine-cytosine pair was also examined in water. For reactions examining the capture of nitrite or ammonium by purines or pyrimidines, 11 mg of pyrimidine or 13 mg of purine were mixed with 70 mg sodium nitrite or 50 mg of ammonium chloride in 1 ml water and were incubated at 60ºC overnight.

A convenient qualitative color test was employed for the fast detection of adenine in the reactions. It consists of adding to the reaction an equal volume of 5% v/v NaOCl and a 1/10 volume of 25% NH4OH. The test is cited in the Merck Index (34) as detecting the presence of cytosine by the appearance of a reddish color, but in our hands it was adenine showing this color instead. Cytosine and to a smaller extent hypoxanthine show a pale yellow color, whereas uracil and water do not develop any color. Guanine does not develop a stable coloration, but starts with a brownish-purple, changing to yellow-green over time and ending colorless after 24 hours. Low guanine concentrations (<5 mg per ml) start with a yellow-green color, becoming colorless in 12 hours. Free ammonia was detected using the Nessler reagent (35). It detects as low as 0.3 μg NH3 in 2 μL by producing a brown precipitate of HgO·Hg(NH2)I.

Alanine produced in the reactions was resolved by paper chromatography with butanol/acetic acid/water 12/3/5 (v/v/v) (alanine retention factor Rf = 0.37) or butanol/acetic acid/water 4/1/1 (v/v/v) (alanine Rf = 0.21) as developing phase. It was detected by briefly dipping the chromatogram into a solution of 0.1 w/v ninhydrin in acetone and heating it at 100ºC for 5-10 min. Purple (mauve) spots, characteristic of amino acid reaction with ninhydrin, were developed at an Rf the same as that for the alanine standard.

The rest of the water soluble reaction products were allowed to dry under reduced pressure, were re-dissolved in 500 μl of water and 20 μl of it were subjected to liquid chromatography-mass spectrometry (LC-MS) using a LCMS-2010EV Shimadzu instrument, equipped with a 4.6 x 150 mm Pathfinder silica 100, 3.5 UM reverse phase HPLC column. The liquid phase consisted of 50% v/v methanol, 50% v/v water and 0.05% v/v formic acid, at a flow rate of 0.4 ml∙ min-1. Detection of the eluted compounds was via a SPD-M20A diode array detector, scanning at all wavelengths from 190 to 800 nm and by a MS detector using an electrospray interface (ESI) in positive ionization mode at full scan acquisition between m/z50-500. The detector voltage was set at 1.4 kV and the nebulizing gas (N2) flow rate was 1.5 L∙ min-1. Identification of the produced alanine or bases was done by comparison of their LC retention time and their UV absorption maxima to those of standard compounds as well as by the size of their molecular ion in the mass spectra. Yields of produced alanine or bases were calculated by integration of their peak areas in the mass spectra and comparison to those of standard alanine of bases (three independent experiments).H

Acknowledgements
The authors would like to thank their colleagues for their comments and suggestions on improving the article

Authors declare no conflicts of interest.

About the Authors
Yannis Gounaris is a Professor of Molecular Biology with over 30 years of research experience in plant and microorganism molecular biology. The mechanism of molecular evolution is his most recent interest.

Constantinos Litinas is a Professor of Organic Chemistry. He has over 30 years of experience in the identification of molecular structures of organic molecules by spectroscopic means and LC-MS.

Eleni Evgenidou is a postdoctoral research associate, experienced in LC-MS and general chemistry techniques for the isolation and identification of organics.

Constantinos Petrotos is Assistant Professor of Biosystem Engineering in the Technological and Educational Institute of Larisa, Greece. He is experienced in isolation of natural products and in their identification by mass spectrometry.

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