Download A hypothesis on the possible contribution of free hypoxanthine and

HYPOTHESIS
Department of Agriculture, University of Thessaly,
New Ionia, Greece.
1
Please cite this article as:
Yannis Gounaris et al., A hypothesis on the possible contribution of free
hypoxanthine and adenine bases in prebiotic amino acid synthesis.
Hypothesis 2015, 13(1): e7, doi:10.5779/hypothesis.v13i1.393
A hypothesis on the possible contribution of free
hypoxanthine and adenine bases in prebiotic
amino acid synthesis
2
Laboratory of Organic Chemistry, Department of
Chemistry, Aristotle University of Thessaloniki,
Thessaloniki, Greece.
3
Department of Biosystems Engineering, Technological and Educational Institute of Larisa, Larisa, Greece.
1/8
Received: 2014/11/25; Accepted: 2014/12/22;
Posted online: 2015/08/05
© 2015 Yannis Gournaris et al., This is an Open Access
article distributed by Hypothesis under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.
*Correspondence:
[email protected]
Yannis Gounaris1*, Constantinos Litinas2, Eleni Evgenidou2, Constantinos Petrotos3
transamination was not observed. In com-
that RNA was formed first by random
synthesis. Thymine is actually 5΄-methyl-
its 6-aminated state, adenine, could not
bination with previous work proving the
polymerization of ribonucleotides
ated uracil and cytosine is uracil aminated
be proven and a different role for the pu-
feasibility of reductive amination of pyru-
the first RNAs are believed to have cata-
at carbon 4.
rine pair had to be sought. An answer to
vate into alanine by cytosine, it is conclud-
lyzed random polypeptide synthesis driv-
ed that the pyrimidine hypoxanthine could
en by the need for stability of this primi-
have
10
and
evolutionarily appeared as an effi-
tive ribosome. In a recent third alternative
cient capturer of inorganic nitrogen spe-
view11, the amino acids may have started
cies, passing them to uracil, for further am-
at some evolutionary point to be synthe-
ination of α-keto acids into amino acids. sized in a way dependent on the bases,
INTRODUCTION It has been proposed which could be the origin of the contemABSTRACT 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
Illustration by Eloïse Kremer
that life originated in a hot, acidic, reduc-
porary interdependence of polypeptide-
ing, anaerobic environment, rich in hy-
nucleic acid synthesis. This eliminates a
drogen gas and nitrogen, sulfur and iron
significant degree of randomness, since
compounds, such as is encountered in
it creates spatial proximity of amino ac-
hydrothermal vents 1-7.
ids to bases, facilitating a subsequent po-
Two different lines of thought are known
concerning
the
prebiotic
evolution-
ary course. According to one of them,
amino acids were formed first and were
subsequently randomly polymerized to
polypeptides6,8,9. The other proposal is
lymerization 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 material12,13, the RNA bases were examined for
any probable catalytic role in amino acid
In a previous work11, it has been shown
that cytosine can reductively aminate the
these two questions is attempted in this
article.
α-keto acid pyruvate into the amino acid RESULTS Reductive amination of pyrualanine. Accordingly, uracil and cytosine
vate into alanine by ammonium or by free
could be considered as the two alterna-
cytosine. A reaction scheme for the
tive states of the pyrimidine ring, with cy-
reductive amination of pyruvate into
tosine donating its 4-amino group for re-
alanine by ammonium or by cyto-
ductive amination of an α-keto acid to
sine in a non-aqueous environment is
the corresponding amino acid, being
shown in Figure 1. The feasibility of the
converted into uracil in the process. Two
reaction with ammonium is shown in
questions arose from this. The first is what
Figure 2. Higher temperatures and reduc-
could be the source of amino groups re-
ing agents, either hydrogen generated
generating cytosine from uracil? The sec-
in situ from HCl and zinc or zinc alone
ond question arising was what could then
favored the reaction. There was no need
be the possible role of the purine bases,
for the presence of uracil. A hydropho-
if the pyrimidines were enough for base-
bic medium, xylene in this case, was
dependent amino acid synthesis to origi-
required. The results show that reduc-
nate? A similar role for hypoxanthine and
tive amination of α-keto acids into amino
HYPOTHESIS
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Gounaris et al.
acids by ammonia directly, without an
of uracil, the red-brown color charac-
Synthesis of adenine from hypoxanthine
intervening stage of cytosine forma-
teristic of the presence of adenine did
and nitrite ions. The possibility was
tion, might have been possible in pre-
not develop upon addition of NaOCl.
examined that nitrogen species less
biotic hydrophobic micro-environments.
Instead a very weak yellow color, char-
reduced than ammonia, such as nitrite ions
About 0.1±0.03% of the original pyruvate
acteristic of the presence of cytosine,
(NO2-), might be utilized as a source of
was transformed into alanine, as calcu-
showed up (Figure 5A), indicating the
amino groups for amino acid synthesis.
lated by comparison of the integrated
deamination of most of the adenine into
The reaction conditions are presented
mass spectrometry of the alanine peaks
hypoxanthine and the amination of some
in Figure 6 and the results are shown in
to those of the standard alanine.
uracil into cytosine. The products were
Figure 7. Nitrite can be reduced to ammo-
confirmed by Liquid Chromatography-
nia by zinc, as indicated by the brown-
The ability of the cytosine exocyclic
amino group to be transferred to pyruvate
for alanine formation had been shown
before11, 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%.
Migration of the adenine exocyclic amino group to uracil. The reaction scheme
used in this and similar transaminaFigure 1 | Reactions employed for the synthesis of alanine by reductive amination of pyruvate
tion experiments is shown in Figure 4
by ammonium or cytosine. Anhydrous conditions were achieved by carrying out the reactions in xylene.
and the results of reactions in which
Reduction of the imine intermediate was done using hydrogen generated in situ from zinc powder and 2M HCl.
purine-pyrimidine
Liberation of the formed alanine from its conjugate to cytosine was effected by sulfonizing the base moiety us-
bated in xylene are shown in Figure 5.
ing 2M sodium bisulfate and leaving the sample at low temperatures for detaching the alanine. For the sake of
When
comparison the sulfonization step was also done on reactions involving ammonium.
enine and a 10-fold molar excess
the
pairs
reaction
were
contained
incuad-
Mass Spectrometry (LC-MS) analysis
ish color development on addition of the
(Figure 5B). The cytosine negative ion
Nessler reagent. Free ammonia was not
(m/z = 110) was detected at elution time
detected when hypoxanthine was also
4.9 min. Hypoxanthine showed as its
present in the reaction. However, ammo-
positive ion with H (m/z = 137) at 4.8
nia not produced from nitrite, but added
min. The yield of transformation of ura-
to the reaction together with hypoxan-
cil into cytosine was about 0.7±0.2%,
thine, cannot be captured by hypoxan-
whereas the deamination of adenine
thine to produce adenine. Hypoxanthine
into hypoxanthine was almost quantita-
was able to become adenine only when
+
tive. These significant losses of ammo-
it was simultaneously present in the
nia during transamination could be at-
aqueous reaction with sodium nitrite
tributed to the far from perfect reducing
and in the presence of a reducing agent,
conditions in the reaction mixtures and
such as zinc powder. An explanation
to the expected loss of liberated vola-
for the observed results could be that
tile ammonia at 60°C, during the imine
any ammonia synthesized by reduction
intermediate step. Nevertheless, the ex-
of the nitrite ion was immediately cap-
periment sufficiently proves the feasibil-
tured by hypoxanthine. However, color
ity of amino group transfer from adenine
developed even in the absence of Zn. It
to uracil. The reverse reaction, contain-
seems probable that the nitrite ion reacts
ing cytosine and a 10-fold molar excess
directly with the hypoxanthine to give a
of hypoxanthine, did not show significant
nitroso product that is subsequently re-
transfer of the amino group from cytosine
duced into adenine. In LC-MS analysis
to hypoxanthine.
of the hypoxanthine-NaNO2 reaction
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products, a negative ion of m/z = 134
mineral iron(III) hydroxide oxide, Fe(OH)
was detected at elution time 4.7 min. This
O18. Glyceraldehyde is considered an in-
fits the adenine negative ion. Its amount
gredient of the prebiotic atmosphere19,20.
is 5±0.8% of the original hypoxanthine. A Also, the α-keto acids pyruvic acid, oxalosecond peak of the same m/z appears
acetic acid and α-ketoglutaric acid have
when the reaction is done in xylene. Its
been identified in carbonaceous mete-
identification has not been pursued fur- orites and in laboratory reactions pyruther, but it fits the hypoxanthine imine
vic acid can produce oxaloacetic acid21.
with ammonia, which is equivalent in mo- Experiments show that pyruvate can form
lecular weight to the adenine tautomers14. by a condensation of a ketene with HCN
Similar reactions between uracil and ni- to give first pyruvonitrile(CH3COCN), a
trite reveal only traces of cytosine (Figure
pyruvate precursor, whereas other nitriles
8), at least an order of magnitude less
lead to various other keto acids22.
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.
Nucleic acids are easily depurinated23
and their N-glycosidic bonds are easily
cleaved24 under hot acidic conditions. It
is therefore probable that in prebiotic hydrothermal vents or similar environments,
DISCUSSION The source of α-keto ac- free bases were encountered initially perids on prebiotic earth has been inves- haps alone and later together with nutigated to a significant extent. Pyruvate
cleic acids. Free nucleic acid bases can
is present in hydrothermal vents, syn- be synthesized from aqueous ammothesized at high temperatures from al- nium cyanide or from a mixture of methkyl thiols and carbon monoxide, under
ane, ammonia and water7, but also from
the catalytic action of iron-sulfur centers
formamide25,26. The presence of H2, am- Figure 2 | Results of reactions for synthesis of the amino acid alanine
temperatures and only traces at RT. The NaHSO3 concentration in the reaction
or other transition metal sulfides15. But
monia and formamide, as well as the low
(ala) from pyruvate (pyr) using ammonium ions. Frames A1 to A4 are nin-
is expected to be lower since this compound reacts with HCl, producing SO2.
pyruvate and other α-keto acids could
pH and high temperature in hydrother- hydrin-stained paper chromatograms of the reaction products. Reactions carried
A4. Optimum conditions for alanine synthesis were employed. Hydrogen
also be derived from α-hydroxy acids
mal vents, are well documented . The
out at room temperature (RT) were compared to those at 60°C. Ala: alanine stan-
was produced in situ from zinc and HCl. Zinc alone also acted as reducer. The
upon oxidation by sulfur/iron sulfite mix- keto acid amination conditions used in
dard. A1. Reactions involving no reduction by Zn + HCl and no NaHSO3 treatment
simultaneous presence of hypoxanthine (hyp) did not significantly alter the
this work are similar to the conditions en- step produced no amino acid product. A2. Reactions involving a NaHSO3 treat-
yield. B. LC-MS chromatogram of the reaction products when 60oC and H2 were
can form by aldehyde rearrangements, countered in hydrothermal vents. In water, ment but no Zn + HCl did produce alanine at higher temperatures, since NaHSO3
used. Upper trace shows the total positive ion current. The lower peak trace shows
tures16,17. These prebiotic α-hydroxy acids
1-3
as is the case of lactic acid synthesis
the bisulfite ion is in chemical equilibrium
is a weak reducer, but only traces of alanine at room temperature. A3. Reactions
from glyceraldehyde, catalyzed by the
with SO2. Bisulfite and SO2 are present in
using only HCl and then NaHSO3 treatment. Some alanine was produced at higher
HYPOTHESIS
the presence of alanine, detected as a positive M+1 ion with proton, at m/z=90.
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magma and volcano emissions27,28, but
cavity, establishing the three-base co-
by the produced adenine has also been
have not been reported for submarine
don as the minimum required size for the
proven.
hydrothermal vents . This is expected
enclosure of α-keto acids and their ami-
could then also be seen as alternative in-
because SO2 is rapidly converted into bi-
nation to amino acids.
2
sulfite 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.
If pyrimidines were sufficient to affect the
amination of α-keto acids into amino acids11, the question arises of what could
be the role of purines and why these
Hypoxanthine
and
adenine
terchanging 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.
bases are constituents of contemporary
A possible order of prebiotic reactions
nucleic acids. As it has been shown in
leading to amino acid synthesis is shown
In the reductive amination of α-keto ac-
this work, adenine cannot effect the for-
in Figure 9. At the earliest evolutionary
ids into amino acids, the imine synthesis
mation of alanine from pyruvate and a
stage α-keto acids could be directly am-
step requires an anhydrous environment,
role for the hypoxanthine-adenine cou-
inated into amino acids by free ammo-
achieved in our experiments by carrying
ple, similar to that of uracil-cytosine, is
nium ions and inorganic reducing pow-
out the reaction in xylene. Xylene is not a
unlikely. Biosynthetically, hypoxanthine
er, as has been shown also before31,32.
prebiotic compound and was used pure-
is the common precursor of adenine
Theoretically, the process would be more
ly to facilitate the heat transfer processes
and guanine. If we accept the view that
efficient if carried out in a hydrophobic
in the reaction mixtures, since the reac-
biochemical pathways are traces of the
environment, such as inside pyrimidine
tion ingredients are poorly soluble in it. In
evolutionary order of appearance of their
or other lipophilic compound aggre-
prebiotic times an anhydrous microen-
individual reactions, it is reasonable to
gates, because the imine formation step
vironment around an α-keto acid could
suggest that the first purine with a bio-
would be facilitated in this case.
be created if several purine or pyrimi-
logical role to appear was hypoxanthine.
dine 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
Figure 3 | In vitro synthesis of alanine via reductive amination of pyruvate by nucleic acid
double helices. As has been proposed
bases. A. Paper chromatogram of the reaction products stained with ninhydrin for amino acid detection.
before29, the formation of ribonucleo-
Only cytosine (C) produced alanine. Transamination by adenine (A) was not successful. B. Scan of the LC-MS
sides and then oligonucleotides would
chromatogram of the cytosine- and uracil-containing reactions for alanine M+H+ (m/z = 90) ion peaks. Alanine
facilitate the bases staying together and
was detected only in the cytosine-containing reaction.
the formation of such a hydrophobic
Reactions of hypoxanthine with ammo-
Nitrite anions and other nitrogen sources captured by uracil, although rather
nium chloride did not synthesize ade-
inefficiently, could produce cytosine af-
nine (Figure 7A). Such an amination is not
ter being reduced by inorganic reduc-
favored thermodynamically, as shown
ers. Cytosine, as all other nucleic acid
by the equilibrium constant Keq = [ino-
bases, is an order of magnitude more ,
sine][NH3] / [adenosine][H2O] = 3830.
soluble in hydrophobic solvents than
Amination of hypoxanthine into adenine
ammonia33,34 and better suited for trans-
by nitrite ions has been shown in this ar-
aminations of keto-acids in a lipophilic
ticle to be possible and more efficient
environment. Hypoxanthine is actually a
than the amination of uracil into cytosine.
modified uracil resulting from the remov-
The transamination of uracil into cytosine
al of the 2-keto group and attachment
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of an imidazole ring. This modified base
to be eventually used for amino acid syn- overnight. Reactions containing NH4Cl
can more efficiently capture and reduce
thesis from α-keto acids.
nitrite, even from aqueous reductive me-
METHODS The reactions for synthesis
dia, 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 before29.
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,
of amino acids via reductive amination
were similarly treated with Zn + HCl and
NaHSO3, except when the effect of no reducing power was to be tested.
of α-keto acids by free nucleic acid bas- In reactions examining the exchange of
es and the analysis and identification of
exocyclic amino groups between purine-
the products were conducted by an ad- pyrimidine base pairs, 11 mg of pyrimiaptation of the methodology described
dine or 13 mg of purine were mixed with
before11. One hundred mg of pyrimidine 10-fold molar excess of the other base in
or purine base were mixed with 100 mg
the pair in 1 ml xylene and were left with
of sodium pyruvate and 100 mg anhy- some zinc powder at 60ºC overnight. The
drous sodium sulfate in 1 ml xylene. The
exchange in the hypoxanthine-cytosine
preparations were left at 60ºC for 24 h to
pair was also examined in water. For re-
achieve imine formation. Sodium sulfate
actions examining the capture of nitrite or
was included to help remove traces of
ammonium by purines or pyrimidines, 11
water present in the reactants as well as
mg of pyrimidine or 13 mg of purine were
water generated during the Schiff base
mixed with 70 mg sodium nitrite or 50 mg
formation. In reactions including them, of ammonium chloride in 1 ml water and
sodium nitrite or ammonium chloride
were included in amounts of 70 mg and
were incubated at 60ºC overnight.
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.
a brownish-purple, changing to yellow- = 0.21) as developing phase. It was de-
A convenient qualitative color test was
green over time and ending colorless af- tected by briefly dipping the chromatoemployed for the fast detection of ade- ter 24 hours. Low guanine concentrations gram into a solution of 0.1 w/v ninhydrin in
water using Na2SO4 was omitted to facilinine in the reactions. It consists of adding (<5 mg per ml) start with a yellow-green acetone and heating it at 100ºC for 5-10
tate some dissolution of the salts into the
to the reaction an equal volume of 5% v/v color, becoming colorless in 12 hours. min. Purple (mauve) spots, characterisxylene solvent. After the imine formation
NaOCl and a 1/10 volume of 25% NH4OH. Free ammonia was detected using the tic of amino acid reaction with ninhydrin,
step, to reduce the imine,10 mg of solid
The test is cited in the Merck Index34 as Nessler reagent35. It detects as low as 0.3 were developed at an Rf the same as that
zinc powder and 10 μl 2M HCl were adddetecting the presence of cytosine by μg NH in 2 μL by producing a brown pre- for the alanine standard.
3
ed under the xylene layer and mixed genthe appearance of a reddish color, but cipitate of HgO·Hg(NH )I.
2
The rest of the water soluble reaction
tly with the zinc-pyruvate-base solid mixin our hands it was adenine showing this
Alanine produced in the reactions was re- products were allowed to dry under reture to generate in situ hydrogen gas at
color instead. Cytosine and to a smaller
solved by paper chromatography with bu- duced pressure, were re-dissolved in 500
60ºC for 1 h. To split the base-amino acid
extent hypoxanthine show a pale yellow
tanol/acetic acid/water 12/3/5 (v/v/v) (ala- μl of water and 20 μl of it were subjectconjugate into free base and amino acid
color, whereas uracil and water do not
nine retention factor Rf = 0.37) or butanol/ ed to liquid chromatography-mass spec300 μl freshly made 2M NaHSO3 were
develop any color. Guanine does not deacetic acid/water 4/1/1 (v/v/v) (alanine Rf trometry (LC-MS) using a LCMS-2010EV
added and the reaction was left at 4ºC
velop a stable coloration, but starts with
50 mg respectively, but the removal of
HYPOTHESIS
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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).
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
Figure 5 | Exocyclic amino group transfer between the free purines and pyrimidines. A. Adenine
in aqueous medium, a peak appears at retention time
(A), hypoxanthine (H), cytosine (C) and uracil (U), alone or in combination, were heated overnight at 60οC in
4.7 min, fitting the adenine ion. A second peak of the
xylene, in the presence of a small amount of zinc powder. The presence of adenine is shown by the develop-
same m/z appears at 4.8 min when the reaction is
ing reddish color using the NaOCl test. In the presence of 10-fold molar excess of uracil the adenine color
done in xylene.
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 45οC. 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.
HYPOTHESIS
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Shimadzu instrument, equipped with a
ABOUT THE AUTHORS Yannis Gounaris 3 Martin W, Baross J, Kelley D, Russell M.
4.6 x 150 mm Pathfinder silica 100, 3.5
is a Professor of Molecular Biology with
UM reverse phase HPLC column. The liq- over 30 years of research experience
uid phase consisted of 50% v/v methanol, in plant and microorganism molecular
50% v/v water and 0.05% v/v formic acid, biology. The mechanism of molecular evo-
Microbiol. 2008;6:805-14.
at a flow rate of 0.4 ml∙ min-1. Detection
primitive earth. In: Rigoutsos I, Stephanopoulos G,
of the eluted compounds was via a SPDM20A 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/z50Figure 8 | LC-MS spectrum of positive ion peaks of m/z=112 among the products of a reaction
500. The detector voltage was set at 1.4
of uracil with sodium nitrite in the presence of Zn powder. Traces of peaks at retention time 4.3-4.7
kV and the nebulizing gas (N2) flow rate
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.
lution 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.
http://dx.doi.org/10.1038/nrmicro1991
4 Miller S, Cleaves H. Prebiotic chemistry on the
editors. Systems Biology, Volume I: Genomics 1.
Oxford: Oxford University Press; 2006. p. 3-56.
5 Miller S, Orgel L. The origins of life on the earth.
Englewood Cliffs (NJ): Prentice-Hall; 1974.
6 Oparin A. The origin of life. New York:: Macmillan;
1938.
Eleni Evgenidou is a postdoctoral re- 7 Schopf J, editor. Earth's earliest biosphere: Its origin
search associate, experienced in LC-MS and evolution. Princeton (NJ): Princeton University
was 1.5 L∙ min-1.Identification of the pro- and general chemistry techniques for the
duced alanine or bases was done by com- isolation and identification of organics.
Press; 1983.
8 Fox S. Self-ordered polymers and propagative cell-
Constantinos Petrotos is Assistant Pro- like systems. Naturwissenschaften. 1969;56:1-9.
UV absorption maxima to those of stan- fessor of Biosystem Engineering in the http://dx.doi.org/10.1007/BF00599584
dard compounds as well as by the size Technological and Educational Institute 9 Miller S. A production of amino acids unparison of their LC retention time and their
of their molecular ion in the mass spectra. of Larisa, Greece. He is experienced in
der possible primitive earth conditions. Science.
Yields of produced alanine or bases were
isolation of natural products and in their 1953;117:528-9.
calculated by integration of their peak
identification by mass spectrometry.
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
Figure 9 | A possible prebiotic role of nucleic acid bases in amino acid synthesis. At the
Hydrothermal vents and the origin of life. Nat Rev
their comments and suggestions on
REFERENCES
http://dx.doi.org/10.1126/science.117.3046.528
10 Gesteland R, Cech T, Atkins J. The RNA world:
The nature of modern RNA suggests a prebiotic RNA
1 Baross J, Hoffman S. Submarine hydrothermal vents
world. Plainview (N.Y): CSHL Press; 2006.
and associated gradient environments as sites for
11 Gounaris Y, Litinas C, Evgenidou E. A possible
the origin and evolution of life. Orig Life Evol Biosph
prebiotic function of cytosine as amino acid synthe-
1985;15:327-45.
sizer. Hypothesis. 2014;12(1).
http://dx.doi.org/10.1007/BF01808177
12 Gilbert W. The RNA world Nature. 1986;319:618.
2 Kingston-Tivey M. Generation of seafloor hydro-
http://dx.doi.org/10.1038/319618a0
earliest evolutionary stage (A), direct reductive amination of α-keto acids into amino acids could be done
improving the article
using ammonia. At stage (B) uracil captures nitrite by reductively transforming it into the exocyclic 6-ami-
CONFLICTS OF INTEREST Authors de- thermal vent fluids and associated mineral deposits.
13 Orgel L. Prebiotic chemistry and the origin of the
no 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
clare no conflicts of interest.
Oceanography. 2007;20:50-65.
RNA world. Crit Rev Biochem Mol. 2004;39:99-123.
http://dx.doi.org/10.5670/oceanog.2007.80
http://dx.doi.org/10.1080/10409230490460765
HYPOTHESIS
Vol.13, No.1 | 2015 | hypothesisjournal.com
amino group to uracil for cytosine formation.
A hypothesis on the possible contribution of free hypoxanthine and adenine bases in prebiotic amino acid synthesis
HYPOTHESIS
8/8
Gounaris et al.
14 Pullman B, Berthod H, Dreyfus M. Amine-imine
pyruvic acid, and other possible metabolism precur-
29 Gounaris Y. An evolutionary theory based on a
tautomerism in adenines. Theoret Chim Acta (Berl).
sors in carbonaceous meteorites. Proc Natl Acad Sci
protein-mRNA co-synthesis hypothesis. J Biol Res-
1969;15:265-8.
U S A. 2011;108:14015-20.
Thessalon. 2011;15:3-16.
http://dx.doi.org/10.1007/BF00526203
http://dx.doi.org/10.1073/pnas.1105715108
30 LWolfenden R. The free energy of hydrolysis of
15 Cody G, Boctor N, Filley T, Hazen R, Scott J,
22 Hagemeyer H. Reactions of ketene. Ind Eng Chem.
adenosine to inosine and ammonia. J Biol Chem.
Sharma A, et al. Primordial carbonylated iron-sulfur
1949;41:765–70
1967;242:4711-4.
compounds and the synthesis of pyruvate. Science.
http://dx.doi.org/10.1021/ie50472a021
31 Hafenbradl D, Keller M, Wächtershäuser G, Stetter
2000;289:1337-40.
23 Lindahl T, Nyberg B. Rate of depurination
K. Primordial amino acids by reductive amination of
http://dx.doi.org/10.1126/science.289.5483.1337
of native deoxyribonucleic acid. Biochemistry.
α-oxo acids in conjunction with the oxidative forma-
16 deAldecoa L, Roldán F, Menor-Salván C.
1972;11:3610-8.
tion of pyrite. Tetrahedron Lett. 1995;36:5179-82.
Pyrrhotite as a catalyst in prebiotic chemical evolu-
http://dx.doi.org/10.1021/bi00769a018
http://dx.doi.org/10.1016/0040-4039(95)01008-6
tion. Life. 2013;3:502-17.
24 March J. Advanced organic chemistry. New York,
PMid:10675357 PMCid:PMC377440
http://dx.doi.org/10.3390/life3030502
Chichester, Brisbane, Toronto, Singapore: John Wiley
32 Huber C, Wächtershäuser G. Primordial re-
17 Wang W, Yang B, Qu Y, Liu X, Su W. FeS/S/
& Sons; 1985.
ductive amination revisited. Tetrahedron Lett.
FeS2 redox system and its oxido reductase-like
25 Basile B, Lazcano A, Oró J. Prebiotic synthe-
2003;44:1695-7.
chemistry in the iron-sulfur world. Astrobiology.
ses of purines and pyrimidines. Adv Space Res.
http://dx.doi.org/10.1016/S0040-4039(02)02863-0
2011;11:471–6.
1984;4:125-31.
33 Brosnan R, Yang L, Milutinovic P, Zhao J,
http://dx.doi.org/10.1089/ast.2011.0624
http://dx.doi.org/10.1016/0273-1177(84)90554-4
Laster M, Eger E, II, et al. Ammonia has anesthetic
18 Weber A. Prebiotic sugar synthesis: Hexose
26 Saladino R, Crestini C, Costanzo G, Negri R, di-
properties. Anesth Analg. 2007;104:1430-3.
and hydroxyl acid synthesis from glyceraldehydes
Mauro E. A possible prebiotic synthesis of purine, ad-
http://dx.doi.org/10.1213/01.ane.0000264072.97705.0f
catalyzed by iron(III) hydroxide oxide. J Mol Evol.
enosine, cytosine and 4(3H)-pyrimidinone from for-
34 O'Neil M, editor. The Merck Index. Whitehouse
1992;35:1-6.
mamide. Implications for the origin of life. Bioorgan
Station (NJ): RSC Publishing; 2013.
http://dx.doi.org/10.1007/BF00160255
Med Chem. 2001;9: 1249-53.
35 Wagenknecht F, Juza R. Potassium triiodomer-
19 Miller S. The formation of organic com-
http://dx.doi.org/10.1016/S0968-0896(00)00340-0
curate(II). In: Brauer G, editor. Handbook of
pounds on the primitive earth. Ann NY Acad Sci.
27 Carroll M, Holloway J. Volatiles in magmas.
Preparative Inorganic Chemistry. 1. New York:
1957;69:260-75.
Washington (DC): Amer Mineral Soc; 1994.
Academic Press; 1963. p. 1100.
http://dx.doi.org/10.1111/j.1749-6632.1957.tb49662.x
28 Métrich N, Bonnin-Mosbah M, Susini J, Menez B,
20 Pinto J, Gladstone G, Yung Y. Photochemical pro-
Galoisy L. Presence of sulfite (SIV) in arc magmas:
duction of formaldehyde in earth's primitive atmo-
Implications for volcanic sulfur emissions. Geophys
sphere. Science. 1980;210:183-5.
Res Lett. 2002;29:33-1-33-4.
http://dx.doi.org/10.1126/science.210.4466.183
http://dx.doi.org/10.1029/2001GL014607
21 Cooper G, Reed C, Nguyen D, Carter M, Wang
Y. Detection and formation scenario of citric acid,
HYPOTHESIS
Vol.13, No.1 | 2015 | hypothesisjournal.com