Guanine, adenine, and hypoxanthine production in UV-irradiated formamide solutions: Relaxation of the requirements for prebiotic purine nucleobase formation

Article abstract of DOI:10.1002/cbic.201000074

Relaxed requirements: We demonstrate the formation of adenine, hypoxanthine, and guanine from heated (130 °C), UV-irradiated formamide solutions in the absence of an inorganic catalyst. Evidence is also provided that "classical" HCN pathways for purine nucleobase production are also active in heated and UV-irradiated formamide reactions. (Chemical Equation Presented)

Full text of DOI:10.1002/cbic.201000074

DOI: 10.1002/cbic.201000074
Guanine, Adenine, and Hypoxanthine Production in UV-Irradiated
Formamide Solutions: Relaxation of the Requirements for Prebiotic Purine
Nucleobase Formation
[
a]
[a]
[a]
[b]
[a]
Hannah L. Barks, Ragan Buckley, Gregory A. Grieves, Ernesto Di Mauro, Nicholas V. Hud,* and
[
a]
Thomas M. Orlando*
[
1]
The RNA-world hypothesis has generated much interest in
finding a plausible prebiotic route to RNA. The abiotic synthe-
sis of the purine nucleobases is of particular interest, given
proposals that early proto-RNA polymers might have contained
with previously proposed HCN chemical pathways to the same
nucleobases.
Thermal reactions of formamide, either neat or in the pres-
ence of calcium carbonate or sodium pyrophosphate, were
carried out at 1308C. Consistent with similar reactions per-
[
2]
only purine nucleobases. Formamide is attractive as a poten-
tial prebiotic starting material for the nucleobases; it contains
the four required elements (C, H, O, N) and stands out because
of its likely prebiotic availability, relative stability, versatile reac-
[3a]
formed at a higher temperature (1608C), purine (C N H ) was
5
4
4
the only nitrogenous base detected in reactions containing no
added inorganic catalyst (Figure 1A). The presence of calcium
[
3]
tivity, and low volatility compared to water. Saladino, Di
Mauro, and co-workers have described formamide thermo-
chemistry in detail and have catalogued many of the potential-
ly prebiotic compounds generated when neat formamide is
heated to 1608C in the presence of various clays and mineral
[
3a,4]
catalysts.
However, the potential for UV photons to pro-
mote nucleobase production from formamide has received
[
4,5]
comparatively little attention;
this is surprising given that
the absence of an ozone layer in the prebiotic atmosphere
would have allowed the passage of photons with wavelengths
[
6]
less than 300 nm. Here, we show for the first time that gua-
nine, adenine, and hypoxanthine can be produced from forma-
mide in a single model prebiotic reaction at lower tempera-
tures than previously reported, if formamide is subjected to UV
irradiation during heating; this observation relaxes the require-
ments for prebiotic purine nucleobase formation. The yield
and diversity of purines produced in heated/UV-irradiated for-
mamide are further enhanced by the presence of inorganic
catalysts, as solids or as dissolved ions. We also analyzed the
products of formamide solutions to which specific hydrogen
[
7]
cyanide (HCN) condensation products were added prior to
heating. These experiments demonstrate that purine nucleo-
base formation through formamide chemistry is compatible
+
+
[
a] H. L. Barks, R. Buckley, G. A. Grieves, Prof. N. V. Hud, Prof. T. M. Orlando
School of Chemistry and Biochemistry
Georgia Institute of Technology
Atlanta, GA 30332-0400 (USA)
Fax: (+1)404-385-6057
E-mail: thomas.orlando@chemistry.gatech.edu
hud@gatech.edu
[b] E. Di Mauro
Dipartimento di Genetica e Biologica Molecolare
Universitꢀ La Sapienza
P. le Aldo Moro 5, 00185 Rome (Italy)
Figure 1. Combined selected ion chromatograms of formamide reactions
with A) no UV irradiation and B) UV irradiation, heated to 1308C for 48 h, in
the presence and absence of inorganic catalysts. Offsets added for clarity.
Products observed include purine (1), adenine (2), hypoxanthine (3), and
guanine (4). Peak labeled (a) is the internal standard 2-aminopurine. Peak
labeled (*) has the same product and precursor ion mass as adenine but is
not adenine. Peak labeled (+) is tentatively assigned as allopurinol based
upon comparison with existing standards. rel. int.=relative intensity.
+
[
] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000074: more detailed information on
experimental techniques, along with additional selected ion and HPLC
chromatograms.
1
240
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 1240 – 1243

carbonate or sodium pyrophosphate enhanced purine yield
and enabled the production of adenine and small amounts of
hypoxanthine; additionally, a trace amount of guanine was
formed in the presence of sodium pyrophosphate (Figure 1A,
inset).
(Figure 2A). This reaction reaches a steady state after 48 hours
and stays constant within experimental error. When these
same reactions are carried out in the presence of UV light, sim-
Irradiation of formamide samples with 254 nm light in-
creased the yield and diversity of purine nucleobases produced
during heating at 1308C for all samples tested. The nucleobas-
es identified included purine, adenine, hypoxanthine, and gua-
nine (Figure 1B). While the yields of all purines identified in-
crease with UV-irradiation, the relative increases in the yields of
hypoxanthine and guanine were most pronounced.
Several pathways have been proposed for the generation of
nucleobases from HCN. As these reactions are also likely in-
volved in the thermal/photoinduced processes in formamide
solutions, we briefly summarize the reaction pathway de-
[
3a,7–9]
scribed in previous studies (Scheme 1).
The primary path-
Figure 2. Yield of adenine by headed formamide solutions A) with no UV ir-
radiation, and B) UV irradiation, as determined by LC-MS (see the Supporting
Information). Solutions of neat formamide (!), and formamide with 10 mm
DAMN (
), 10 mm AICN (*), or 10 mm AICA (~) were maintained at 1308C;
samples in (B) were irradiated with 254 nm light.
&
Scheme 1. Putative pathways for production of nucleobases from forma-
mide (1). Energy input in the form of heat and/or UV irradiation isomerizes
formamide to formamidic acid (2), which is then converted into hydrogen
cyanide and/or hydrogen isocyanide and water (3). Further thermal or pho-
tochemical transformations are believed to generate the reactive intermedi-
ates diaminomaleonitrile (DAMN, 4), diaminofumaronitrile (DAFN, 5a), ami-
noimidazolecarbonitrile (AICN, 5b), and aminoimidazolecarboxamide (AICA,
ilar adenine yields are observed for samples spiked with AICN.
In the UV-irradiated AICN-containing solution, there appears to
be a photoinduced decrease in adenine yield at longer reac-
tion times; after 48 hours, AICN or adenine might undergo
photodecomposition, or the formation of higher molecular
weight chemical species might be stimulated. In cases in
which 10 mm DAMN is initially present, irradiation results in an
approximately 15-fold increase in adenine yield—from 20 mg
adenine per gram of formamide (0.2 mm) to about 300 mg per
gram of formamide (2.5 mm; Figure 2B). This observation is
consistent with a photochemical step in the production of ade-
nine from DAMN-containing formamide solutions; this is likely
the formation of DAFN, which is the trans isomer of DAMN. UV
irradiation of samples containing 10 mm AICA did not result in
an appreciable increase in adenine production.
5
c). These intermediates react further to form purine nucleobases including
purine (6a), adenine (6b), hypoxanthine (6c), and guanine (6d).
ways are thought to involve diaminomaleonitrile (DAMN, 4),
diaminofumaronitrile (DAFN, 5a), aminoimidazolecarbonitrile
(AICN, 5b), and aminoimidazolecarboxamide (AICA, 5c) as in-
termediates. In these pathways, DAMN and DAFN are pro-
posed precursors to purine (6a) and adenine (6b), AICN to
adenine, and AICA to hypoxanthine (6c) and guanine (6d).
As a means to explore the possible intermediates of the UV-
enabled purine nucleobase formation process, DAMN, AICN,
and AICA were each added to neat formamide (10 mm) before
heating and UV irradiation. The progression of selected prod-
uct yields for these reactions and for neat formamide was
monitored over a 96 hour period. Heating 10 mm DAMN and
Heating neat formamide or a formamide solution containing
10 mm AICN to 1308C without irradiation yields very little hy-
poxanthine by a purely thermal process; in contrast, a solution
containing 10 mm AICA yields appreciable hypoxanthine (Fig-
ure 3A). The yield of hypoxanthine rises to approximately
240 mg hypoxanthine per gram of formamide (2 mm) in
10 mm AICA in neat formamide for 48 hours without UV irradi-
ation at 1308C yields only modest quantities of adenine; heat-
ing 10 mm AICN in neat formamide yields greater amounts
ChemBioChem 2010, 11, 1240 – 1243
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1241

in the formamide reactions. Representative chromatograms
from LC-MS and LC-UV, which provide some illustration of the
breadth of other compounds generated, are provided in the
Supporting Information (Figures S1 and S2).
We have worked with solutions of neat formamide because,
for practical experimental considerations, it is the obvious
starting point to study the effects of UV-irradiation on nucleo-
base production from formamide. However, it is difficult to en-
vision a situation in which neat formamide was collected on
the prebiotic Earth, as water, which is miscible with formamide,
[10]
was most likely present
and more abundant than forma-
mide. Nevertheless, given the lower volatility of formamide
and the fact that azeotropic mixtures are not formed by water
[11]
and formamide, one could imagine scenarios in which pri-
marily aqueous pools that initially contained small amounts of
formamide would lose water through evaporation, during hot
and dry periods, to give rise to concentration solutions of
formamide.
We have performed experiments to test whether this
“
drying pool” model can give rise to solutions of formamide
that also produce nucleobases. In one such experiment, 10
mol% formamide in water was heated to 1008C for an extend-
ed period of time. Our results indicate the loss of water, the
hydrolysis of formamide to ammonium formate at a rate of
approximately 1% per 24 h (Figure S3), and the formation of
purine, adenine, and hypoxanthine in detectable amounts over
the course of 96 h (Figure 4).
Figure 3. Yield of hypoxanthine by formamide solutions A) with no UV irradi-
ation and B) UV irradiation, as determined by LC-MS (SI). Solutions of neat
formamide (!), and formamide with 10 mm DAMN (
1
2
&
), 10 mm AICN (*),
0 mm AICA (~) were maintained at 1308C in A) and B) and irradiated with
54 nm light in B) only.
12 hours; it then remains approximately constant with sus-
tained heating.
Hypoxanthine yields for all UV-irradiated and heated forma-
mide solutions studied reach a plateau of approximately
100 mg hypoxanthine per gram of formamide (1 mm; Fig-
ure 3B), whether spiked with a putative intermediate or not.
Formamide solutions initially with 10 mm AICA exhibited great-
er hypoxanthine yield (similar to the thermal-only experi-
ments), but yields declined over time in the UV-irradiated sam-
ples; this suggests that approximately 100 mg hypoxanthine
per gram of formamide (about 1 mm) is a steady-state condi-
tion. This observation is consistent with an increased impor-
tance of pathways mediated by DAMN and AICN and de-
creased importance of the AICA-mediated pathways with
regard to hypoxanthine production in irradiated reactions.
Overall, our observations for the effects of DAMN, AICN, and
AICA on purine nucleobase formation in formamide are consis-
tent with these molecules being possible intermediates under
our reaction conditions. We note that LC-MS/MS and HPLC
analysis did not show the presence of DAMN, AICN, or AICA as
final products in any reactions investigated; this suggests that
if these molecules are truly intermediates in purine production,
their conversion to purines is fast relative to their production
from formamide.
Figure 4. Selected ion chromatogram demonstrating nucleobase production
after heating a 10 mol% formamide/90% water mixture at 1008C for 96 h.
Products observed include purine (1), adenine (2), and hypoxanthine (3).
The peak labeled (+) has the same mass as hypoxanthine and is tentatively
identified as allopurinol based on comparison with an authentic standard.
While the yields of these nucleobases are lower than those
observed for the neat formamide reactions, the confirmation
of nucleobase production from an initially mixed water–forma-
mide solution supports the prebiotic relevance of the experi-
ments presented above, and further relaxes the requirements
for prebiotic purine nucleobase production. We also note that
the mixed-solvent, inorganic catalyst-free, nonirradiated reac-
tion produced hypoxanthine and adenine; under the same re-
action conditions these products were not observed in neat
formamide reactions. Given the previously reported observa-
tion that ammonium formate facilitates adenine produc-
While the purine nucleobases were the focus of this study,
these compounds represent a subset of all products generated
1
242
www.chembiochem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 1240 – 1243

[
8a,d,12]
tion
and our observation that ammonium formate was
Sample analysis: LC-MS was carried out by using a Merck SeQuant
ZIC-HILIC HPLC column on an Agilent 1100 binary HPLC system
coupled to a Micromass Quattro LC triple quadrupole mass spec-
trometer. Information on temperature and gradient is given in the
SI. Reverse-phase HPLC was performed on an Agilent 1200 Series
RRLC system with a diode array detector. A Phenomenex Synergi
Polar RP column was used for separations.
produced over the course of heating and water evaporation, it
is possible that the in situ formation of ammonium formate is
an important factor in nucleobase production in this mixed-
solvent system.
The observation that 254 nm UV light induces the formation
of adenine, hypoxanthine, and guanine from neat formamide,
suggests that formamide is a photoactive entity. The electronic
absorption spectrum of formamide nominally begins at
Acknowledgements
[
13]
2
06 nm, which corresponds to a higher energy than that of
The authors gratefully acknowledge support from the NSF (CHE-
the photons used in the present study. However, the tail of the
UV absorption spectrum of a formamide solution extends to
long enough wavelengths that the extinction coefficient of
0739189), NASA Exobiology (NNG05GP20G, NNX08A014G) and
the European Space Agency. We thank Heather D. Bean and
Irena Mamajanov for helpful discussions.
À1 À1
formamide at 254 nm is nonzero (approximately 0.6 cm m ).
Recently, Duvernay and co-workers outlined a photochemical
pathway from formamide (1) to formimidic acid (2), which
Keywords: formamide
photochemistry · prebiotic syntheses · purines
·
nitrogen
heterocycles
·
[
9]
then decomposes to HCN/HNC and water (3). HCN and HNC
are believed to then undergo polymerization reactions to pro-
duce DAMN, AICN, and AICA under thermal and photochemi-
cal/thermal conditions (Scheme 1). Assuming that optical exci-
tation of formamide is one of the important photochemical
transitions, we can see from Duvernay’s results that incident
photons could make water available even in neat formamide
to help initiate and facilitate the chemical steps leading to nu-
[
[
1] R. F. Gesteland, T. R. Cech, J. F. Atkins, The RNA World, 3rd ed., Cold
Spring Harbor Laboratory Press, New York, 1999.
2] a) F. H. C. Crick, J. Mol. Biol. 1968, 38, 367–379; b) T. R. Battersby, M. Al-
balos, M. J. Friesenhahn, Chem. Biol. 2007, 14, 525–531; c) B. D. Heu-
berger, C. Switzer, ChemBioChem 2008, 9, 2779–2783.
[
3] a) R. Saladino, C. Crestini, F. Ciciriello, G. Costanzo, E. Di Mauro, Chem.
Biodiversity 2007, 4, 694–720; b) K. T. Suzuki, H. Yamada, M. Hirobe, J.
Chem. Soc. Chem. Commun. 1978, 485; c) H. Yamada, M. Hirobe, K. Higa-
shiyama, H. Takahashi, K. T. Suzuki, Tetrahedron Lett. 1978, 19, 4039.
[
9]
cleobase formation.
Here, we have demonstrated the production of adenine, hy-
poxanthine, and guanine in UV-irradiated formamide solutions.
These “one-pot” reactions occur due to the synergy of thermal
and photochemical processes. The reactions’ yields and prod-
uct distributions are enhanced in cases in which inorganic cat-
alysts (as solids or dissolved ions) are present. The lower tem-
perature at which our study was conducted combined with
the equalizing effect that UV light has on purine nucleobase
production (regardless of the presence of inorganic catalyst),
further relax the chemical and environmental requirements for
the formation of biopolymer building blocks on the prebiotic
Earth.
[4] R. Saladino, U. Ciambecchini, C. Crestini, G. Costanzo, R. Negri, E. Di
Mauro, ChemBioChem 2003, 4, 514–521.
[
[
[
5] S. D. Senanayake, H. Idriss, Proc. Natl. Acad. Sci. USA 2006, 103, 1194–
198.
6] W. J. Schopf, Earth’s Earliest Biosphere: Its Origin and Evolution, Princeton
University Press, Princeton, 1983.
7] a) J. Orꢁ, Nature 1961, 191, 1193–1194; b) J. P. Ferris, L. E. Orgel, J. Am.
Chem. Soc. 1965, 87, 4976–4977; c) J. P. Ferris, L. E. Orgel, J. Am. Chem.
Soc. 1966, 88, 1074; d) R. A. Sanchez, J. P. Ferris, L. E. Orgel, J. Mol. Biol.
1
1
967, 30, 223–253.
[8] a) E. Yonemitsu, T. Isshiki, Y. Kijima, US Patent 4,059,582, 1975; b) A. B.
Voet, A. W. Schwartz, Bioorg. Chem. 1983, 12, 8–17; c) M. Levy, S. L.
Miller, J. Oro, J. Mol. Evol. 1999, 49, 165–168; d) G. Zubay, T. Mui, Origins
Life Evol. Biosphere 2001, 31, 87–102; e) I. M. Lagoja, P. Herdewijn,
Chem. Biodiversity 2004, 1, 106–111; f) L. E. Orgel, Origins Life Evol. Bio-
sphere 2004, 34, 361–369.
[
9] F. Duvernay, A. Trivella, F. Borget, S. Coussan, J. P. Aycard, T. Chiavassa, J.
Phys. Chem. A 2005, 109, 11155–11162.
Experimental Section
[
10] a) E. B. Watson, T. M. Harrison, Science 2005, 308, 841–844; b) T. M. Har-
rison, J. Blichert-Toft, W. Muller, F. Albarede, P. Holden, S. J. Mojzsis, Sci-
ence 2006, 312, 1139b.
Reaction conditions: Formamide solutions that contained calcium
carbonate or sodium pyrophosphate had these inorganic catalysts
present at 4.4% w/w. Formamide solutions that contained putative
purine intermediates (DAMN, AICA, or AICN), had these com-
pounds present at 10 mm. Reactions described as UV-irradiated
were exposed to a low pressure mercury lamp (Pen-Ray, primary
emission at 254 nm; no emission below 200 nm), during heating.
All reactions were heated at 1308C for 48 h, unless otherwise
noted.
[
[
11] V. Campos, A. C. G. Marigliano, H. N. Solimo, J. Chem. Eng. Data 2008,
5
3, 211–216.
12] A. Hill, L. E. Orgel, Origins Life Evol. Biosphere 2002, 32, 99–102.
[13] J. M. Gingell, N. J. Mason, H. Zhao, I. C. Walker, M. R. F. Siggel, Chem.
Phys. 1997, 220, 191–205.
Received: March 6, 2010
Published online on May 21, 2010
ChemBioChem 2010, 11, 1240 – 1243
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1243