Reprint
Supported by
www.chemeurj.org
Prebiotic Chemistry C. Menor-Salv n,* M. R. Mar n-Yaseli . . . . . . . . . . 6488 – 6497 A New Route for the Prebiotic Synthesis of Nucleobases and Hydantoins in Water/Ice Solutions Involving the Photochemistry of Acetylene
2013 – 19/20
Greatness from small beginnings: The ultraviolet irradiation of a urea solution subjected to freeze–thaw cycles under an acetylene atmosphere leads to the formation of pyrimidines, purines, and hydantoin (see scheme).
This work describes the possible pathways to different nitrogen heterocycles from these precursors. This reaction completes the model of the abiotic origin of nucleobases in our solar system and on prebiotic Earth.
DOI: 10.1002/chem.201204313
A New Route for the Prebiotic Synthesis of Nucleobases and Hydantoins in Water/Ice Solutions Involving the Photochemistry of Acetylene C¦sar Menor-Salv n* and Margarita R. Mar n-Yaseli[a] Dedicated to Professor Juan P¦rez-Mercader Abstract: The origin of nucleobases and other heterocycles is a classic question in the chemistry of the origins of life. The construction of laboratory models for the abiotic synthesis of nitrogen heterocycles in plausible natural conditions also aids the understanding and prediction of chemical species in the Solar System. Here, we report a new explanation for the origin of hydantoins, purines, and pyrimidines in eutectic water/ice/urea solutions driven by ultraviolet irradiation (in the 185– 254 nm range, UVC) of acetylene
under anoxic conditions. An analysis of the products indicates the synthesis of hydantoin and 5-hydroxyhydantoin, the purines uric acid, xanthine, and guanine, and the pyrimidines uracil and cytosine. The synthesis occurred together with the photo-oxidation of bases in a complex process for which possible pathways are proposed. In conclusion, Keywords: heterocycles nucleobases · photochemistry prebiotic chemistry · urea
Introduction It is likely that the genetic code has been a feature of life on Earth since the existence of the first organisms,[1] but the origin of the nucleic acids in the first life-forms remains unknown. From a chemical perspective, we could surmise that regardless of whether RNA is prebiotic,[2] the origin of informational polymers was preceded by the synthesis of a stock of nucleobases and related molecules in a chemical evolutionary process. This premise is attractive because the retrosynthetic analyses of nucleobases leads to small precursors whose prebiotic existence is plausible. The prebiotic stock of nitrogen heterocycles could also have been imported from extraterrestrial sources by meteoritic bombardment. From this point of view, models for the natural abiotic synthesis of nitrogen heterocycles could explain the origin of the nucleobases found in meteorites and their potential presence as icy grains in dense molecular clouds or planetary environments.[3] Since the prebiotic synthesis of adenine, achieved in 1961 by hydrogen cyanide polymerization,[4] three main routes for
· ·
an acetylene-containing atmosphere could contribute to the origin of nucleobases in the presence of a urea/water system by an HCN-independent mechanism. The presence of ice has a dual role as a favorable medium for the synthesis of nucleobases and protection against degradation and as a source of free radicals for the synthesis of highly oxidized heterocycles. A mechanism for the origin of hydantoins and uracil from urea in plausible conditions for prebiotic chemistry is also proposed.
the prebiotic synthesis of pyrimidines and purines have been identified (Scheme 1): 1) the formation of purines by HCN oligomerization,[5] 2) the formation of pyrimidines from cyanoacetylene or b-alanine and urea or guanidine[6] and 3) the synthesis of both purines and pyrimidines by using formamide as a precursor in the presence of mineral catalysts.[7] If we support the idea of a prebiotic origin of nucleobases, a
[a] Dr. C. Menor-Salv n, M. R. Mar n-Yaseli Centro de Astrobiolog a (CSIC-INTA) Ctra. Torrejýn-Ajalvir km. 4 28850 Torrejýn de Ardoz (Spain) E-mail: menorsc@cab.inta-csic.es Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201204313.
6488
Scheme 1. Summary of the main currently identified prebiotic pathways to purines and pyrimidines.
Õ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 6488 – 6497
FULL PAPER “cold origin” or “ice world” is the preferred prebiotic environment because the nucleobases would be stable under cold conditions, and such an environment supports the relevance of eutectic water solutions of reactants in an ice matrix.[8] In a previous publication, we demonstrated that the synthesis of pyrimidines and purines is possible in an ice matrix using eutectic urea solutions by sparking a methane/nitrogen atmosphere.[9] We explained this synthesis of nitrogen heterocycles by invoking the two classic mechanisms of cyanoacetylene and hydrogen cyanide formation because both reactive molecules are products of the heating or sparking of CH4/N2 atmospheres.[10] In our analysis of the gas mixture obtained after sparking a CH4/N2 mixture, the main product was acetylene, followed by unsaturated hydrocarbons and HCN, but no cyanoacetylene was found.[11] Additionally, acetylene could be formed by the photolysis of methane[12] and is present as one of the minor components in the atmosphere of Titan[13] and possibly in other icy bodies in the solar system.[14] Acetylene could thus be a source material for the abiotic formation of polycyclic aromatic hydrocarbons and polymeric photopolymers that could constitute the haze observed in the atmospheres of Titan and Jupiter.[15] Surprisingly, acetylene has received comparatively less attention in the construction of laboratory models for prebiotic chemical evolution. The photochemistry of acetylene is a complex process in which ultraviolet irradiation in the presence of water leads to the formation of glyoxal, formaldehyde, oxalic acid, glyoxylic acid, and formic acid in a mechanism driven by hydroxyl radicals generated from the photolysis of water.[16] Glyoxal is a precursor of hydantoin, a common prebiotic molecule, and glyoxylic acid is a prebiotic precursor of increasing interest.[17] Despite these findings, the photolysis of acetylene in the presence of water under anoxic conditions has not been widely studied, and we cannot disregard the formation of other precursors that could lead to nitrogen heterocycles by condensation with urea and ring-closure. In this work, the potential relevance of acetylene photooxidation in the origin of nucleobases was explored. We investigated if an ice matrix could be a favorable environment for the synthesis of nucleobases and explored the pathways for their abiotic formation in the context of secondary photo-oxidation processes in a plausible prebiotic scenario.
Results and Discussion Following the previous experimental model,[9] a solution of urea (0.1 m) in ultrapure water was subjected to freeze–thaw cycles (beginning from the eutectic point of the urea/water system, ¢21 C, to 5 C) in a sealed reactor under a pure acetylene atmosphere for three weeks (Figure S1, the Supporting Information). The system was irradiated with UV light (254 nm) for the first 72 h. After the end of the experiment, the reactor content was allowed to warm to room temperature, collected in sealed vials and stored at ¢80 C. Chem. Eur. J. 2013, 19, 6488 – 6497
For the analysis of nitrogen heterocycles, the remaining urea was separated, and the fractions containing heterocycles were collected using cation exchange resin. The retained compounds were eluted and analyzed by gas chromatography-mass spectrometry (GC-MS) after derivatization as trimethylsilyl ethers/esters using total ion count (TIC) mode and selected ion mode (SIM). The ions at m/z 254, 257, and 270 were monitored for cytosine and the hydroxylated and reduced forms of pyrimidines. The reaction products (Figure 1 and Figure 2) contain hydantoins, pyrimidines and purines, including uracil, uric acid, xanthine, guanine, and adenine. The observation of purines as their 8-hydroxy derivatives did not necessarily indicate a photo-oxidation process and could be an artifact from the formation of trimethylsilyl derivatives,[18] The highest yields corresponded to 5-hydroxyhydantoin, 2-oxo-4,5-dihydroxyimidazolidine, hydantoin, uracil, parabanic acid, and uric acid (Table 1). Additionally, cytosine, 6-methyluracil (not Table 1. Selected yields and mass spectrometric data for purines, pyrimidines, and hydantoins as their trimethylsilyl derivatives. Product
Yield[a] m/z ([%])
hydantoin
66
5-hydroxyhydantoin uracil
128 27
cytosine[b]
3.6
dihydroorotic acid[c] uric acid[d] xanthine guanine
4.1 33 16 14
244(20), 229(100), 147(88), 100(15), 86(40), 73(50) 332(3), 317(24), 202(20), 189(33), 174(9), 147(100), 100(12), 73(80) 256(52), 255(50), 241(100), 147(42), 113(26), 99(60), 73(41) 254(100), 240(89), 170(19), 147(12), 112(25), 98(39), 73(41) 359(10), 331(5), 257(28), 147(30), 100(59), 73(100) 456(51), 441(57), 383(15), 147(18), 73(100) 368(12), 353(30), 294(9), 147(31), 73(100) 367(15), 352(34), 264(9), 147(15), 99(9), 73(100)
[a] Yield defined as mg of product per gram of acetylene used in the experiment. [b] Expressed as sum of cytosine and dihydrocytosine. [c] TriTMS derivative. [d] Tetra-TMS derivative.
visible in the TIC analysis due to coelution), and iso-orotic and orotic acids were found in significant quantities by using SIM analysis at m/z 254 (Figure S2, the Supporting Information). The formation of dihydrocytosine and dihydroorotic acid was observed in the SIM GC/MS analysis at m/z 257 (Figure S2, the Supporting Information). The expected formation of solid photopolymers[16] was observed in an acetylene/water irradiation control experiment (Figure S3, the Supporting Information) but was inhibited in the presence of urea, indicating that urea acted as a sink for reactive species generated during irradiation. The major aqueous products found, after UV irradiation of acetylene in the presence of water subjected to freeze–thaw cycles under anoxic conditions, were glycolic acid and succinic acid. Significant quantities of acetic acid, glyoxylic acid, oxalic acid, and benzoic acid were also observed. A control experiment was performed entirely at low temperature in eutectic conditions of the urea/water system
Õ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6489
C. Menor-Salv n and M. R. Mar n-Yaseli
Figure 1. Total ion chromatogram of the unambiguously identified TMS-derivatized products after UV irradiation of a urea solution subjected to freeze– thaw cycles under an acetylene atmosphere.
(¢21 C, a solid solution at our standard urea concentration). Under these conditions, we found that yields of hydantoins and uracil, the major products identified, were significantly lower than those found for the freeze–thaw cycle experiments. Glycolic, glyoxylic and benzoic acids, together with evidences of uracil photoproducts, were also found. Cytosine and purines fell under the detection limit. The synthesis of purines (Scheme 2) can be achieved following two pathways. In the first pathway, guanine and adenine are formed first, followed by deamination to yield xan-
Scheme 2. Synthesis of purines by irradiation of urea/acetylene.
6490
www.chemeurj.org
thine and hypoxanthine. Then, uric and parabanic acids are produced as the final photoproducts of guanine[19] . We hypothesized a second pathway involving hydantoin as the precursor of the purine ring. To test this possibility, an experiment was performed by using urea (0.1 m) and hydantoin (0.025 m) subjected to freeze–thaw cycles and UV irradiation under an inert (argon) atmosphere (Figure S4, the Supporting Information). The products found in the TIC GC-MS chromatogram were 5-hydroxyhydantoin, an oxidation product of hydantoin, and parabanic acid. Significant quantities of uric acid and its photoproducts, allantoin, 5-hydroxy-hydantoin-5-carboxamide, and alloxanic acid were also found.[20] Based on mass spectrum interpretation, we also tentatively identified 5-hydroxyisouric acid, which yields allantoin or 5-hydroxy-hydantoin-5-carboxamide upon hydrolysis and loss of ammonia and carbon dioxide. This result suggested the formation of the purine ring from hydantoin followed by a purine photo-alteration pathway similar to the non-enzymatic uric acid degradation associated with biological processes.[20] This pathway is initiated by uric acid, an efficient scavenger of the free radicals generated by UV irradiation. The hydroxyl radical-addition to the purine
Õ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 6488 – 6497
Prebiotic Synthesis of Nucleobases and Hydantoins
FULL PAPER
Figure 2. Continuation of the chromatogram in Figure S2 (the Supporting Information), showing the TMS derivatives of purines obtained after UV irradiation of a urea solution subjected to freeze–thaw cycles under an acetylene atmosphere. Allantoin was detected as tri- and tetra-(trimethylsilyl) derivatives. Adenine was not found, however, a peak for 8-hydroxyadenine was noted instead. 8-hydroxyadenine, along with 8-oxoguanine, could be derivatization artifacts of adenine and guanine and not real compounds synthesized in the experiment.[18] These compounds are therefore considered to be indirect evidence of the formation of purine nucleobases.
ring then yields the hydroxylated form of uric acid, which degrades to allantoin through ring-opening and decarboxylation, or to alloxanic acid through oxidation, hydrolysis, and loss of urea (Scheme 3). Parabanic acid has not yet been observed in the irradiation of hydantoin solution subjected to freeze–thaw cycles under an inert atmosphere (Figure S5, the Supporting Information). In the absence of urea, hydantoin irradiation mainly afforded 2-oxo-5-hydroxyimidazole, and 5-hydroxyhydantoin. This finding suggests that parabanic acid was generated in the water/ice solution as the final photo-alteration product of purines. The mechanisms of formation of uric acid from urea and hydantoin in our experimental model are not currently well-understood. However, we identified the formation of glycine (a hydrolysis product of hydantoin) and a very small proportion of N-formylglycine. This result indicates the formation of single carbon active molecules or an organocatalytic carbodiimide/urea cycle.[21] The formation of carbodiimide by dehydration of urea in the eutectic, concentrated brines in an ice matrix and the acidic medium generated during the reaction leads to a highly electrophilic reactant that activates the carboxyl of glycine and favors formylation. How the formation of urea Chem. Eur. J. 2013, 19, 6488 – 6497
Scheme 3. Synthesis of uric acid from hydantoin and urea under an inert atmosphere.
Õ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6491
C. Menor-Salv n and M. R. Mar n-Yaseli
derivatives, single carbon active species, and carbodiimide dantoin identified in the experiment performed by using drives the formation of the purine ring from hydantoin is deuterated acetylene were originated by the photo-oxidation unclear and is currently under investigation. of pyrimidines, a major proportion of deuterium-free 5-hyHydantoin, together with 5-hydroxyhydantoin and paradroxyhydantoin should be observed. However, the photobanic acid, is a product of the degradation of pyrimidines by oxidation of thymine leads to the formation of 5-methyl-5hydroxyl radicals[22] or other reactive-oxygen species[23] genhydroxyhydantoin,[26] indicating that C5 in the hydantoin ring comes from C5 in the pyrimidine ring. If the real mecherated by the UV irradiation of water.[16c,d] Under our condianism of photo-oxidation of pyrimidines preserves the hytions, the irradiation of aqueous cytosine and uracil soludrogen at the C5 position, then the high yield of 5-hydroxytions did not yield hydantoin (Figure S6, the Supporting Inhydantoin-5[D] obtained would be consistent with the degformation). As a consequence, photo-oxidation of previously radation of pyrimidines. By comparison, the photo-oxidation generated nucleobases does not explain the presence of a of hydantoin leads preferentially to the formation of 2-oxorelatively high amount of hydantoin and 2-oxo-4,5-dihydrox5-hydroxyimidazole and a minor amount of deuterium-free yimidazolidine. However, given that glyoxal is one of the 5-hydroxyhydantoin, possibly originating from the photo-oxmajor products of acetylene irradiation through the addition idation of uric acid or other purines. Anyway, the yield of 5of hydroxyl radicals,[16, 24] the formation of hydantoin by the hydroxyhydantoin observed after the irradiation of cytosine reaction of glyoxal with urea could be achieved under our and uracil is low (estimated 13 % of transformation of cytoexperimental conditions. To provide evidence supporting sine and uracil) compared with the yield of the main experithis hypothesis, two experiments were performed. In the ment, suggesting a direct synthetic pathway. Consequently, first, a solution of urea (0.1 m) and glyoxal (0.01 m) (simulatthe main possible source for 5-hydroxyhydantoin-5[D] is ing the quantitative transformation of acetylene into glyoxring-closure of the a-hydroxyhydantoic acid, produced by al) was submitted to freeze–thaw cycles under an acetylene condensation of urea and glyoxylic acid, favored by the atmosphere without UV irradiation. After the given reaction acidic medium generated by acetylene irradiation time, high yields of 2-oxo-4,5-dihydroxyimidazolidine and (Scheme 5). Glyoxylic acid is a product of the UV irradiahydantoin were found, but the photo-oxidation products 5tion of acetylene or acetic acid in presence of water, as well hydroxyhydantoin and 2-oxo-5-hydroxyimidazole were not as a product of oxidation of glyoxal. By using deuterated observed (Figure S7, the Supporting Information). Second, acetylene, the reaction would result in the a-deuterated the irradiation of dideuteroacetylene over a solution of urea form of a-hydroxyhydantoic acid and, hence, deuterated 5(0.1 m) subjected to freeze–thaw cycles yielded 2-oxo-4,5-dihydroxyhydantoin should be obtained. To confirm this pathhydroxyimidazolidine-4,5[D2] and hydantoin-5,5[D2] with a way, a solution of urea (0.1 m) and glyoxylic acid (0.01 m) minor amount of deuterium-free hydantoin. On the basis of was submitted to freeze–thaw cycles under an inert (argon) these experiments, the probable mechanism for the synthesis of hydantoin is through a pinacol/pinacolone-type rearrangement of 2-oxo-4,5-dihydroxyimidazolidine (Scheme 4). The experimentation with deuterated acetylene led to the formation of 5-hydroxyhydantoin-5[D], which could be the result of a direct synthesis by condensation of acetylene photoproducts and urea or the result of the photo-oxidation of pyrimidines and, to a lesser extent, hydantoin (See Figure S8 (the Supporting Information) for a summary of the deuterated products found). The mechanism for the formation of 5-hydroxyhydantoin by photo-oxidation of pyrimidines is unclear, but the most accepted explanation is the formation of dialuric acid or alloxan by hydroxyl radical-addition, followed by loss of carbon.[25] In that case, if the 5-hydroxyhy- Scheme 4. Synthesis of hydantoin from acetylene and a water/urea solution. 6492
www.chemeurj.org
Õ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 6488 – 6497
Prebiotic Synthesis of Nucleobases and Hydantoins
Scheme 5. Synthesis of hydantoins in a urea/ice matrix under an acetylene atmosphere and UV irradiation. The route to 5-hydroxyhydantoin by ring-closure of 2-hydroxyhydantoic acid is prevalent against the formation of pyrimidine ring in the isolated glyoxylic-urea system.
atmosphere with UV irradiation. After the given reaction time, high yields of 5-hydroxyhydantoin and possible photooxidation products were found (Figure S9, the Supporting Information). The reaction between urea and glyoxylic acid could also be the origin of uracil by the Davidson and Baudisch synthesis.[27] Although the reaction originally occurred between malic acid and urea in concentrated sulfuric acid, the key step in uracil formation was the condensation of urea and glyoxylic acid, generated from malic acid dehydration in a sulfuric acid solution. The freezing cycles leading to brines with concentrated organic solutes would favor these final condensation steps. The experiment performed by ultraviolet irradiation of a water/ice solution of urea and glyoxylic acid under an inert atmosphere led to the formation of a low yield of uracil, partially confirming this scheme. However, the low quantity of uracil found does not explain the yield observed in the main experiment, suggesting additional formation mechanisms or the lack of key reactants or catalysts in the complex mixture formed by the irradiation acetylene atmosphere. The formation of cytosine-5,6[D2] was observed in the same experiment as evidenced by the two mass unit increases of the [M¢1] + and [M¢15] + ions in the mass spectrum. The presence of uracil-6[D] was inferred on the basis of the net shift of the same ions by one unit and the unaltered m/z 99 ion in the silylated uracil mass spectrum (C4H7OSi + , the fragment includes the C4- and C5-ring carbons). These results indicate that the C5 position was unsubstituted in the pyrimidine ring. Additionally, the formation of deuterated 5-hydroxyuracil and undeuterated 6-hydroxyuracil confirmed this structure. This conclusion suggests that the synthesis of pyrimidines follows three pathways. The first pathway is the synthesis of cytosine and subsequent formation of 5,6-dihydrocytosine and 5-hydroxycytosine (although this could be an artifact, see ref. [18]). The second pathway is the aforementioned synthesis of uracil from glyoxylic acid and urea by the Davidson and Baudich process. Third, the uracil synthesis could proceed in a pathway parallel to the Chem. Eur. J. 2013, 19, 6488 – 6497
FULL PAPER biological synthesis of orotic acid:[28] ureido-intermediates are formed by the reaction of urea with reactive carbonyl compounds generated by acetylene/water irradiation[29] and the ring is closed to produce dihydroorotic acid and dihydrouracil. This reaction would be followed by dehydrogenation to orotic acid and photochemical decarboxylation to dihydrouracil and uracil,[30] which coexist in an equilibrium.[31] The detection of undeuterated orotic acid, dihydroorotic6[D], and malic acid support this mechanism. Other pathways[32] that could yield uracil are the condensation of a,b-unsaturated acids with urea or the Fischer and Roeder synthesis of dihydrouracil from urea and acrylic acid, which could be a photochemical product of acetylene or its derivatives acetaldehyde and acrolein,[33] followed by photochemical dehydrogenation to uracil. Therefore, one possible mechanism for the formation of iso-orotic acid and 6-methyluracil, deuterated at C6 and the methyl group, respectively, is that both compounds could be derivatives of uracil after the addition of formaldehyde or acetate at C6.[34, 6b] Thymine was not found in the mixture, and the reason for the preference for addition at C6 was not clear. Thus, the direct synthesis of iso-orotic acid from urea and other intermediates derived from acetylene, followed by decarboxylation to uracil cannot be disregarded. Taking these results together with the addition of the hydroxyl radical and the photochemical alteration of the products,[20a] a general scheme could be suggested for the chemistry of pyrimidines in an acetylene/urea/ice system (Scheme 6). Overall, the low temperature of the reaction and the formation of eutectic urea solutions in the ice matrix favored the formation of nitrogen heterocycles through the photolysis of acetylene, formation of carbonyl compounds and condensation with urea, and the protection of both purines and pyrimidines against degradation (Figure S10, the Supporting Information). The polymerization of acetylene with incorporation of nitrogen from cyanic acid has been hypothesized for the origin of nitrogen heterocycles in condensates under interstellar space conditions.[16d] Here, we demonstrate that nitrogen heterocycles could be formed by ultraviolet irradiation of acetylene-containing atmospheres. This formation depends on the presence of an efficient condensing agent and a nitrogen source, such as urea, whose presence in a planetary environment at a pre-biochemical evolutionary stage is supported by its facile synthesis from plausible precursors.[35] The difference between this route and that from synthesis under astrophysical conditions, in which the formation of pyrimidines could proceed through hydroxyl addition to pyrimidine or the reaction of cumulene species with urea,[36] is the role of water. UV irradiation of acetylene in the presence of water leads to reactive carbonyl compounds even in the absence of atmospheric oxygen. A major difference with spark-discharge experiments[9] when considering the role of urea is the lack of s-triazines. We explained the formation of s-triazine by the decomposition of urea in ammonia and isocyanate and reaction of the isocyanate with biuret, also generated by sparking. This find-
Õ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6493
C. Menor-Salv n and M. R. Mar n-Yaseli
the radiation source.[38] Another possible route is the uptake of acetylene into the water/ice phase, in which the generation of reactive-oxygen species under UV radiation could be favored at lower energies. The high-energy ultraviolet radiation, that is, vacuum ultraviolet radiation (VUV) or extreme ultraviolet radiation (EUV), has been used usually in the study of the photochemical origin of organic molecules from an astrochemical point of view. The irradiation with VUV of acetylene/water mixtures at ¢263 C and ultrahigh vacuum leads to hydroxylated photoproducts.[39] However, the production of organic molecules driven by the Scheme 6. Summary of prebiotic chemistry of pyrimidines in a urea/ice matrix initiated by the UV irradiation of acetylene. photodissociation of water at lower photon energies and higher pressures has been scarcely studied. The ultraviolet irradiation of water/ice at ing is consistent with the results observed under UV irradiaatmospheric pressure and at wavelengths of more than tion, in which triazines were absent, indicating that the for200 nm generates molecular oxygen, hydrogen peroxide, hymation of ammonia, isocyanate, and biuret from urea was droxyl radicals, and atomic hydrogen.[38, 40] At higher presnegligible. This fact also discards possible HCN formation [37] by UV irradiation of ammonia and acetylene. To test this sures, the energy necessary for the water photodissociation shows a redshift and, in the GPa range, the generation of possibility, a control experiment was performed with a soluhydroxyl radical from water/ice has been reported at 350 nm tion of ammonia subjected to freeze–thaw cycles and UV irirradiation.[41] In our conditions, the combination of higherradiation under acetylene. Analysis of the products did not reveal purines or pyrimidines but did contain 1H-imidazole, energy photons in the gas phase and the photoreactions 1H-imidazole-2-carboxaldehyde, and a small amount (< 1 % driven by lower energy photons absorbed in the condensed of total imidazoles) of hydantoin (Figure S11, the Supportphase, which could generate radical initiators from water ing Information). Other indications of the unavailability of and/or organic solutes, could explain the importance of the HCN from irradiation of urea/acetylene is the low yield of reactive-oxygen species in the system. One of these species adenine with respect to guanine and uric acid and the lack is molecular oxygen, which could be photochemically generof 2,4-diaminopyrimidine, a common product found in preated in water/ice, as well as being present as a trace in the biotic chemistry experiments involving the cyanide pathexperimental setup. Oxygen is an interesting initiator; way.[3a, 9] In addition, we did not detect any 4-aminoimidathrough the O2 dissociation by irradiation at 185 nm, followed by hydroxyl radical generation in presence of water zoles, such as 4-aminoimidazole-5-carboxamide (AICA), or ozone formation and subsequent photolysis at which are classic intermediates in the synthesis of purines by 254 nm.[16e, 42] It could be interesting from a prebiotic evoluHCN polymerization. Water also plays an essential role in the formation of photion point of view to establish how small proportions of motoproducts under UV irradiation. The atmospheric processlecular oxygen[43] influenced the photochemical production ing of acetylene in the presence of water in gas phase leads of relevant organic molecules. to the formation of reactive intermediates as glyoxal, which Although the reaction products studied in the water solucan enter the liquid/solid water pool readily due to its high tion were dominated by the reaction between acetylene and HenryÏs law constant. Once in the water pool, glyoxal could reactive-oxygen species, the photodissociation of acetylene react with urea or could be oxidized by hydroxyl radical has been evidenced by the presence of benzoic acid in the generated in the ice matrix to yield glyoxylic acid. This reaction products. Further work is necessary to study the alroute depends on the reactive-oxygen species generated by ternative routes initiated by acetylene-derived reactive spephotodissociation of water, which, moreover, are involved to cies, as ethynyl or vinylidene radicals. Independently of the explain the deuterated products found by using C2D2. Water first photochemical steps, our overall results show that the water/ice system irradiation with UVC constitutes a source photodissociation yielding the hydroxyl radical could take of reactive-oxygen species, evidenced by the generation of place in the gas phase, initiated by the 185 nm photons from 6494
www.chemeurj.org
Õ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 6488 – 6497
Prebiotic Synthesis of Nucleobases and Hydantoins
FULL PAPER Conclusion
Scheme 7. Reconstruction of the photochemistry of acetylene in the water/ice system under UVC irradiation, based on the products found in water/urea- and pure water experiments.
oxalic, glyoxylic and glycolic acids, which have been regarded as products of the hydroxyl radical-mediated photooxidation of glyoxal and acetic acid[44] (Scheme 7). Our results show that the glyoxal is a precursor of larger dicarboxylic acids, as succinic acid (Figure S7, the Supporting Information). An alternative route could be the recombination product of radicals generated by UVC irradiation of the organic acids previously formed.[45] The production of glyoxylic acid from glyoxal (or by the photooxidation of other acetylene-derived precursors, as acetic acid), in the presence of urea leads to the formation of 5-hydroxyhydantoin and pyrimidines in our experimental model. This result could be connected with the glyoxylate scenario proposed by Eschenmoser, wherein the glyoxylic acid is the starting point towards several molecules with prebiotic interest, including sugars.[46, 17c] The possible role of glyoxylic acid as a mimic of phosphate in a plausible RNA ancestor[47] and as starting point to a primordial metabolism, strengthens the prebiotic interest of the production of glyoxylic acid and nucleobases in the same experimental model. This paves the way to further research in the glyoxylate/ice scenario. The photochemical behavior of the nucleobases and hydantoin in the water/ice matrix, which has not been studied apart from experiments under astrophysical conditions, showed that the photoproducts found are comparable to those obtained in the biochemistry of bases and reveals pathways to form nucleobases that are compatible with the chemomimesis concept,[48] such as the synthesis of uric acid or uracil through dihydroorotic acid or iso-orotic acid. This new alternative mechanism minimizes the contribution of cytosine deamination to uracil synthesis and instead indicates a role for acetylene, a molecule thought to be accessible to planetary atmospheric chemistry and astrochemistry.
Chem. Eur. J. 2013, 19, 6488 – 6497
A cold environment (liquid water and ice) with cyclic freezing and thawing was exposed to an acetylene-rich atmosphere under anoxic conditions. Ultraviolet irradiation of the system led to the formation of reactive intermediates that enriched the water pool in organic acids including acetic, glyoxylic, glycolic, and succinic acids. If the water solution contained urea, UV irradiation led to the formation of nitrogen heterocycles such as hydantoin, pyrimidines, and purines, with 5-hydroxyhydantoin, hydantoin, uric acid, and uracil being the most abundant products. Analysis of the reaction products suggests that hydantoin was a product of the direct synthesis from acetylene-derived glyoxal and urea. 5-hydroxyhydantoin was a product of both the direct synthesis from glyoxylic acid and urea, and, to a lesser extent, from the photodegradation of purines and pyrimidines. The water/ice matrix played a dual role as a protective medium and a source of radicals for the photo-oxidation of purines and pyrimidines. The results further suggest that the synthesis of the nucleobases proceeded through a HCN- or nitrile-independent pathway. Also, the formation of cytosine and uracil could run by independent pathways, being negligible the deamination of cytosine in our experimental conditions. The availability of acetylene and urea in a prebiotic world and the yields of hydantoins recorded here could explain the origin of these molecules in prebiotic and astrochemistry laboratory simulations. Our results also contribute to the understanding of chemical evolution in cold, water-rich planetary environments.
Experimental Section Urea solution freeze–thaw cycles under an UV-irradiated acetylene atmosphere: Urea (0.1 m, 50 mL; Fluka Biochemika, Sigma Aldrich GmbH, Germany) in ultrapure, degassed water was frozen at ¢5 C in a sealed and thermostated glass reactor (Figure S1, the Supporting Information) under acetylene at atmospheric pressure. The acetylene was generated by reaction of calcium carbide (Panreac SA, Spain) with ultrapure water. The acetylene generated contained significant quantities of vinyl acetylene, divinylsulfide, and acetic acid, as well as traces of phosphine, sulfur dioxide, and other unsaturated hydrocarbons. The acetylene stream was purified by passing through a glass column filled with granular silica gel impregnated with 60 % phosphoric acid and heated to 175 C in a custom-made cylindrical furnace. Next, acetylene was deoxygenated and acetic and phosphoric acids removed by bubbling in a solution of pyrogallol in KOH (1 m). The purity of acetylene in the reactor was assessed by the injection of the gas (50 mL) into a PLOT-Q column mounted on a Perkin–Elmer Autosystem XL-Turbomass Gold quadrupole GC/MS system in scan EI mode in the range 20–200 amu. The analysis showed pure acetylene with traces of vinylacetylene and acetic acid. Once the reactor was filled with purified acetylene (approximately 2.3 g or 0.09 mol of gas), freeze–thaw cycles were established by varying the temperature between ¢21 and 5 C (two hours at ¢21 C, ramping to 5 C at 0.1 C min¢1 and two hours at 5 C) by using a Haake Phoenix II programmable cryostat (Thermo Electron Corporation). The acetylene atmosphere was irradiated with UV radiation by using a Spectroline 11SC1 Hg vapor discharge lamp. This lamp emits photons corresponding to Hg emission lines with a main wavelength centered at (253.7 10) nm
Õ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6495
C. Menor-Salv n and M. R. Mar n-Yaseli
with an estimated flux of 5.8 × 1014 photons cm¢2 s¢1 from 1“ of the lamp surface and a wavelength centered at (185 5) nm that constitutes approximately 5 % of the UVC emission. The irradiance spectrum of the lamp shows small peaks in the near-UV and visible regions. The irradiation was performed for 72 h. After this period, the lamp was disconnected and the reactor was kept sealed, which then underwent active freeze– thaw cycles for three weeks. The system was then allowed to warm to room temperature and the yellowish and slightly acidic (mean pH 3.5) aqueous solution was stored in sealed headspace vials under an inert atmosphere and frozen in liquid nitrogen. The frozen samples were freezedried and stored at ¢20 C until analysis. For the control experiments using an inert atmosphere, argon of the maximum purity available (99.9995 %, Praxair SA, Spain) was used. Deuterated-acetylene experiments: To explore the mechanism involved in our experiments, identical conditions were employed, but C2D2 was used instead of acetylene. Deuterated acetylene was generated by reaction of calcium carbide with deuterium oxide (containing 99 % deuterium; Aldrich Chemical Company, Inc. WI, USA). Ion-exchange separation: The experiment products contained an excess of urea, which could interfere with uracil determination by GC-MS. To remove excess urea, the dried sample was dissolved in HCl (10 mL, 0.1 m) and stirred for 1 hour with Dowex 50W × 8–400 cation exchange resin in the H form. After stirring, the solution containing pure urea was removed and the resin, suspended in water, was transferred to a glass column. The solution was eluted and column washed sequentially with water, NH3 (0.1 m), and HCl (0.1 m) until the effluent was slightly acidic. The eluted fractions were combined and freeze-dried prior to GCMS analysis. Gas chromatography/mass spectrometry: The solid, freeze-dried samples were derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) with 1 % trimethylchlorosilane (TMCS, provided by Pierce, Rockford, IL, USA). Briefly, the dried sample (1 mg) was combined with BSTFA TMCS (0.1 mL) in a dry glass vial, stirred and heated at 60 C for 3 h. After that, the sample (1 mL) was transferred to an Agilent GC 6850A gas chromatograph in the splitless mode with the injection port at 290 C. The analysis was performed using an HP-5 MS (Agilent), 5 % phenyl-95 % methylsiloxane capillary column (30 m × 0.25 mm i.d., 0.25 mm film). Helium was used as the carrier gas at a flow rate of 1.1 mL min¢1. The oven temperature, initially 40 C for 1.5 min, was ramped to 130 C at a rate of 5 C min¢1. Then, the temperature was ramped to 180 C at 10 C min¢1 and held for 10 min. Next, the temperature was raised to 220 C at 20 C min¢1 and held for 15 min. In the last step, the temperature was raised to 300 C at 10 C min¢1 and held for 18 min. Mass spectrometric analysis was performed using an Agilent 5975C VL MSD quadrupole in EI mode with an ionization energy of 70 eV. The ion source temperature was set at 230 C and the quadrupole at 290 C. Identification of compounds was performed in scan mode with a range of 45–650 amu. The determination of cytosine and related compounds was performed by using scan fragmentograms and selected ion monitoring (SIM) mode at 254 and 257 amu. When available, the identified compounds were confirmed against authentic standard (provided by Sigma–Aldrich) mass spectra and retention times. Controls were performed by using mixtures of standards to identify possible derivatization artifacts and to avoid ambiguities. Other organic compounds were identified by searching their mass spectra in the NIST database, or comparison of published mass spectra data and mass spectrum interpretation. For identification purposes, we considered only peaks with a signal-to-noise ratio over 20. Those peaks whose match probability in the database were below 90 % and/or tentatively or ambiguously identified were considered unidentified and not discussed in this paper.
6496
www.chemeurj.org
Acknowledgements This work was supported by grant AYA2009–13920-C02–01 from the Ministerio de Ciencia e Innovaciýn (Spain) and the Instituto Nacional de T¦cnica Aerospacial “Esteban Terradas”.
[1] J. P. Dworkin, A. Lazcano, S. L. Miller, J. Theor. Biol. 2003, 222, 127 – 134. [2] a) J. L. Bada, Earth Planet. Sci. Lett. 2004, 226, 1 – 15; b) C. Anastasi, F. F. Buchet, M. A. Crow, A. L. Parkes, M. W. Powner, J. M. Smith, J. D. Sutherland, Chem. Biodiversity 2007, 4, 721 – 739. [3] a) M. P. Callahan, K. E. Smith, H. J. Cleaves, J. Ruzicka, J. C. Stern, D. P. Glavin, C. H. House, J. P. Dworkin, Proc. Natl. Acad. Sci. USA 2011, 108, 13995 – 13998; b) Z. Martins, O. Botta, M. L. Fogel, M. A. Sephton, D. P. Glavin, J. S. Watson, J. P. Dworkin, A. W. Schwartz, P. Ehrenfreund, Earth Planet. Sci. Lett. 2008, 270, 130 – 136; c) S. A. Sandford, M. P. Bernstein, L. A. Allamandola, Astrophys. J. 2004, 607, 346 – 360. [4] a) J. Orý, A. P. Kimball, Arch. Biochem. Biophys. 1961, 94, 217 – 225; b) J. Orý, A. P. Kimball, Arch. Biochem. Biophys. 1962, 96, 293 – 313. [5] a) A. W. Schwartz, H. Joosten, A. B. Voet, BioSystems 1982, 15, 191 – 193. [6] a) K. E. Nelson, M. P. Robertson, M. Levy, S. L. Miller, Orig. Life Evol Biosph. 2001, 31, 221 – 229; b) A. W. Schwartz, G.JF. Chittenden, Biosystems 1977, 9, 87 – 92. [7] a) R. Saladino, M. Barontini, C. Cossetti, E. Di Mauro, C. Crestini, Orig. Life Evol. Biosph. 2011, 41, 317 – 330; b) R. Saladino, C. Crestini, F. Ciciriello, G. Constanzo, E. Di Mauro, Chem. Biodiversity 2007, 4, 694 – 720. [8] a) A. Eschenmoser, Angew. Chem. 2011, 123, 12618 – 12681; Angew. Chem. Int. Ed. 2011, 50, 12412 – 12472; b) C. Menor-Salv n, M. R. Mar n-Yaseli, Chem. Soc. Rev. 2012, 41, 5404 – 5415 and references therein. [9] C. Menor-Salv n, M. Ruiz-Bermejo, M. I. Guzm n, S. Osuna-Esteban, S. Veintemillas-Verdaguer, Chem. Eur. J. 2009, 15, 4411 – 4418. [10] L. E. Orgel, Orig. Life Evol. Biosph. 2004, 34, 361 – 369. [11] M. Ruiz-Bermejo, C. Menor-Salv n, J. L. de La Fuente, E. MateoMart , S. Osuna-Esteban, J. A. Mart n-Gago, S. Veintemillas-Verdaguer, Icarus 2009, 204, 672 – 680. [12] A. Bar-Nun, Icarus 1979, 38, 180 – 191. [13] a) W. Zheng, D. Jewitt, R. I. Kaiser, J. Phys. Chem. A 2009, 113, 11174 – 11181; b) R. K. Khanna, Icarus 2005, 178, 165 – 170; c) D. W. Clarke, J. P. Ferris, Icarus 1997, 127, 158 – 172. [14] C. Tornow, E. Kîhrt, U. Motschmann, Astrobiology 2005, 5, 632 – 650. [15] a) C. Menor-Salv n, M. Ruiz-Bermejo, S. Osuna-Esteban, G. MuÇoz-Caro, S. Veintemillas-Verdaguer, Chem. Biodiversity 2008, 5, 2729 – 2739; b) F. Cataldo, J. Photochem. Photobiol. A 1996, 99, 75 – 81. [16] a) D. Cremer, R. Crehuet, J. Anglada, J. Am. Chem. Soc. 2001, 123, 6127 – 6141; b) S. Hatakeyama, N. Washida, H. Akimoto, J. Phys. Chem. 1986, 90, 173 – 178; c) D. J. Peeble, S. Das, C. von Sonntag, J. Phys. Chem. 1985, 89, 57 – 84; d) M. Nuevo, S. N. Milam, S. A. Sandford, Astrobiology 2012, 12, 295 – 314; e) A. G. Carlton, B. J. Turpin, K. E. Altieri, S. Seitzinger, A. Reff, H. J. Lim, B. Ervens, Atmos. Environ. 2007, 41, 7588 – 7602. [17] a) P. D. de Marcellus, Astrobiology 2011, 11, 847 – 854; b) A. Eschenmoser, Chem. Biodiversity 2007, 4, 554 – 573; c) E. Ware, Chem. Rev. 1950, 46, 403 – 470. [18] a) J. L. Little, J. Chromatogr. A 1999, 844, 1 – 22; b) T. G. England, A. Jenner, O. I. Aruoma, B. Halliwell, Free Rad. Res. 1998, 29, 321 – 330. [19] C. Vialas, C. Claparols, G. Pratviel, B. Meunier, J. Am. Chem. Soc. 2000, 122, 2157 – 2167. [20] a) K. J. Volk, R. A. Yost, A. Brajler-Toth, Anal. Chem. 1989, 61, 1709 – 1717; b) K. Kahn, P. Serfoto, P. A. Tipton, J. Am. Chem. Soc. 1997, 119, 5435 – 5442; c) S. K. Tyagi, G. Dryhurst, J. Electroanal.
Õ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 6488 – 6497
Prebiotic Synthesis of Nucleobases and Hydantoins
[21] [22]
[23]
[24]
[25] [26]
[27] [28]
[29] [30] [31] [32]
Chem. 1987, 216, 137 – 156; d) H. Kaur, B. Hallywell, Chem-Biol. Interactions 1990, 73, 235. R. Saladino, G. Botta, S. Pino, G. Constanzo, E. Di Mauro, Chem. Soc. Rev. 2012, 41, 5526 – 5565. a) J. Hong, D. G. Kim, C. Cheong, K. J. Paeng, Microchem. J. 2001, 68, 173 – 182; b) T. J. Yu, R. G. Sutherland, R. E. Verrall, Can. J. Chem. 1980, 58, 1909 – 1915. a) J. R. Wagner, J. Cadet, Acc. Chem. Res. 2010, 43, 564 – 571; b) J. RiviÀre, F. Bergeron, S. Tremblay, D. Gasparutto, J. Cadet, J. Am. Chem. Soc. 2004, 126, 6548 – 6549; c) E. Gilbert, S. HoffmannGlewe, Water Res. 1992, 26, 1533 – 1540. a) R. Volkamer, P. J. Ziemann, M. J. Molina, Atmos. Chem. Phys. 2009, 9, 1907 – 1928; b) F. Wittrock, A. Richter, H. Oetjen, J. P. Burrows, M. Kanakidou, S. Myriokefalitakis, R. Volkamer, S. Beirle, U. Platt, T. Wagner, Geophys. Res. Lett. 2006, 33, L 16804. G. Baccolini, C. Boga, C. Delpivo, G. Micheletti, Tetrahedron Lett. 2011, 52, 1713 – 1717. M. Redrejo-Rodr guez, C. Saint-Pierre, S. Couve, A. Mazouzi, A. A. Ishchenko, D. Gasparutto, M. Saparbaev, PLoS ONE 2011, 6, e21039. D. Davidson, O. Baudisch, J. Am. Chem. Soc. 1926, 48, 2379 – 2383. Y. Yamagata, K. Sasaki, O. Takaoka, S. Sano, K. Inomata, K. Kanemitsu, Y. Inoue, I. Matsumoto, Orig. Life Evol. Biosph. 1990, 20, 389 – 399. M. Terasaki, S. Nomoto, H. Mita, A. Shimoyama, Orig. Life Evol. Biosph. 2002, 32, 91 – 98. a) A. Lazcano, Orig. Life Evol. Biosph. 2010, 40, 161 – 167; b) J. A. Ferris, P. C. Joshi, J. Org. Chem. 1979, 44, 2133 – 2137. a) G. J. F. Chittenden, A. W. Schwartz, Nature 1976, 263, 350 – 351; b) A. J. Varghese, Biochemistry 1971, 10, 4283 – 4290. I. M. Lagoja, Chem. Biodiversity 2005, 2, 1 – 50.
Chem. Eur. J. 2013, 19, 6488 – 6497
FULL PAPER [33] J. P. Ferris, Orig. Life Evol. Biosph. 1992, 22, 109 – 134. [34] R. F. Cline, R. M. Fink, K. Fink, J. Am. Chem. Soc. 1959, 81, 2521 – 2527. [35] a) R. Navarro-Gonz lez, A. Negrýn-Mendoza, E. Chacýn, Orig. Life Evol. Biosph. 1989, 19, 109 – 118. [36] J. A. Kaye, D. F. Strobel, Icarus 1983, 54, 417 – 433. [37] a) P. P. Bera, M. Nuevo, S. N. Milam, S. A. Sandford, T. J. Lee, J. Chem. Phys. 2010, 133, 104303; b) T. Wang, J. H. Bowie, Org. Biomol. Chem. 2012, 10, 652 – 662. [38] A. Yabushita, T. Hama, M. Kawasaki, J. Photochem. Photobiol C: Photochem. Rev. 2013, DOI:10.1016/j.jphotochemrev.2013.01.001. [39] C. Y. Robert Wu, D. L. Judge, B. M. Cheng, W. H. Shih, T. S. Yih, W. H. Ip, Icarus 2002, 156, 456 – 473. [40] a) P. Kl n, I. Holoubeck, Chemosphere 2002, 46, 1201 – 1210; b) C. C. Wang, L. I. Davis Jr, J. Chem. Phys. 1975, 62, 53 – 55. [41] M. Ceppatelli, R. Bini, V. Schettino, Proc. Natl. Acad. Sci. USA 2009, 106, 11454 – 11459. [42] M. I. Stefan, J. R. Bolton, Environ. Sci. Technol. 1999, 33, 870 – 873. [43] J. Kasting, Nat. Geosci. 2013, 6, 9 – 10. [44] D. H. Parker, Acc. Chem. Res. 2000, 33, 563 – 571. [45] K. E. Altieri, S. P. Seitzinger, A. G. Carlton, B. J. Turpin, G. C. Klein, A. G. Marshall, Atmos. Environ. 2008, 42, 1476 – 1490. [46] V. N. Sagi, P. Venkateshwarlu, F. Hu, G. Meher, R. Krishnamurthy, J. Am. Chem. Soc. 2012, 134, 3577 – 3589. [47] H. D. Bean, F. A. L. Anet, I. R. Gould, N. V. Hud, Orig. Life Evol. Biosph. 2006, 36, 39 – 63. [48] J. Peretý, Chem. Soc. Rev. 2012, 41, 5394 – 5403.
Õ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: December 4, 2012 Revised: February 15, 2013 Published online: March 27, 2013
www.chemeurj.org
6497