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ARTICLE TYPEPrebiotic chemistry in eutectic solutions at the water-ice matrixa

César Menor-Salván*b and Margarita R. Marín-Yaseli

Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XXDOI: 10.1039/b000000x

A crystalline ice matrix at subzero temperatures can maintain a liquid phase where organic solutes and salts concentrates to form eutectic solutions. This concentration effect converts the confined reactant solutions in the ice matrix, sometimes making condensation and polymerisation reactions occur more favourably. These reactions occur at significant high rates from a prebiotic chemistry standpoint, and the labile products can be preserved from degradation. The experimental study of the synthesis of nitrogen heterocycles at the ice-water system showed the efficiency of this scenario and could explain the origin of nucleobases in the inner Solar System bodies, including meteorites and extra-terrestrial ices, and on the early Earth. The same conditions can also favour the condensation of monomers to form ribonucleic acid and peptides. Together with the synthesis of these monomers, the ice world (i.e., the chemical evolution in the range between freezing point of water and the limit of stability of liquid brines, ≈273 to 210 K) is an under-explored experimental model in prebiotic chemistry.

IntroductionLife as we know it depends on interfacial redox and transport processes between liquid water and a system of lipid membranes with the associated protein machinery. It seems logical to assume that life emerged from liquid water solutions where relatively simple raw materials were synthesised or accumulated. These solutions could be subjected to water-mineral matrix interfacial chemistry or concentration and compartmentalisation process, which ultimately leads to the emergence of life in a complexity increasing process. Consequently, to determine the possible compositions of the raw materials for the plausible first steps of abiotic evolution, pioneering experiments on prebiotic chemistry have been conducted in water-saturated atmospheres and liquid solutions1, which are largely supported by a reductive atmosphere model.

The criticisms regarding an efficient atmospheric-liquid water origin for the organic components of the first biochemical processes on Earth arise from the lack of a universally accepted geochemical model for the Archean atmosphere. Additionally, the classic prebiotic chemistry approach deals with the problem of the concentration and stability in liquid water of the plausible prebiotic reactants. These criticisms and the lack of experimental evidence supporting a model for the origin of biochemical pathways have led to two main schools of thought:

The first concept is the possibility of an in situ origin on Earth, which focus on either water-mineral interfacial processes as a way for concentration and compartmentalisation of environmentally synthesised reactants2 or on the origin of chemoautotrophic pre-biochemical systems3. The second concept argues that amino acids, nitrogen heterocycles and simple organic molecules and monomers could be synthesised by irradiation at very low temperatures in extra-

terrestrial ice layers composed of water and other condensates4. Ice is the most abundant form of water beyond the asteroid belt5. The chemistry of ices at low temperatures followed by the delivery of the organic molecules on Earth by comets, meteorites and dust particles could have been an important source of organics on the prebiotic Earth and could have played a key role in early chemical evolution. The photochemistry and radiochemistry of outer solar system bodies and interstellar ices has received substantial attention6.

Despite the research into the photochemical transformations in ice from an astrochemical point of view, the study of the chemistry in the range of stability of the ice-water interface has not received much attention. This may be due to the scarcity of the defined conditions in the Solar System during the epoch of active prebiotic chemistry or the difficulties for demonstrating that these cold conditions existed in Hadean Earth.

The evidence for a liquid water subsurface ocean on Saturn’s moon Europa7 and the possible presence of water-ammonia eutectic brines or even a subsurface ocean in other outer giant planet satellites such as Titan8 or Enceladus9 rekindled the interest in liquid water prebiotic chemistry. Moreover, the subsequent proposed steps for the emergence of cellular life have a limited temperature range, and a hot prebiotic Earth was regarded to be an unlikely environment for the origin of life by some authors10. Miller and Orgel stated in 1974 that the emergence of biological organisation could only occur at temperatures below the melting point of the polynucleotide structure. After observing the instability of organic compounds in the prebiotic stages, these authors concluded that a temperature of 273 K would have been beneficial and that temperatures near the eutectic point of NaCl solutions (251.3 K) would have been even better11.

The low temperatures in planetary surface ices could be more conductive to the origin and the preservation of molecules that

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could be relevant for the emergence of life. In 1994, in one of the first explorations of the idea of an ice world-based origin of the life raw materials, Bada et al.12 suggested that ice formations on early Earth could have preserved organic compounds against hydrolysis or photochemical degradation. Under plausible planetary conditions, the presence of liquid water at T<273 K within an ice matrix creates a potential reactor where the synthesis or polymerisation of molecules of biological interest could occur. Herein, we will review our current knowledge of the chemical models that simulate possible prebiotic synthetic pathways in liquid water interfacial ice. The experimental approaches developed in the literature are primarily focused on the RNA-world hypothesis of an abiotic origin of nucleic acids, as these studies provide experimental evidence for the abiotic synthesis and polymerisation of nitrogen heterocycles and nucleotides. Apart from the molecular evolutionary perspective for the emergence of life, exploring the chemistry in liquid inclusions confined in an ice matrix could explain and predict the composition of objects in the inner Solar System and icy planetary bodies.

The ice/ liquid water system and its presence on the early Earth and in the Solar SystemThe ice-liquid water system has not received much attention in the literature, including the chemical physics and astrochemical/astrobiological literature. In the latter case, the experimental efforts are focused on low temperature condensates, where there is no evidence of a liquid interface and the ice is in its amorphous crystalline state. In the inner Solar System, including on Earth, ice occurs naturally in the crystalline form with two primary polymorphs, which are cubic and hexagonal. The crystallisation of water under current Earth surface conditions results in hexagonal ice Ih. The ice formed from liquid or heated from amorphous ice at temperatures between 100 and 130K is crystalline, with a diamond-type cubic structure Ic

13. Cubic ice is metastable at T<70 K and undergoes a transformation to the amorphous state (the stable form at these temperatures) via cosmic ray bombardment and ultraviolet irradiation14. The irradiation diminishes the kinetic barrier between the metastable cubic ice form and stable amorphous ice form at lower temperatures15. The crystallisation of ice Ih leads to the formation of various interfaces, such as ice-ice, ice-atmosphere and water-ice, as well as water-ice-mineral, which results from crystallisation of solutes by ice matrix exclusion or the presence of suspended mineral grains16. The ice-ice and ice-atmosphere interfaces are not a distinct transition. Nuclear magnetic resonance studies of ice crystals indicate the existence of a liquid transition between the crystals or between the ice and the atmosphere. The thickness of this liquid phase becomes monomolecular at T< 243 K and is thickened by dissolved solutes excluded from the ice matrix to the interface during crystallisation16.

The unexpected presence of crystalline ice in the Quaoar object at the Kuiper Belt, on Enceladus and its suggested presence in Titan17 imply that the evolution of ices are subject to occasional heating events. If crystalline ice and if even fluid water solutions are unambiguously present, the conditions for the increase in organic complexity from reactions between precursors

such as cyanide or cyanoacetylene may exist. The young and active surface of the Jovian moon Europa suggests the possibility of a subsurface water ocean from the observations of the Voyager mission and strengthened by the observations with the Galileo spacecraft18. Recently, it has been stated that Europa possesses and active dynamic ice-water system with cycles of melting and refreezing. In addition, a lenticular body of liquid brine in the Thera Macula region of approximately 20.000-60.000 km3 has been predicted19. The composition of Europa’s subsurface water, underlying an ice crust, could be rich in sulphate salts, the source of surface evaporite deposits20. The details on water composition and temperature are unknown, but estimations suggest a Mg-SO4-Na(K) rich water with temperatures in the range 210-270 K21. A model for the formation of liquid ammonia-water pockets that cause episodic cryomagmatism and a subsurface eutectic water-ammonia solution has been proposed for the Saturn moon Titan22. Within this context, both Titan and Europa constitute important astrobiological targets for direct exploration and laboratory simulations to predict the chemistry that will be found and to test our experimental prebiotic chemistry models23. A complex prebiotic chemistry has been predicted for Titan that includes the formation of nucleobases24 and the possibility of a methane-acetylene based chemical or biochemical evolution25. From these hypotheses based on atmospheric or surface chemistry, the prebiotic possibilities of liquid water brines entrapped under ice has received less attention and is the object of speculative discussion regarding possible biochemical evolution and the presence of chemoautotrophic life26.

Some models suggest a Hadean terrestrial atmosphere composed primarily of high pressure carbon dioxide. If liquid water were present in oceans over a basaltic crust, a CO2

atmosphere would be unstable and could be depleted as carbonates in a period of approximately 10 million years due to hydrothermal circulation and reaction of the CO2 with the crustal rock. Under these conditions, together with the Hadean faint Sun, the model developed by Sleep and Zahnle27 agrees with the ideas suggested by J. Bada in 199412, predicting ice-covered oceans and an average surface temperature of approximately 220 K, with freeze-thaw episodes motivated by occasional warming provoked by high energy impacts. These cold conditions would be prevented if a methane-rich atmosphere were present during the Hadean, as methane is a potent greenhouse gas. Evidence thus far does not support an atmosphere with a high enough concentration of methane to avoid freezing of the ocean surface. This model would be amenable for the development of prebiotic chemistry in an ice matrix based on HCN, cyanoacetylene, acetylene, urea or cyanate precursors synthesised on Earth or brought in via extraterrestrial input28.

The freezing of ocean water is a complex process. Modern sea water begins to freeze at 271.2 K and crystals of pure ice (Ih) begin to grow, surrounded by liquid brine with sodium chloride concentrations up to 25%. The liquid solution is concentrated within the ice structure in channels, which have been observed in stained samples under the microscope, with diameters ranging from 10 to 100 µm29. Based on observations of microscopic ice layers, it is estimated that 1 m3 of sea ice has a network of channels with a combined surface area of 105 to 106 m2. The volume of ice occupied by the brine channels and the brine

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ARTICLE TYPEconditions within the channels are directly proportional to the temperature; at 267 K, the brine salinity in sea ice is 100 (on the practical salinity scale, i.e., dimensionless units that are equivalent to the ratio between the sample solution and a standard KCl solution; normal ocean water has a salinity range of 30-35); at 263 K, the salinity rises to 145, and at 252 K, the salinity reaches a maximum of 21630. In sea ice, the presence of interstitial channels filled with liquid water and concentrated solutes has been observed over a range of temperatures down to 243 K. Sea ice can lead to the formation of solid mineral phases from the crystallisation of dissolved salts. During freezing or thawing events, the temperature gradients and density changes in the ice matrix lead to pressure gradients and motion of the trapped liquid water that fills the channels and pores. The freezing process led to the formation of potential gradients, with pH variations of up to 3 units31.

The boundary between liquid and solid water has a different refractive index and reveals an interface. Measurements of the zeta potential (electric potential difference between the fluid brine and the stationary liquid layer attached to the ice crystals) showed that the interfacial properties of an ice-water system are comparable to the interface with hydrophobic and nonionogenic solids, such as diamond or hydrocarbons32. These properties could be essential for the solute exclusion from the interstitial brines in ice and the formulation of a freeze-concentration model for explaining the prebiotic chemistry observed in the ice matrix. Observation of the behaviour of stains in ice shows that organic molecules are excluded from the ice matrix and concentrated in the interstitial brine, where chromatographic separation has been noted. Another important property of the behaviour of organic molecules in ice is that a dilute starting solution of a given solute always reaches the same molal concentration in the interstitial solution, which is determined by the final incubation temperature33. For example, a freezing dilute urea solution tends to form an interstitial eutectic 8 m solution with a melting point of 261K. These properties of the ice-water interface convert the ocean ices, at temperatures within the range of existence of the interface with liquid brines, into a potential reactor for the first steps responsible for the emergence of life.

Prebiotic synthesis of nucleobases and other nitrogen heterocycles in the ice matrixNucleobases are a small group of one-ring (pyrimidines) and two-ring (purines) nitrogen heterocycles that, together with sugars and phosphate, compose nucleic acids. The pyrimidines include uracil, thymine and cytosine and purines include adenine and guanine. Other heterocycles belonging to both groups are important intermediates in the biochemistry, including xanthine, hypoxanthine and orotic acid. It is generally assumed that the earliest living forms on Earth used a genetic code based on nucleobases34. In addition, nitrogen heterocycles could have been involved in the first metabolic pathways as cofactors35.

Regardless of the controversy regarding whether life began with a replicator, as suggested by the RNA-world hypothesis, or with metabolism, as suggested by later authors36, there is no evidence to discard the hypothesis of a prebiotic source of nucleobases or cofactors for the first living system. The first logical hypothesis considers that the prebiotic synthesis took place on Earth, although it is not clear if the environmental conditions were consistent with efficient in situ synthesis37. The second logical hypothesis is the delivery of nitrogen heterocycles to Earth by comets, meteorites and dust particles. This extra-terrestrial delivery could compensate for a possible lack of availability from in situ synthesis. Analysis of carbonaceous chondrites, a class of meteorites rich in organic carbon and water38, has demonstrated the presence of N-heterocycles. These heterocycles include adenine, guanine and triazines (ammeline and melamine), which were found in the Orgeil meteorite by Hayatsu in 196439. Subsequent analyses performed from 1965-197540 show that the extraction conditions and sample treatments determine the analytical results. However, the presence of nucleobases in carbonaceous chondrites is widely accepted. In 2008, Martins et al. demonstrated41 the extra-terrestrial origin of xanthine and adenine in a Murchinson meteorite sample using carbon isotope measurements. Recently, Callahan et al. demonstrated that the suite of purines found in carbonaceous chondrites is consistent with those obtained using ammonium cyanide chemistry42. The questions that arise from these results include how were the nitrogen heterocycles synthesised on Earth or other bodies in Solar System, and how could the ice-water interface play a role in this process?

Synthesis based on hydrogen cyanide

The synthesis of nucleobases and other nitrogen heterocycles in the parent body of a meteorite could be a process that is dependent on the water content and irradiation of precursors. The seminal work of Juan Oró and co-workers demonstrated that adenine can be easily synthesised from hydrogen cyanide (Scheme 1)43. A prebiotic origin for the nucleobases was thereafter regarded as a realistic possibility44. Additionally, a Fischer-Tropsch type synthetic mechanism catalysed by mineral phases at high temperature has been suggested for the origin of N-heterocycles in meteorites45, but its actual significance is unclear46 and currently is not a widely accepted route.

Cyanide is the primary precursor involved in our current models for prebiotic synthesis of nitrogen heterocycles and a possible precursor to the organic molecules that gave rise to biochemistry. Cyanide could be generated photochemically or by spark discharges in methane/nitrogen planetary atmospheres47. In addition, free HCN and cyanide polymers have been observed in comets, dust particles48 and the Titan atmosphere49. The mechanism of synthesis of adenine from HCN implies that the first step is polymerisation to the HCN-tetramer diaminomaleonitrile (DAMN; Scheme 1). This intermediate

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could undergo further polymerisation to form dark brown solid polymers, which upon hydrolysis release nitrogen heterocycles, including adenine50. This hydrolysis could take place in the ice-water interface in the parent body of comets or meteorites during their journey in the inner Solar System or after these objects impacted the Earth. Another possible mechanism is the reaction of DAMN with formamidine51 to afford a 4-amino-5-cyanoimidazole (AICN) intermediate. This reaction yields adenine through the coupling of HCN or formamidine. The hydrolysis of AICN leads to 4-aminoimidazole-5-carboxamide (AICA), which could be a xanthine and hypoxanthine precursor52 (Scheme 1). Formamidine has also been identified as an organic precursor found in comets53 and prebiotic chemistry laboratory simulations54. A possible major mechanism for the formation of adenine from HCN, which was elucidated by Voet and Schwartz in 1982, is the reaction between the HCN tetramer and its cyanoimino tautomer or diiminosuccinonitrile (an oxidation product of HCN tetramer) to yield the carbamimidoyl cyanide derivative. This molecule cyclises to 4-amino-2-cyanoimidazole-5-carbimidoylcyanide. Further addition of the cyanoimino derivative and ring closure, affords adenine-8-carboxamide (Scheme 1)55. This product is quantitatively converted to adenine by hydrolysis. The above mechanism was supported by the structural elucidation of 4-amino-2-cyanoimidazole-5-carboxamide and its hydrolysis product, 4-aminoimidazole-2,5-dicarboxamide. However, the adenine-8-carboxamide has not yet been identified in HCN oligomerisation experiments.

The last proposed mechanism is the UV-induced photo-isomerisation of the HCN tetramer to 4-amino-5-cyanoimidazole. The reaction of this imidazole with HCN or with its hydrolysis product ammonium formate in a melt directly yields adenine56. Because it is the key reaction in the pathway, the formation of the HCN tetramer requires a high HCN concentration to avoid the volatilisation or hydrolysis to ammonium formate, which competes with the formation of diaminomaleonitrile in dilute solutions. Therefore, it would have been impossible to reach sufficiently high HCN concentrations in the open oceans or by water evaporation57.

One solution to this problem could be to consider alternatives to aqueous HCN chemistry. The formation of nucleobases from formamide in the presence of inorganic catalysts at high temperature creates a robust pathway for adenine, hypoxanthine, uracil and cytosine among other N-heterocycles 58. One solution to this problem could be concentrating HCN using the liquid-ice interfacial properties. During the first attempt to test this possibility, Sanchez et al. (1966) showed that HCN concentrates in a frozen eutectic solution. The eutectic solution, which has a mole fraction of 70 to 80% in HCN, is formed at 249 K and deposits a dark HCN polymer59. Considering the activation energy of the HCN polymerisation and the rate constants, the formation of the HCN tetramer in eutectic fluids should be complete in a few years. At 173 K, the reaction occurs over the order of hundreds of millions of years60. The advantageously stable conditions in a water-ice interface could surpass the handicap of prebiotic synthesis at low temperatures and the problem of concentration and stability at high temperatures.

Additionally, the freezing of dilute glycolonitrile solutions, produced by addition of HCN and formaldehyde, produces

adenine in low yield (0.004%)61. In a long duration experiment, Miyakawa et al. maintained a frozen solution of ammonium cyanide at 195 K over 27 years and at the end of this time period, identified adenine as well as other purine and pyrimidine products62. Although the HCN pathway has been extensively studied for the synthesis of purines, it has been demonstrated that the polymerisation of cyanide could provide a pathway for the formation of the pyrimidines including uracil, 5-hydroxyuracil and orotic acid63. The freezing of cyanide solutions could also provide a source of amino acids. In 1972, another long-term experiment involved a solution of NH4CN prepared from HCN and NH3. These reagents were frozen and subjected to variable temperatures of 253K and 195K for 25 years. The analysis indicated the formation of glycine and small amounts of alanine and aspartic acid64. The mechanism for the cold synthesis of amino acids from HCN has not been elucidated, but may include the hydrolysis of HCN polymers65 and the hydrolysis of 2-aminoacetonitrile, which is formed during HCN tetramer evolution, to glycine (Scheme 1).

Prebiotic laboratory synthesis from frozen cyanide solutions could be a model for the prebiotic synthesis of nucleobases. This synthesis could also explain the chemistry observed in ice-covered objects within the inner solar system, such as asteroids and comets during their closest passage to the sun, and in objects with complex chemistry, including Titan or Enceladus. To efficiently serve both goals, more experimental work should be performed to elucidate the mechanisms involved in frozen HCN solution, to test if the classic pathway through cyanoimidazole derivatives is reproducible in the ice matrix scenario and to determine if alternative pathways should also be examined.

Synthesis based on cyanoacetylene/acetylene and the role of urea

Cyanoacetylene is the other primary precursor considered for the synthesis of nucleobases. Cyanoacetylene can be obtained in the laboratory from methane/nitrogen mixtures by spark discharges66 by irradiation with short-wave ultraviolet radiation at 185 and 254 nm67; the spectrum of this molecule has been observed in the interstellar medium68 and by the Voyager mission in Titan’s atmosphere69, where crystalline condensates of cyanoacetylene with acetylene may exist70.

The potential prebiotic relevance of cyanoacetylene in origin of life studies were pointed out by Ferris, Sanchez and Orgel in 1968. They observed that the reaction of cyanoacetylene with aqueous 1 M sodium cyanate or 1 M urea gave cytosine in up to 5% yield (Scheme 2)71. The prebiotic availability of cyanate could be explained by the hydrolysis of cyanogen and urea, which may also be present in cometary and interstellar ices72.

The mechanism of this reaction could be explained by cyanoacetaldehyde, generated by hydrolysis of cyanoacetylene. The Miller research demonstrated the eutectic concentration and reaction of cyanoacetaldehyde with urea in an ice matrix at 253K to give cytosine and uracil in 0.005% and 0.02% yields, respectively73. In the same report, cyanoacetaldehyde reacted with guanidine at 253K to give cytosine in 0.05% yield and uracil in 10.8% yield, as well as lesser amounts of isocytosine and 2,4-diaminopyrimidine after 2 months74. This reaction may proceed through the cyanoacetaldehyde dimer, 4-(hydroxymethylene)

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ARTICLE TYPEpentenedinitrile, easily formed by concentrating the cyanoacetaldehyde solutions (Scheme 2)72.

The basis of these experiments is the freezing of a urea or guanidine solution. This process provides a concentration mechanism because the crystalline ice excludes the solute and a eutectic is formed. At 262K, urea forms an 8 m eutectic solution in water. This effect could be significant from a prebiotic point of view, despite the slower reaction rates, as has been shown in recent experiments.

An unresolved issue with the cyanoacetylene pathway in the synthesis of nucleobases is its reactivity to nucleophiles75, which suggests a high number of competitive reactions that lead to the formation of amino- or hydroxyacrylonitriles and subsequent polymers or hydrolysis products; On the other hand, the prebiotic origin of cytosine was questioned, at least in the liquid water medium, because its spontaneous and rapid deamination to uracil76. In part, the reactions in the water-ice interface could overcome the problem of dilution and degradation associated with solutions in liquid water pools.

Although much time has elapsed since the first proposal in 1966 of a low temperature prebiotic environment for the origin of nucleobases, it was not until 2000 that the product of the classic approach of spark discharges in methane/nitrogen based atmosphere was subjected to eutectic freezing77 at 253K for 5 years. The frozen spark discharge product showed a more extensive mixture of amino acids and the presence of adenine, which was absent in the control experiment at room temperature. The first experimental simulation of prebiotic synthesis in ice-liquid water directly from nitrogen/methane atmosphere by spark discharges was performed in 200978. The sparking on a freezing dilute urea solution under a nitrogen/methane atmosphere leads to the formation of cytosine, uracil and 2,4,6-trihydroxypyrimidine (barbituric acid) as the main identified pyrimidines, in addition to adenine. The experiments showed that using the freeze-thaw conditions, the observed sequence of pyrimidine yield obtained was cytosine > uracil > 2,4-diaminopyrimidine > 2,4,6-trihydroxypyrimidine. The formation or pyrimidines by oxidative alteration of cytosine (UV irradiation, hydroxyl radical addition or other free radical mechanism and further oxidation to barbituric acid) could explain the results observed79. The formation of cytosine as the main pyrimidine suggests that the low temperature conditions could reduce the rate of deamination to uracil and favour subsequent chemical evolution steps, as suggested by Bada12.

The triazine series (cyanuric acid, ammelide, ammeline and melamine) are also obtained in high yields (Scheme 3). The formation of triazines appears to be dependent on the freezing of urea solution. The triazines are not biological compounds, but they could mimic nucleobases behaviour in nucleic acids and their potential prebiotic role has been discussed80. Their presence in meteorites remains contentious81.

The key factor appears to be the freezing process itself and not

the temperature of the final ice obtained, as the temperature was selected to be right below the freezing point of 0.1 M urea. In a liquid urea solution at room temperature, there is no evidence of nucleobases. Instead, the formation of hydantoins, nitriles and tholins (reddish-brown, insoluble, heteropolymeric or macromolecular materials formed by sparking or irradiation of simple carbon sources, as methane) is prevalent.

The behaviour of urea in the ice-water interface is the key factor because urea tends to form dimers or oligomers in a concentration-dependent manner82. Urea molecules in aqueous fluids tend to form hydrogen bonds with neighbouring water molecules at both the amino and the carbonyl groups83. Spectroscopic studies show that at urea concentrations higher than 1 M, the urea-urea molecular interactions are significant. The urea-urea molecular interaction with subsequent formation of dimers or clusters of urea molecules becomes dominant at eutectic concentration84. During freezing, the urea is segregated from pure ice to accumulate in supercooled microfluid inclusions of a supersaturated solution. This system is governed by dehydration and association of solute molecules85. Thus, the extent of urea dimerisation (18% in 0.1 M urea solution at standard temperature86) is expected to increase and to become quantitatively a few degrees below the onset of freezing. Consequently, we expect an apparently paradoxical similarity between the process observed in molten urea84 and urea clusters entrapped in an ice matrix when the latter are subjected to direct sparking or irradiation.

This behaviour could explain the sequence of products obtained (cyanuric acid > ammelide > ammeline > melamine), which is the same sequence observed when urea is heated above its melting point. The spark discharges into the ice, then, could thermally decompose urea clusters in ammonium cyanate. Further decomposition of ammonium cyanate leads cyanic acid. The cyanic acid reacts with urea to form the biuret and with the formed biuret to form cyanuric acid (a cyanic acid trimer), which is the main triazine observed84. Several routes to ammelide are possible: reaction of cyanuric acid and ammonia or cyanic acid and urea or biuret. The process, in which the decomposition products accelerate the formation of triazines, could explain the high concentration of cyanuric acid obtained in these conditions. Other parallel pathway is the formation of melamine by cyanamide polymerization. The melamine hydrolysis yield cyanuric acid (Scheme 3). These pathways and the same reaction sequence, with the same relative abundance of triazines, have been studied in molten urea87 at temperatures between 406 and 460K. In this case, an alternative route for forming purines could result from the condensation of amino acids and biuret, a reaction that occurs at high temperature88; however, this alternative still has not been studied in ice-water systems and could be an unlikely possibility because of the high activation energy of such condensations. We also cannot discard other alternative pathways parallel to the polymerisation of concentrated urea solutions. For

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example, the production of cyanic acid during atmospheric discharges or thermal alteration of tholins89 and the subsequent reaction in freezing urea solutions could be an alternative source of cyanuric acid. Additional laboratory studies are necessary for clarifying the mechanisms involved in the cold synthesis of triazines and purines in the ice matrix.

The effect of concentration of solutes in the ice matrix, together with the low availability of water vapour could explain the preferential synthesis of polycyclic aromatic hydrocarbons (PAHs) by sparking methane/nitrogen atmosphere over an ice matrix90. The model of PAH synthesis is interesting because it could confirm the theoretical synthesis of aromatics by acetylene insertion mechanisms proposed for the Titan’s atmosphere91. In laboratory experiments at sub-zero temperatures65, the acetylene addition mechanism could explain the preferential formation of aromatics and poly(triacetylene) polymers (Scheme 4) by two possible mechanisms. First, a single aromatic ring could be generated from acetylene and vinyl radical and PAH growth by H abstraction and acetylene addition (Berthelot synthesis, similar to PAHs formation in flames). The second mechanism involves polyyne growth. The presence of water ice induces oxidations leading to the formation of aromatic polar species as benzaldehyde or acetophenone. The reaction in ice, in contrast to the dry high temperature synthesis of PAHs, leads to hydroxyl-rich poly(triacetylene) based polymers. Overall, these ice-water laboratory experiments reveal the expected chemical species in surface or subsurface ices on solar system objects or extrasolar planetary bodies.

The activation of methane/nitrogen atmospheres by spark discharges could lead to various chemistries involving reactive intermediates, including HCN, cyanoacetylene and acetylene. The preference for the hydantoins in liquid urea solutions at room temperature versus pyrimidines in frozen solution experiments could be due to the acetylene formation and subsequent alteration by means of ozone and hydroxyl radicals at higher temperatures to form alpha-dicarbonyl compounds as glyoxal92. The reaction of glyoxal with urea in mild acidic conditions yields hydantoin93, whose further oxidation yields 5-hydroxyhydantoin and parabanic acid (Scheme 3). These three hydantoins are always found together in all the experiments reported in the literature. Its formation could be explained also as alteration products of uracil by hydroxyl and other free radicals generated in water solutions by photolysis or irradiation94,79. At lower temperatures, the degradation of pyrimidines to hydantoins and the oxidation of acetylene could be diminished, due to the lower availability of reactive oxygen species generated from the excitation of water. In consequence, hydantoins could be the final products of alteration of pyrimidines in prebiotic conditions subjected to UV-irradiation or other energetic processes. Regarding acetylene, the polymerisation could be the preferred reaction pathway, as shown by the formation of poly-triacetylene and aromatic hydrocarbons at the ice-water matrix previously described. In this environment, the HCN or cyanoacetylene pathways could dominate, as long with other alternative mechanisms, as the synthesis of uracil by reaction of urea with acetylene dicarboxylic acid95. This acid is the aqueous hydrolysis product of dicyanoacetylene, which is an exotic product of methane/nitrogen atmospheres observed in the Titan atmosphere96. The role of acetylene derivatives has not been

studied in the ice-water scenario, and further experiments are necessary to explore the possible alternative pathways related to acetylene in prebiotic synthesis in an ice matrix and to put it in context with the classic mechanisms involving cyanide and cyanoacetylene. The products identified in the simulations of methane/nitrogen atmospheres over the ice-water interface include dicarboxylic and hydroxycarboxylic acids, amino acids and pyrazines, suggesting an additional mechanism to those suggested above.

In summary, the advantages of an ice-water interface in prebiotic synthesis include the reduction in the formation of polymers and tholin with a preference for ring systems (nitrogen heterocycles or aromatic rings) by the effect of concentration of diluted reactants such as HCN, urea or cyanate. Combined with other rocks or minerals, the freezing of liquid water solutions could favour mineral surface-organic solute interactions97

The ice-water system in the origin of nucleic acidsThe ice matrix is an appropriate environment for the synthesis

of nitrogen heterocycles, as demonstrated by the synthesis of triazines and nucleobases in freezing urea solutions. Could the ice-water interface be a favourable environment for the assembly of the first biologically relevant informational polymers?

The success in the synthesis of nucleobases from a feedstock of active nitrogen species available prebiotically led to the establishment of a similar retrosynthetic analysis for RNA and to the search for prebiotically plausible syntheses of a primordial informational, self-replicating polymer. If the discovery of an abiotic pathway to the origin of the first nucleotides and the constitutional self-assembly of RNA is achieved, the RNA-world hypothesis (a term coined by Walter Gilbert in 1986)98, which proposes a molecular evolutionary step involving autocatalytic RNA molecules prior to the origin of protein synthesis and metabolic machinery, will be strengthened. The current state of prebiotic chemistry does not provide a complete model for an abiotic origin of RNA, and the first formulations of an RNA world have been re-evaluated99. However, some argue that it may be premature to conclude that the prebiotic RNA world is unlikely to be a step in the emergence of life100.

In this context, the ice-water interface has been evaluated thoroughly as a matrix for the polymerisation of highly activated nucleotides. The first demonstration of this possibility was performed by Gryaznov and Letsinger in 1993101. In their experiment, the coupling of an alpha-bromoacyl-activated oligonucleotide (bromoacetylamino-3’-desoxythimidine in the 3’-terminus) with another oligonucleotide with a phosphorothioate group in the 5’-terminus proceeded without a template in a frozen saline solution at 255 K in 5 days. The reaction was explained as a result of the high local concentration of reactants in the fluid cavities in the ice matrix.

The enhancing effect of the ice matrix in the formation of RNA oligomers was demonstrated by Kanavarioti et al. in a very remarkable experiment in which oligouridylates up to 22 bases long were synthesised by incubating a uridine 5´-monophosphorimidazolide solution at 255 K at a pH range between 6 and 8 in the presence of magnesium and lead cations102:

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The study of the ribonuclease A digestion products showed that the oligomers obtained are mainly linear and that 30% carry at least one 3´-5´ linkage. The fluorescence microscopy observation of an ice layer in the experimental conditions with acridine orange staining indicated that the organic solutes were concentrated in the eutectic lattice structure included in the ice matrix. The authors concluded that the formation of eutectic solutions of reactants in the ice matrix facilitated the oligomerisation. The polymerisation most likely occurs in the liquid concentrated solutions between the ice crystals, and not by the adsorption of reactants onto the ice surfaces, as previously suggested by Stribling and Miller103, who studied the template directed synthesis of poly(U) in diluted solutions concentrated by freezing close to the NaCl eutectic. The ice also has an effect on the metal catalysis. The reaction in the ice-water medium requires Pb2+ as a catalyst and not Mg2+. This phenomenon is different from reactions in solution, which require both magnesium and lead cations. A possible interpretation of this observation is that the molecular associations in an ice matrix tend to be more stable than the corresponding ones in solution. An open question that arises is the role of certain metal cations (for example lead) as prebiotic catalysts. The lead catalysis in the polymerisation of activated nucleotides could be related to the mechanism of leadzymes104 and suggests that metal ion catalysis is central in a hypothetical RNA world.

If pyrimidine and purine-activated nucleotides are used in the water-ice interface at 255 K during 38 days in the presence of Mg2+ and Pb2+, a mixed-sequence polynucleotide with approximately the same proportion of purine and pyrimidines residues is obtained105. Monnard and Szostak106 studied the template-directed RNA polymerisation in water-ice at 256.4 K, a temperature that permits the maintenance of a stable water-ice interface for long periods of time. They found that lead and magnesium ions catalyse the elongation of a RNA hairpin with a 5’-overhang as a template.

Similarly, the non-enzymatic synthesis of polyadenosine in a sea-ice matrix, directed by poly(U) was performed, using adenosine-5´-monophosphate (2-methyl) imidazolide as monomer. Temperature fluctuations established the freeze-partial thaw cycles during one year. The results show high molecular weight poly(A) formation, with chain lengths of as many as 420 residues107:

Scheme 6 (uncaptioned)

The freezing-concentration model could also govern the

conformational rearrangement pathway of the formed polymers. Freezing a 21-nt RNA hairpin solution at 203 K followed by incubation at 263 K results in the conversion to the duplex dimer

form108. The formation of frozen microenvironments during prebiotic evolution could be a key factor in the possible prebiotic evolution of informational polymers.

The formation of peptides in the ice matrixThe linking of monomer units to form simple polymers likely

defined an important step in the origins of life, and many conditions have been proposed, including dehydration agents109, sulphide minerals110, melting111 or hydrothermal systems112. Further studies suggest important roles for catalytic surfaces, such as clays, or interfaces created by wet–dry cycling of monomers on mineral surfaces113.

Based on this idea, Schwendinger and Rode found a particularly simple process of salt-induced peptide formation, using 40-50 mM amino acid solutions where NaCl at concentrations above 3 M can act as a dehydrating or condensation agent, using dissolved Cu(II) as a catalyst114. Experiments carried out by Fox demonstrated that the melting of amino acids at temperatures in the range of 400 to 433 K, to allow melting without decomposition, produces a type of polymer called ‘proteinoids’. This phenomenon will occur provided that acidic or basic amino acids are present in excess115. However, the so-called ‘proteinoids’ are mainly heteropolymers containing only very small quantities of peptide bonds116. The melting of a mixture of urea and alanine yields the dipeptide Ala-Ala81.

The largest number of proposals and related experiments performed in order to model the prebiotic peptide formation in solution involves the postulated existence of coadjutant condensation reagents in a homogenous catalytic process. These reagents include cyanamide and cyanoguanidine, which may act as prebiotically plausible condensing agents117.

A problem associated with high temperature processes is the decomposition of amino acids and the hydrolysis of peptides, which constitutes a limitation for the organisation of larger polymers118. The synthesis in freezing solutions could prevent undesirable side reactions, hydrolysis of the formed peptide bond, and the decomposition of amino acids as well as reduce the rate of amino acid racemisation119. This idea is connected to a different approach to the problem of amino acid condensation that was introduced years ago: the salt-induced peptide formation reaction. Salty brines could have played a role in the polymerisation of amino acids. However, the formation of a peptide bond is not straightforward at low temperatures without condensing agents, and the experiments performed were carried out at high temperatures in drying conditions. Could freezing of the primitive oceans have produced the concentrated salty brines with the associated condensing agents needed to promote the salt-induced polymerisation process? Orgel et al. studied the oligomerisation of beta-amino acids in aqueous solutions under eutectic conditions using activation by the water-soluble reagents EDAC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) and carbonyl-

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diimidazole120. The oligomerisation of beta-amino acids (L-Aspartic acid, beta-amino adipic acid, beta-glutamic acid) using these condensing agents proceeds efficiently at 253 K (under eutectic freezing), even from dilute solutions of the substrates. This reaction produces peptides in the range 15 to 20 units (maximum: 45) in length with a yield of over 50%. The efficiency of polymerisation and the length distribution of the oligomers was almost unaffected by the solute concentration over a broad range of 0.1 to 100 mM at 253K. According to these results, the EDAC reagent constitutes the model of a group of activating agents whose function is the direct reaction with the carboxyl group of amino acids. Cyanogen, cyanamide and cyanoguanidine are prebiotically plausible members of this group. The elucidation of the pathway shows that the first step is the direct attack of the carboxyl group on the carbodiimide to form an O-acylisourea. The free amino group of another amino acid attacks this activated species to form a peptide bond. In the case of alpha amino acids, the carboxyl group of the dipeptide can be activated and then cyclise efficiently to give a diketopiperazine, thus inhibiting oligomerisation121. Cyclisation of an activated dimer of beta-amino acids is not straightforward because an eight-membered ring does not form readily.

In 1996, Vajda et al. synthesised four protected dipeptides and a protected tripeptide in frozen dioxane and other organic solvents122. The data demonstrated that the coupling rates in frozen dioxane at 254 K exceed by approximately one order of magnitude the rates in liquid solution at 313 K. Vajda suggested that enhanced reaction rates and/or yields, diminution of racemisation, and the suppression of side reactions can be expected in frozen systems, and these possibilities substantially increase the importance of peptide formation in eutectic frozen solutions123. However, no further investigation on these possibilities has been performed.

Concluding remarksPrebiotic chemistry in the range of stability of a liquid water-ice interface (277 to 243K in common laboratory conditions) has been proposed since the pioneering experiment in the field. These ideas were proposed to overcome the concentration and stability problems associated with liquid water prebiotic chemistry. The experiments performed demonstrated that the synthesis of aromatic hydrocarbons, purines and pyrimidines and other nitrogen heterocycles of potential prebiotic interest (such as triazines) are favoured in the ice matrix by classic cyanide and cyanoacetylene pathways following a freezing-concentration model. Despite these results, the experimental prebiotic chemistry in the solute-concentrated solutions that fill the space confined by ice matrix has received relatively little attention in the elaboration of the models for the origin of organics in Solar System bodies and prebiotic evolution. Consequently, it is necessary to clarify the mechanisms involved and the role of reactants as well as to perform more experiments under plausible prebiotic conditions, especially if geochemical models support stable icy environments on the prebiotic Earth. The concentration of reactant solutions by freezing also enhances the polymerisation of activated nucleotides and the formation of small peptides in the presence of an activating agent. The prebiotic relevance of these polymerisation reactions and the gap

between the nucleobase synthesis and the organisation of the first biopolymers is a matter for discussion. Nevertheless, the ice world constitutes an interesting prebiotic chemistry scenario that awaits further investigation.

AcknowledgementsWe acknowledge the Centro de Astrobiologia (CSIC-INTA) and the grants of the project AYA2009-13920-C02-01 from the Ministerio de Ciencia e Innovación (MICINN, Spain).

Notes and referencesa Part of the prebiotic chemistry themed issueb Centro de Astrobiología (INTA-CSIC), INTA, E-28850 Torrejón de Ardoz, Spain. Tel: 32 91520 6458; E-mail: menorsc@cab.inta-csic.es

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SCHEME CAPTIONS

Scheme 1 Synthesis of purines by polymerisation of cyanide to HCN tetramer and formation of cyanoimidazole derivatives. The related formation of glycine, formamidine and glycolonitrile, were observed in ice-water experiments.

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Scheme 2 Cyanoacetylene as a precursor for pyrimidines. The reaction ofcyanoacetylene with urea or ammonium cyanate yields cytosine, whose deamination leads to uracil. The reaction with guanidine directly forms 2,4-

diaminopyrimidine. Is possible that reaction goes through a pentanedinitrile intermediate.

Scheme 3 Urea as precursor of nitrogen heterocycles. Possible pathways to pyrimidines, hydantoins and triazines in frozen urea solution under a methane/nitrogen atmosphere.

Scheme 4 Polycyclic aromatic hydrocarbons and acetylene polymers detected from sparking a methane/nitrogen atmosphere on water-ice matrix.

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