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Prebiotic 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 20XX DOI: 10.1039/b000000x

5 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 10 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 15 brines, ≈273 to 210 K) is an under-explored experimental model in prebiotic chemistry.

Introduction prebiotic reactants. These criticisms and the lack of experimental evidence supporting a model for the origin of biochemical Life as we know it depends on interfacial redox and transport pathways have led to two main schools of thought: processes between liquid water and a system of lipid membranes 40 The first concept is the possibility of an in situ origin on Earth, with the associated protein machinery. It seems logical to assume which focus on either water-mineral interfacial processes as a 20 that life emerged from liquid water solutions where relatively way for concentration and compartmentalisation of simple raw materials were synthesised or accumulated. These environmentally synthesised reactants2 or on the origin of solutions could be subjected to water-mineral matrix interfacial chemoautotrophic pre-biochemical systems3. chemistry or concentration and compartmentalisation process, 45 The second concept argues that amino acids, nitrogen which ultimately leads to the emergence of life in a complexity heterocycles and simple organic molecules and monomers could 25 increasing process. Consequently, to determine the possible be synthesised by irradiation at very low temperatures in extra- compositions of the raw materials for the plausible first steps of terrestrial ice layers composed of water and other condensates4. abiotic evolution, pioneering experiments on prebiotic chemistry Ice is the most abundant form of water beyond the asteroid belt5. have been conducted in water-saturated atmospheres and liquid 50 The chemistry of ices at low temperatures followed by the solutions1, which are largely supported by a reductive atmosphere delivery of the organic molecules on Earth by comets, meteorites 30 model. and dust particles could have been an important source of The criticisms regarding an efficient atmospheric-liquid water organics on the prebiotic Earth and could have played a key role origin for the organic components of the first biochemical in early chemical evolution. The photochemistry and processes on Earth arise from the lack of a universally accepted 55 radiochemistry of outer solar system bodies and interstellar ices geochemical model for the Archean atmosphere. Additionally, the has received substantial attention6. 35 classic prebiotic chemistry approach deals with the problem of Despite the research into the photochemical transformations in the concentration and stability in liquid water of the plausible ice from an astrochemical point of view, the study of the This journal is © The Royal Society of Chemistry [year][journal], [year], [vol], 00–00 | 1 CREATED USING THE RSC ARTICLE TEMPLATE (VER. 3.0) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS ARTICLE TYPE www.rsc.org/xxxxxx | XXXXXXXX

chemistry in the range of stability of the ice-water interface has transformation to the amorphous state (the stable form at these not received much attention. This may be due to the scarcity of temperatures) via cosmic ray bombardment and ultraviolet the defined conditions in the Solar System during the epoch of60 irradiation14. The irradiation diminishes the kinetic barrier active prebiotic chemistry or the difficulties for demonstrating between the metastable cubic ice form and stable amorphous ice 15 5 that these cold conditions existed in Hadean Earth. form at lower temperatures . The crystallisation of ice Ih leads to The evidence for a liquid water subsurface ocean on Saturn’s the formation of various interfaces, such as ice-ice, ice- moon Europa7 and the possible presence of water-ammonia atmosphere and water-ice, as well as water-ice-mineral, which eutectic brines or even a subsurface ocean in other outer giant65 results from crystallisation of solutes by ice matrix exclusion or planet satellites such as Titan8 or Enceladus9 rekindled the interest the presence of suspended mineral grains16. The ice-ice and ice- 10 in liquid water prebiotic chemistry. Moreover, the subsequent atmosphere interfaces are not a distinct transition. Nuclear proposed steps for the emergence of cellular life have a limited magnetic resonance studies of ice crystals indicate the existence temperature range, and a hot prebiotic Earth was regarded to be of a liquid transition between the crystals or between the ice and an unlikely environment for the origin of life by some authors1070. the atmosphere. The thickness of this liquid phase becomes Miller and Orgel stated in 1974 that the emergence of biological monomolecular at T< 243 K and is thickened by dissolved solutes 15 organisation could only occur at temperatures below the melting excluded from the ice matrix to the interface during point of the polynucleotide structure. After observing the crystallisation16. instability of organic compounds in the prebiotic stages, these The unexpected presence of crystalline ice in the Quaoar authors concluded that a temperature of 273 K would have been75 object at the Kuiper Belt, on Enceladus and its suggested beneficial and that temperatures near the eutectic point of NaCl presence in Titan17 imply that the evolution of ices are subject to 20 solutions (251.3 K) would have been even better11. occasional heating events. If crystalline ice and if even fluid The low temperatures in planetary surface ices could be more water solutions are unambiguously present, the conditions for the conductive to the origin and the preservation of molecules that increase in organic complexity from reactions between precursors could be relevant for the emergence of life. In 1994, in one of the80 such as cyanide or cyanoacetylene may exist. The young and first explorations of the idea of an ice world-based origin of the active surface of the Jovian moon Europa suggests the possibility 25 life raw materials, Bada et al.12 suggested that ice formations on of a subsurface water ocean from the observations of the Voyager early Earth could have preserved organic compounds against mission and strengthened by the observations with the Galileo hydrolysis or photochemical degradation. Under plausible spacecraft18. Recently, it has been stated that Europa possesses planetary conditions, the presence of liquid water at T<273 K85 and active dynamic ice-water system with cycles of melting and within an ice matrix creates a potential reactor where the refreezing. In addition, a lenticular body of liquid brine in the 30 synthesis or polymerisation of molecules of biological interest Thera Macula region of approximately 20.000-60.000 km3 has could occur. Herein, we will review our current knowledge of the been predicted19. The composition of Europa’s subsurface water, chemical models that simulate possible prebiotic synthetic underlying an ice crust, could be rich in sulphate salts, the source pathways in liquid water interfacial ice. The experimental90 of surface evaporite deposits20. The details on water composition approaches developed in the literature are primarily focused on and temperature are unknown, but estimations suggest a Mg- 35 the RNA-world hypothesis of an abiotic origin of nucleic acids, SO4-Na(K) rich water with temperatures in the range 210-270 as these studies provide experimental evidence for the abiotic K21. A model for the formation of liquid ammonia-water pockets synthesis and polymerisation of nitrogen heterocycles and that cause episodic cryomagmatism and a subsurface eutectic nucleotides. Apart from the molecular evolutionary perspective95 water-ammonia solution has been proposed for the Saturn moon for the emergence of life, exploring the chemistry in liquid Titan22. Within this context, both Titan and Europa constitute 40 inclusions confined in an ice matrix could explain and predict the important astrobiological targets for direct exploration and composition of objects in the inner Solar System and icy laboratory simulations to predict the chemistry that will be found planetary bodies. and to test our experimental prebiotic chemistry models23. A 100 complex prebiotic chemistry has been predicted for Titan that The ice/ liquid water system and its presence on includes the formation of nucleobases24 and the possibility of a the early Earth and in the Solar System methane-acetylene based chemical or biochemical evolution 25. From these hypotheses based on atmospheric or surface 45 The ice-liquid water system has not received much attention in chemistry, the prebiotic possibilities of liquid water brines the literature, including the chemical physics and105 entrapped under ice has received less attention and is the object of astrochemical/astrobiological literature. In the latter case, the speculative discussion regarding possible biochemical evolution experimental efforts are focused on low temperature condensates, and the presence of chemoautotrophic life26. where there is no evidence of a liquid interface and the ice is in Some models suggest a Hadean terrestrial atmosphere 50 its amorphous crystalline state. In the inner Solar System, composed primarily of high pressure carbon dioxide. If liquid including on Earth, ice occurs naturally in the crystalline form 110 water were present in oceans over a basaltic crust, a CO2 with two primary polymorphs, which are cubic and hexagonal. atmosphere would be unstable and could be depleted as The crystallisation of water under current Earth surface carbonates in a period of approximately 10 million years due to conditions results in hexagonal ice Ih. The ice formed from liquid hydrothermal circulation and reaction of the CO2 with the crustal 55 or heated from amorphous ice at temperatures between 100 and rock. Under these conditions, together with the Hadean faint Sun, 13 130K is crystalline, with a diamond-type cubic structure Ic115. the model developed by Sleep and Zahnle27 agrees with the ideas Cubic ice is metastable at T<70 K and undergoes a

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5 ARTICLE TYPE suggested by J. Bada in 199412, predicting ice-covered oceans and be essential for the solute exclusion from the interstitial brines in an average surface temperature of approximately 220 K, with ice and the formulation of a freeze-concentration model for freeze-thaw episodes motivated by occasional warming provoked explaining the prebiotic chemistry observed in the ice matrix. by high energy impacts. These cold conditions would be Observation of the behaviour of stains in ice shows that 5 prevented if a methane-rich atmosphere were present during the50 organic molecules are excluded from the ice matrix and Hadean, as methane is a potent greenhouse gas. Evidence thus far concentrated in the interstitial brine, where chromatographic does not support an atmosphere with a high enough concentration separation has been noted. Another important property of the of methane to avoid freezing of the ocean surface. This model behaviour of organic molecules in ice is that a dilute starting would be amenable for the development of prebiotic chemistry in solution of a given solute always reaches the same molal 10 an ice matrix based on HCN, cyanoacetylene, acetylene, urea or55 concentration in the interstitial solution, which is determined by cyanate precursors synthesised on Earth or brought in via the final incubation temperature33. For example, a freezing dilute extraterrestrial input28. urea solution tends to form an interstitial eutectic 8 m solution The freezing of ocean water is a complex process. Modern sea with a melting point of 261K. These properties of the ice-water

water begins to freeze at 271.2 K and crystals of pure ice (Ih) interface convert the ocean ices, at temperatures within the range 15 begin to grow, surrounded by liquid brine with sodium chloride60 of existence of the interface with liquid brines, into a potential concentrations up to 25%. The liquid solution is concentrated reactor for the first steps responsible for the emergence of life. within the ice structure in channels, which have been observed in stained samples under the microscope, with diameters ranging Prebiotic synthesis of nucleobases and other from 10 to 100 µm29. Based on observations of microscopic ice nitrogen heterocycles in the ice matrix 20 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 Nucleobases are a small group of one-ring (pyrimidines) and two- volume of ice occupied by the brine channels and the brine65 ring (purines) nitrogen heterocycles that, together with sugars and conditions within the channels are directly proportional to the phosphate, compose nucleic acids. The pyrimidines include temperature; at 267 K, the brine salinity in sea ice is 100 (on the uracil, thymine and cytosine and purines include adenine and 25 practical salinity scale, i.e., dimensionless units that are guanine. Other heterocycles belonging to both groups are equivalent to the ratio between the sample solution and a standard important intermediates in the biochemistry, including xanthine, KCl solution; normal ocean water has a salinity range of 30-35);70 hypoxanthine and orotic acid. It is generally assumed that the at 263 K, the salinity rises to 145, and at 252 K, the salinity earliest living forms on Earth used a genetic code based on 34 reaches a maximum of 21630. In sea ice, the presence of nucleobases . In addition, nitrogen heterocycles could have been 35 30 interstitial channels filled with liquid water and concentrated involved in the first metabolic pathways as cofactors . solutes has been observed over a range of temperatures down to Regardless of the controversy regarding whether life began 243 K. Sea ice can lead to the formation of solid mineral phases75 with a replicator, as suggested by the RNA-world hypothesis, or 36 from the crystallisation of dissolved salts. with metabolism, as suggested by later authors , there is no During freezing or thawing events, the temperature gradients evidence to discard the hypothesis of a prebiotic source of 35 and density changes in the ice matrix lead to pressure gradients nucleobases or cofactors for the first living system. The first and motion of the trapped liquid water that fills the channels and logical hypothesis considers that the prebiotic synthesis took pores. The freezing process led to the formation of potential80 place on Earth, although it is not clear if the environmental 37 gradients, with pH variations of up to 3 units31. conditions were consistent with efficient in situ synthesis . The The boundary between liquid and solid water has a different second logical hypothesis is the delivery of nitrogen heterocycles 40 refractive index and reveals an interface. Measurements of the to Earth by comets, meteorites and dust particles. This extra- zeta potential (electric potential difference between the fluid brine terrestrial delivery could compensate for a possible lack of and the stationary liquid layer attached to the ice crystals) showed85 availability from in situ synthesis. Analysis of carbonaceous that the interfacial properties of an ice-water system are chondrites, a class of meteorites rich in organic carbon and 38 comparable to the interface with hydrophobic and nonionogenic water , has demonstrated the presence of N-heterocycles. These 45 solids, such as diamond or hydrocarbons32. These properties could heterocycles include adenine, guanine and triazines (ammeline and melamine), which were found in the Orgeil meteorite by

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Hayatsu in 196439. Subsequent analyses performed from 1965- structural elucidation of 4-amino-2-cyanoimidazole-5- 197540 show that the extraction conditions and sample treatments60 carboxamide and its hydrolysis product, 4-aminoimidazole-2,5- determine the analytical results. However, the presence of dicarboxamide. However, the adenine-8-carboxamide has not yet nucleobases in carbonaceous chondrites is widely accepted. In been identified in HCN oligomerisation experiments. 5 2008, Martins et al. demonstrated41 the extra-terrestrial origin of The last proposed mechanism is the UV-induced photo- xanthine and adenine in a Murchinson meteorite sample using isomerisation of the HCN tetramer to 4-amino-5-cyanoimidazole. carbon isotope measurements. Recently, Callahan et al.65 The reaction of this imidazole with HCN or with its hydrolysis demonstrated that the suite of purines found in carbonaceous product ammonium formate in a melt directly yields adenine56. chondrites is consistent with those obtained using ammonium Because it is the key reaction in the pathway, the formation of the 10 cyanide chemistry42. The questions that arise from these results HCN tetramer requires a high HCN concentration to avoid the include how were the nitrogen heterocycles synthesised on Earth volatilisation or hydrolysis to ammonium formate, which or other bodies in Solar System, and how could the ice-water70 competes with the formation of diaminomaleonitrile in dilute interface play a role in this process? solutions. Therefore, it would have been impossible to reach sufficiently high HCN concentrations in the open oceans or by Synthesis based on hydrogen cyanide water evaporation57. 15 The synthesis of nucleobases and other nitrogen heterocycles in One solution to this problem could be to consider alternatives the parent body of a meteorite could be a process that is75 to aqueous HCN chemistry. The formation of nucleobases from dependent on the water content and irradiation of precursors. The formamide in the presence of inorganic catalysts at high seminal work of Juan Oró and co-workers demonstrated that temperature creates a robust pathway for adenine, hypoxanthine, adenine can be easily synthesised from hydrogen cyanide uracil and cytosine among other N-heterocycles 58. One solution 20 (Scheme 1)43. A prebiotic origin for the nucleobases was to this problem could be concentrating HCN using the liquid-ice thereafter regarded as a realistic possibility44. Additionally, 80 a interfacial properties. During the first attempt to test this Fischer-Tropsch type synthetic mechanism catalysed by mineral possibility, Sanchez et al. (1966) showed that HCN concentrates phases at high temperature has been suggested for the origin of in a frozen eutectic solution. The eutectic solution, which has a N-heterocycles in meteorites45, but its actual significance is mole fraction of 70 to 80% in HCN, is formed at 249 K and 25 unclear46 and currently is not a widely accepted route. deposits a dark HCN polymer59. Considering the activation Cyanide is the primary precursor involved in our current85 energy of the HCN polymerisation and the rate constants, the models for prebiotic synthesis of nitrogen heterocycles and a formation of the HCN tetramer in eutectic fluids should be possible precursor to the organic molecules that gave rise to complete in a few years. At 173 K, the reaction occurs over the biochemistry. Cyanide could be generated photochemically or by order of hundreds of millions of years60. The advantageously 30 spark discharges in methane/nitrogen planetary atmospheres47. In stable conditions in a water-ice interface could surpass the addition, free HCN and cyanide polymers have been observed in90 handicap of prebiotic synthesis at low temperatures and the comets, dust particles48 and the Titan atmosphere49. problem of concentration and stability at high temperatures. The mechanism of synthesis of adenine from HCN implies that Additionally, the freezing of dilute glycolonitrile solutions, the first step is polymerisation to the HCN-tetramer produced by addition of HCN and formaldehyde, produces 35 diaminomaleonitrile (DAMN; Scheme 1). This intermediate adenine in low yield (0.004%)61. In a long duration experiment, could undergo further polymerisation to form dark brown solid95 Miyakawa et al. maintained a frozen solution of ammonium polymers, which upon hydrolysis release nitrogen heterocycles, cyanide at 195 K over 27 years and at the end of this time period, including adenine50. This hydrolysis could take place in the ice- identified adenine as well as other purine and pyrimidine water interface in the parent body of comets or meteorites during products62. Although the HCN pathway has been extensively 40 their journey in the inner Solar System or after these objects studied for the synthesis of purines, it has been demonstrated that impacted the Earth. Another possible mechanism is the reaction100 the polymerisation of cyanide could provide a pathway for the of DAMN with formamidine51 to afford a 4-amino-5- formation of the pyrimidines including uracil, 5-hydroxyuracil cyanoimidazole (AICN) intermediate. This reaction yields and orotic acid63. The freezing of cyanide solutions could also adenine through the coupling of HCN or formamidine. The provide a source of amino acids. In 1972, another long-term

45 hydrolysis of AICN leads to 4-aminoimidazole-5-carboxamide experiment involved a solution of NH4CN prepared from HCN 52 (AICA), which could be a xanthine and hypoxanthine precursor105 and NH3. These reagents were frozen and subjected to variable (Scheme 1). Formamidine has also been identified as an organic temperatures of 253K and 195K for 25 years. The analysis precursor found in comets53 and prebiotic chemistry laboratory indicated the formation of glycine and small amounts of alanine simulations54. A possible major mechanism for the formation of and aspartic acid64. The mechanism for the cold synthesis of 50 adenine from HCN, which was elucidated by Voet and Schwartz amino acids from HCN has not been elucidated, but may include in 1982, is the reaction between the HCN tetramer and 110 its the hydrolysis of HCN polymers65 and the hydrolysis of 2- cyanoimino tautomer or diiminosuccinonitrile (an oxidation aminoacetonitrile, which is formed during HCN tetramer product of HCN tetramer) to yield the carbamimidoyl cyanide evolution, to glycine (Scheme 1). derivative. This molecule cyclises to 4-amino-2-cyanoimidazole- Prebiotic laboratory synthesis from frozen cyanide solutions 55 5-carbimidoylcyanide. Further addition of the cyanoimino could be a model for the prebiotic synthesis of nucleobases. This derivative and ring closure, affords adenine-8-carboxamide115 synthesis could also explain the chemistry observed in ice- (Scheme 1)55. This product is quantitatively converted to adenine covered objects within the inner solar system, such as asteroids by hydrolysis. The above mechanism was supported by the

5 ARTICLE TYPE and comets during their closest passage to the sun, and in objects synthesis of nucleobases is its reactivity to nucleophiles75, which with complex chemistry, including Titan or Enceladus. To suggests a high number of competitive reactions that lead to the efficiently serve both goals, more experimental work should be formation of amino- or hydroxyacrylonitriles and subsequent performed to elucidate the mechanisms involved in frozen HCN polymers or hydrolysis products; On the other hand, the prebiotic 5 solution, to test if the classic pathway through cyanoimidazole50 origin of cytosine was questioned, at least in the liquid water derivatives is reproducible in the ice matrix scenario and to medium, because its spontaneous and rapid deamination to determine if alternative pathways should also be examined. 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. Synthesis based on cyanoacetylene/acetylene and the role of 10 urea 55 Although much time has elapsed since the first proposal in 1966 of a low temperature prebiotic environment for the origin of Cyanoacetylene is the other primary precursor considered for the nucleobases, it was not until 2000 that the product of the classic synthesis of nucleobases. Cyanoacetylene can be obtained in the approach of spark discharges in methane/nitrogen based 66 laboratory from methane/nitrogen mixtures by spark discharges atmosphere was subjected to eutectic freezing77 at 253K for 5 by irradiation with short-wave ultraviolet radiation at 185 and60 years. The frozen spark discharge product showed a more 67 15 254 nm ; the spectrum of this molecule has been observed in the extensive mixture of amino acids and the presence of adenine, 68 interstellar medium and by the Voyager mission in Titan’s which was absent in the control experiment at room temperature. 69 atmosphere , where crystalline condensates of cyanoacetylene The first experimental simulation of prebiotic synthesis in ice- 70 with acetylene may exist . liquid water directly from nitrogen/methane atmosphere by spark The potential prebiotic relevance of cyanoacetylene in origin65 discharges was performed in 200978. The sparking on a freezing 20 of life studies were pointed out by Ferris, Sanchez and Orgel in dilute urea solution under a nitrogen/methane atmosphere leads to 1968. They observed that the reaction of cyanoacetylene with the formation of cytosine, uracil and 2,4,6-trihydroxypyrimidine aqueous 1 M sodium cyanate or 1 M urea gave cytosine in up to (barbituric acid) as the main identified pyrimidines, in addition to 71 5% yield (Scheme 2) . The prebiotic availability of cyanate could adenine. The experiments showed that using the freeze-thaw be explained by the hydrolysis of cyanogen and urea, which may70 conditions, the observed sequence of pyrimidine yield obtained 72 25 also be present in cometary and interstellar ices . was cytosine > uracil > 2,4-diaminopyrimidine > 2,4,6- The mechanism of this reaction could be explained by trihydroxypyrimidine. The formation or pyrimidines by oxidative cyanoacetaldehyde, generated by hydrolysis of cyanoacetylene. alteration of cytosine (UV irradiation, hydroxyl radical addition The Miller research demonstrated the eutectic concentration and or other free radical mechanism and further oxidation to reaction of cyanoacetaldehyde with urea in an ice matrix at 253K75 barbituric acid) could explain the results observed 79. The 30 to give cytosine and uracil in 0.005% and 0.02% yields, formation of cytosine as the main pyrimidine suggests that the 73 respectively . In the same report, cyanoacetaldehyde reacted with low temperature conditions could reduce the rate of deamination guanidine at 253K to give cytosine in 0.05% yield and uracil in to uracil and favour subsequent chemical evolution steps, as 10.8% yield, as well as lesser amounts of isocytosine and 2,4- suggested by Bada12. 74 diaminopyrimidine after 2 months . This reaction may proceed80 The triazine series (cyanuric acid, ammelide, ammeline and 35 through the cyanoacetaldehyde dimer, 4-(hydroxymethylene) melamine) are also obtained in high yields (Scheme 3). The pentenedinitrile, easily formed by concentrating the formation of triazines appears to be dependent on the freezing of 72 cyanoacetaldehyde solutions (Scheme 2) . urea solution. The triazines are not biological compounds, but The basis of these experiments is the freezing of a urea or they could mimic nucleobases behaviour in nucleic acids and guanidine solution. This process provides a concentration85 their potential prebiotic role has been discussed80. Their presence 40 mechanism because the crystalline ice excludes the solute and a in meteorites remains contentious81. eutectic is formed. At 262K, urea forms an 8 m eutectic solution The key factor appears to be the freezing process itself and not in water. This effect could be significant from a prebiotic point of the temperature of the final ice obtained, as the temperature was view, despite the slower reaction rates, as has been shown in selected to be right below the freezing point of 0.1 M urea. In a recent experiments. 90 liquid urea solution at room temperature, there is no evidence of 45 An unresolved issue with the cyanoacetylene pathway in the

nucleobases. Instead, the formation of hydantoins, nitriles and (PAHs) by sparking methane/nitrogen atmosphere over an ice tholins (reddish-brown, insoluble, heteropolymeric or60 matrix90. The model of PAH synthesis is interesting because it macromolecular materials formed by sparking or irradiation of could confirm the theoretical synthesis of aromatics by acetylene simple carbon sources, as methane) is prevalent. insertion mechanisms proposed for the Titan’s atmosphere91. In 5 The behaviour of urea in the ice-water interface is the key laboratory experiments at sub-zero temperatures65, the acetylene factor because urea tends to form dimers or oligomers in a addition mechanism could explain the preferential formation of concentration-dependent manner82. Urea molecules in aqueous65 aromatics and poly(triacetylene) polymers (Scheme 4) by two fluids tend to form hydrogen bonds with neighbouring water possible mechanisms. First, a single aromatic ring could be molecules at both the amino and the carbonyl groups 83. generated from acetylene and vinyl radical and PAH growth by H 10 Spectroscopic studies show that at urea concentrations higher abstraction and acetylene addition (Berthelot synthesis, similar to than 1 M, the urea-urea molecular interactions are significant. PAHs formation in flames). The second mechanism involves The urea-urea molecular interaction with subsequent formation of70 polyyne growth. The presence of water ice induces oxidations dimers or clusters of urea molecules becomes dominant at leading to the formation of aromatic polar species as eutectic concentration84. During freezing, the urea is segregated benzaldehyde or acetophenone. The reaction in ice, in contrast to 15 from pure ice to accumulate in supercooled microfluid inclusions the dry high temperature synthesis of PAHs, leads to hydroxyl- of a supersaturated solution. This system is governed by rich poly(triacetylene) based polymers. Overall, these ice-water dehydration and association of solute molecules85. Thus, the75 laboratory experiments reveal the expected chemical species in extent of urea dimerisation (18% in 0.1 M urea solution at surface or subsurface ices on solar system objects or extrasolar standard temperature86) is expected to increase and to become planetary bodies. 20 quantitatively a few degrees below the onset of freezing. The activation of methane/nitrogen atmospheres by spark Consequently, we expect an apparently paradoxical similarity discharges could lead to various chemistries involving reactive between the process observed in molten urea84 and urea clusters80 intermediates, including HCN, cyanoacetylene and acetylene. The entrapped in an ice matrix when the latter are subjected to direct preference for the hydantoins in liquid urea solutions at room sparking or irradiation. temperature versus pyrimidines in frozen solution experiments 25 This behaviour could explain the sequence of products could be due to the acetylene formation and subsequent alteration obtained (cyanuric acid > ammelide > ammeline > melamine), by means of ozone and hydroxyl radicals at higher temperatures which is the same sequence observed when urea is heated above85 to form alpha-dicarbonyl compounds as glyoxal92. The reaction of its melting point. The spark discharges into the ice, then, could glyoxal with urea in mild acidic conditions yields hydantoin 93, thermally decompose urea clusters in ammonium cyanate. Further whose further oxidation yields 5-hydroxyhydantoin and parabanic 30 decomposition of ammonium cyanate leads cyanic acid. The acid (Scheme 3). These three hydantoins are always found cyanic acid reacts with urea to form the biuret and with the together in all the experiments reported in the literature. Its formed biuret to form cyanuric acid (a cyanic acid trimer), which90 formation could be explained also as alteration products of uracil is the main triazine observed84. Several routes to ammelide are by hydroxyl and other free radicals generated in water solutions possible: reaction of cyanuric acid and ammonia or cyanic acid by photolysis or irradiation94,79. At lower temperatures, the 35 and urea or biuret. The process, in which the decomposition degradation of pyrimidines to hydantoins and the oxidation of products accelerate the formation of triazines, could explain the acetylene could be diminished, due to the lower availability of high concentration of cyanuric acid obtained in these conditions.95 reactive oxygen species generated from the excitation of water. In Other parallel pathway is the formation of melamine by consequence, hydantoins could be the final products of alteration cyanamide polymerization. The melamine hydrolysis yield of pyrimidines in prebiotic conditions subjected to UV-irradiation 40 cyanuric acid (Scheme 3). These pathways and the same reaction or other energetic processes. Regarding acetylene, the sequence, with the same relative abundance of triazines, have polymerisation could be the preferred reaction pathway, as shown been studied in molten urea87 at temperatures between 406 and100 by the formation of poly-triacetylene and aromatic hydrocarbons 460K. In this case, an alternative route for forming purines could at the ice-water matrix previously described. In this environment, result from the condensation of amino acids and biuret, a reaction the HCN or cyanoacetylene pathways could dominate, as long 45 that occurs at high temperature88; however, this alternative still with other alternative mechanisms, as the synthesis of uracil by has not been studied in ice-water systems and could be an reaction of urea with acetylene dicarboxylic acid95. This acid is unlikely possibility because of the high activation energy of such105 the aqueous hydrolysis product of dicyanoacetylene, which is an condensations. We also cannot discard other alternative pathways exotic product of methane/nitrogen atmospheres observed in the parallel to the polymerisation of concentrated urea solutions. For Titan atmosphere96. The role of acetylene derivatives has not been 50 example, the production of cyanic acid during atmospheric studied in the ice-water scenario, and further experiments are discharges or thermal alteration of tholins89 and the subsequent necessary to explore the possible alternative pathways related to reaction in freezing urea solutions could be an alternative source110 acetylene in prebiotic synthesis in an ice matrix and to put it in of cyanuric acid. Additional laboratory studies are necessary for context with the classic mechanisms involving cyanide and clarifying the mechanisms involved in the cold synthesis of cyanoacetylene. The products identified in the simulations of 55 triazines and purines in the ice matrix. methane/nitrogen atmospheres over the ice-water interface The effect of concentration of solutes in the ice matrix, include dicarboxylic and hydroxycarboxylic acids, amino acids together with the low availability of water vapour could explain115 and pyrazines, suggesting an additional mechanism to those the preferential synthesis of polycyclic aromatic hydrocarbons suggested above.

5 ARTICLE TYPE In summary, the advantages of an ice-water interface in45 monophosphorimidazolide solution at 255 K at a pH range prebiotic synthesis include the reduction in the formation of between 6 and 8 in the presence of magnesium and lead polymers and tholin with a preference for ring systems (nitrogen cations102: heterocycles or aromatic rings) by the effect of concentration of 5 diluted reactants such as HCN, urea or cyanate. Combined with Scheme 5 (uncaptioned) other rocks or minerals, the freezing of liquid water solutions 97 could favour mineral surface-organic solute interactions 50 The study of the ribonuclease A digestion products showed that the oligomers obtained are mainly linear and that 30% carry at The ice-water system in the origin of nucleic acids least one 3´-5´ linkage. The fluorescence microscopy observation The ice matrix is an appropriate environment for the synthesis of an ice layer in the experimental conditions with acridine 10 of nitrogen heterocycles, as demonstrated by the synthesis of orange staining indicated that the organic solutes were triazines and nucleobases in freezing urea solutions. Could the55 concentrated in the eutectic lattice structure included in the ice ice-water interface be a favourable environment for the assembly matrix. The authors concluded that the formation of eutectic of the first biologically relevant informational polymers? solutions of reactants in the ice matrix facilitated the The success in the synthesis of nucleobases from a feedstock oligomerisation. The polymerisation most likely occurs in the 15 of active nitrogen species available prebiotically led to the liquid concentrated solutions between the ice crystals, and not by establishment of a similar retrosynthetic analysis for RNA and to60 the adsorption of reactants onto the ice surfaces, as previously 103 the search for prebiotically plausible syntheses of a primordial suggested by Stribling and Miller , who studied the template informational, self-replicating polymer. If the discovery of an directed synthesis of poly(U) in diluted solutions concentrated by abiotic pathway to the origin of the first nucleotides and the freezing close to the NaCl eutectic. The ice also has an effect on 20 constitutional self-assembly of RNA is achieved, the RNA-world the metal catalysis. The reaction in the ice-water medium requires 2+ 2+ hypothesis (a term coined by Walter Gilbert in 1986)98, which65 Pb as a catalyst and not Mg . This phenomenon is different proposes a molecular evolutionary step involving autocatalytic from reactions in solution, which require both magnesium and RNA molecules prior to the origin of protein synthesis and lead cations. A possible interpretation of this observation is that metabolic machinery, will be strengthened. The current state of the molecular associations in an ice matrix tend to be more stable 25 prebiotic chemistry does not provide a complete model for an than the corresponding ones in solution. An open question that abiotic origin of RNA, and the first formulations of an RNA70 arises is the role of certain metal cations (for example lead) as world have been re-evaluated99. However, some argue that it may prebiotic catalysts. The lead catalysis in the polymerisation of be premature to conclude that the prebiotic RNA world is activated nucleotides could be related to the mechanism of 104 unlikely to be a step in the emergence of life100. leadzymes and suggests that metal ion catalysis is central in a 30 In this context, the ice-water interface has been evaluated hypothetical RNA world. thoroughly as a matrix for the polymerisation of highly activated75 If pyrimidine and purine-activated nucleotides are used in the nucleotides. The first demonstration of this possibility was water-ice interface at 255 K during 38 days in the presence of 2+ 2+ performed by Gryaznov and Letsinger in 1993101. In their Mg and Pb , a mixed-sequence polynucleotide with experiment, the coupling of an alpha-bromoacyl-activated approximately the same proportion of purine and pyrimidines 105 106 35 oligonucleotide (bromoacetylamino-3’-desoxythimidine in the 3’- residues is obtained . Monnard and Szostak studied the terminus) with another oligonucleotide with a phosphorothioate80 template-directed RNA polymerisation in water-ice at 256.4 K, a group in the 5’-terminus proceeded without a template in a frozen temperature that permits the maintenance of a stable water-ice saline solution at 255 K in 5 days. The reaction was explained as interface for long periods of time. They found that lead and a result of the high local concentration of reactants in the fluid magnesium ions catalyse the elongation of a RNA hairpin with a 40 cavities in the ice matrix. 5’-overhang as a template. The enhancing effect of the ice matrix in the formation of85 Similarly, the non-enzymatic synthesis of polyadenosine in a RNA oligomers was demonstrated by Kanavarioti et al. in a very sea-ice matrix, directed by poly(U) was performed, using remarkable experiment in which oligouridylates up to 22 bases adenosine-5´-monophosphate (2-methyl) imidazolide as long were synthesised by incubating a uridine 5´- monomer. Temperature fluctuations established the freeze-partial

thaw cycles during one year. The results show high molecular Orgel et al. studied the oligomerisation of beta-amino acids in weight poly(A) formation, with chain lengths of as many as 420 aqueous solutions under eutectic conditions using activation by residues107: the water-soluble reagents EDAC (1-ethyl-3-(3- 60 dimethylaminopropyl)-carbodiimide) and carbonyl- Scheme 6 (uncaptioned) diimidazole120. The oligomerisation of beta-amino acids (L- 5 Aspartic acid, beta-amino adipic acid, beta-glutamic acid) using The freezing-concentration model could also govern the these condensing agents proceeds efficiently at 253 K (under conformational rearrangement pathway of the formed polymers. eutectic freezing), even from dilute solutions of the substrates. Freezing a 21-nt RNA hairpin solution at 203 K followed by65 This reaction produces peptides in the range 15 to 20 units incubation at 263 K results in the conversion to the duplex dimer (maximum: 45) in length with a yield of over 50%. The 10 form108. The formation of frozen microenvironments during efficiency of polymerisation and the length distribution of the prebiotic evolution could be a key factor in the possible prebiotic oligomers was almost unaffected by the solute concentration over evolution of informational polymers. a broad range of 0.1 to 100 mM at 253K. According to these 70 results, the EDAC reagent constitutes the model of a group of The formation of peptides in the ice matrix activating agents whose function is the direct reaction with the carboxyl group of amino acids. Cyanogen, cyanamide and The linking of monomer units to form simple polymers likely cyanoguanidine are prebiotically plausible members of this 15 defined an important step in the origins of life, and many group. The elucidation of the pathway shows that the first step is conditions have been proposed, including dehydration agents10975, the direct attack of the carboxyl group on the carbodiimide to sulphide minerals110, melting111 or hydrothermal systems112. form an O-acylisourea. The free amino group of another amino Further studies suggest important roles for catalytic surfaces, acid attacks this activated species to form a peptide bond. In the such as clays, or interfaces created by wet–dry cycling of case of alpha amino acids, the carboxyl group of the dipeptide 20 monomers on mineral surfaces113. can be activated and then cyclise efficiently to give a Based on this idea, Schwendinger and Rode found a80 diketopiperazine, thus inhibiting oligomerisation121. Cyclisation particularly simple process of salt-induced peptide formation, of an activated dimer of beta-amino acids is not straightforward using 40-50 mM amino acid solutions where NaCl at because an eight-membered ring does not form readily. concentrations above 3 M can act as a dehydrating or In 1996, Vajda et al. synthesised four protected dipeptides and 25 condensation agent, using dissolved Cu(II) as a catalyst 114. a protected tripeptide in frozen dioxane and other organic Experiments carried out by Fox demonstrated that the melting of85 solvents122. The data demonstrated that the coupling rates in amino acids at temperatures in the range of 400 to 433 K, to frozen dioxane at 254 K exceed by approximately one order of allow melting without decomposition, produces a type of polymer magnitude the rates in liquid solution at 313 K. Vajda suggested called ‘proteinoids’. This phenomenon will occur provided that that enhanced reaction rates and/or yields, diminution of 30 acidic or basic amino acids are present in excess115. However, the racemisation, and the suppression of side reactions can be so-called ‘proteinoids’ are mainly heteropolymers containing90 expected in frozen systems, and these possibilities substantially only very small quantities of peptide bonds116. The melting of a increase the importance of peptide formation in eutectic frozen mixture of urea and alanine yields the dipeptide Ala-Ala81. solutions123. However, no further investigation on these The largest number of proposals and related experiments possibilities has been performed. 35 performed in order to model the prebiotic peptide formation in solution involves the postulated existence of coadjutant Concluding remarks condensation reagents in a homogenous catalytic process. These reagents include cyanamide and cyanoguanidine, which may act95 Prebiotic chemistry in the range of stability of a liquid water-ice as prebiotically plausible condensing agents117. interface (277 to 243K in common laboratory conditions) has 40 A problem associated with high temperature processes is the been proposed since the pioneering experiment in the field. These decomposition of amino acids and the hydrolysis of peptides, ideas were proposed to overcome the concentration and stability which constitutes a limitation for the organisation of larger problems associated with liquid water prebiotic chemistry. The polymers118. The synthesis in freezing solutions could prevent100 experiments performed demonstrated that the synthesis of undesirable side reactions, hydrolysis of the formed peptide bond, aromatic hydrocarbons, purines and pyrimidines and other 45 and the decomposition of amino acids as well as reduce the rate nitrogen heterocycles of potential prebiotic interest (such as of amino acid racemisation119. This idea is connected to a triazines) are favoured in the ice matrix by classic cyanide and different approach to the problem of amino acid condensation that cyanoacetylene pathways following a freezing-concentration was introduced years ago: the salt-induced peptide formation105 model. Despite these results, the experimental prebiotic chemistry reaction. Salty brines could have played a role in the in the solute-concentrated solutions that fill the space confined by 50 polymerisation of amino acids. However, the formation of a ice matrix has received relatively little attention in the elaboration peptide bond is not straightforward at low temperatures without of the models for the origin of organics in Solar System bodies condensing agents, and the experiments performed were carried and prebiotic evolution. Consequently, it is necessary to clarify out at high temperatures in drying conditions. 110 the mechanisms involved and the role of reactants as well as to Could freezing of the primitive oceans have produced the perform more experiments under plausible prebiotic conditions, 55 concentrated salty brines with the associated condensing agents especially if geochemical models support stable icy environments needed to promote the salt-induced polymerisation process? on the prebiotic Earth.

5 ARTICLE TYPE The concentration of reactant solutions by freezing also enhances10 We acknowledge the Centro de Astrobiologia (CSIC-INTA) and the polymerisation of activated nucleotides and the formation of the grants of the project AYA2009-13920-C02-01 from the small peptides in the presence of an activating agent. The Ministerio de Ciencia e Innovación (MICINN, Spain). prebiotic relevance of these polymerisation reactions and the gap 5 between the nucleobase synthesis and the organisation of the first biopolymers is a matter for discussion. Nevertheless, the ice Notes and references world constitutes an interesting prebiotic chemistry scenario that 15 a Part of the prebiotic chemistry themed issue awaits further investigation. b Centro de Astrobiología (INTA-CSIC), INTA, E-28850 Torrejón de Ardoz, Spain. Tel: 32 91520 6458; E-mail: [email protected] Acknowledgements

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SCHEME CAPTIONS

75 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. Scheme 2 Cyanoacetylene as a precursor for pyrimidines. The reaction of cyanoacetylene 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. 5 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. 10