This article was published as part of the

Prebiotic chemistry themed issue

Guest editors Jean-François Lambert, Mariona Sodupe and Piero Ugliengo

Please take a look at the issue 16 2012 table of contents to access other reviews in this themed issue

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5404 Chem. Soc. Rev., 2012, 41, 5404–5415 This journal is c The Royal Society of Chemistry 2012

Cite this: Chem. Soc. Rev., 2012, 41, 5404–5415

Prebiotic chemistry in eutectic solutions at the water–ice matrixw

Cesar Menor-Salvan* and Margarita R. Marın-Yaseli

Received 29th February 2012

DOI: 10.1039/c2cs35060b

A crystalline ice matrix at subzero temperatures can maintain a liquid phase where organic

solutes and salts concentrate 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 significantly high rates from a prebiotic

chemistry standpoint, and the labile products can be protected 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 the 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.

Introduction

Life 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 compartmentalisa-

tion processes, which ultimately leads to the emergence of life

in a complexity increasing process. Consequently, to deter-

mine the possible compositions of the raw materials for the

plausible first steps of abiotic evolution, pioneering experi-

ments on prebiotic chemistry have been conducted in water-

saturated atmospheres and liquid solutions,1 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

Centro de Astrobiologıa (INTA-CSIC), INTA,E-28850 Torrejon de Ardoz, Spain. E-mail: menorsc@cab.inta-csic.es;Tel: +32 91520 6458w Part of the prebiotic chemistry themed issue.

Cesar Menor-Salvan

Cesar Menor-Salvan studiedChemistry at the Universityof Alcala and obtained hisPhD degree in Biochemistryin 2004, working on themetabolism and toxicology ofthiolated purine bases. Since2007 he has been a ResearchScientist at the Centro deAstrobiologia (CAB) andstarted a line devoted to theprebiotic chemistry of nitrogenheterocycles and the origin ofcofactors and proto-metabolicpathways. His researchinterests include Prebiotic

Chemistry, the origins of biochemistry and the organic markersof biological evolution on Earth.

Margarita R. Marın-Yaseli

Margarita Roig Marın-Yaseliobtained her degrees inPharmacy and Biochemistry atthe University of Zaragoza. Nowshe is a PhD student at theCentro de Astrobiologia, focusedon the Prebiotic Chemistry ofnitrogen heterocycles.

Chem Soc Rev Dynamic Article Links

www.rsc.org/csr TUTORIAL REVIEW

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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 focuses on either water–mineral interfacial pro-

cesses as a way for concentration and compartmentalisation of

environmentally synthesised reactants2 or on the origin of

chemoautotrophic pre-biochemical systems.3

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

condensates.4 Ice is the most abundant form of water beyond

the asteroid belt.5 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 have received substantial attention.6

Despite the research into the photochemical transforma-

tions 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 authors.10 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 better.11

The low temperatures in planetary surface ices could be more

conductive to the origin and the preservation of molecules that

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, Bada12 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 o 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 pers-

pective 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 System

The 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

130 K is crystalline, with a diamond-type cubic structure Ic.13

Cubic ice is metastable at T o 70 K and undergoes a

transformation to the amorphous state (the stable form at

these temperatures) via cosmic ray bombardment and ultra-

violet irradiation.14 The irradiation diminishes the kinetic

barrier between the metastable cubic ice form and stable

amorphous ice form at lower temperatures.15 The crystalli-

sation 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

grains.16 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 To 243 K and

is thickened by dissolved solutes excluded from the ice matrix

to the interface during crystallisation.16

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 is 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 observa-

tions of the Voyager mission and strengthened by the observa-

tions with the Galileo spacecraft.18 Recently, it has been stated

that Europa possesses an active dynamic ice–water system

with cycles of melting and refreezing. In addition, a lenticular

body of liquid brine in the TheraMacula region of approximately

20000–60000 km3 has been predicted.19 The composition of

Europa’s subsurface water, underlying an ice crust, could be rich

in sulphate salts, the source of surface evaporite deposits.20

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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 K.21 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

Titan.22 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

models.23 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 biochem-

ical evolution.25 From these hypotheses based on atmospheric

or surface chemistry, the prebiotic possibilities of liquid water

brines entrapped under ice have received less attention and

are the object of speculative discussion regarding possible

biochemical evolution and the presence of chemoautotrophic

life.26

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 1994,12 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 input.28

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 mm.29 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 conditions within the channels are directly propor-

tional 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 216.30 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 units.31

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 hydrocarbons.32

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 temperature.33 For example, a freezing

dilute urea solution tends to form an interstitial eutectic 8 m

solution with a melting point of 261 K. 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 matrix

Nucleobases 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 nucleobases.34 In addition, nitrogen hetero-

cycles could have been involved in the first metabolic pathways

as cofactors.35

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 authors,36 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 synthesis.37 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,

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a class of meteorites rich in organic carbon and water,38 has

demonstrated the presence of N-heterocycles. These hetero-

cycles include adenine, guanine and triazines (ammeline and

melamine), which were found in the Orgeil meteorite by

Hayatsu in 1964.39 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 chemistry.42 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 Oro 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 possibility.44 Additionally,

a Fischer–Tropsch type synthetic mechanism catalysed by

mineral phases at high temperature has been suggested for the

origin of N-heterocycles in meteorites,45 but its actual signifi-

cance 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 atmospheres.47

In addition, free HCN and cyanide polymers have been observed

in comets, dust particles48 and in the Titan atmosphere.49

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

could undergo further polymerisation to form dark brown

solid polymers, which upon hydrolysis release nitrogen hetero-

cycles, including adenine.50 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 mecha-

nism 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-carbox-

amide (AICA), which could be a xanthine and hypoxanthine

precursor52 (Scheme 1). Formamidine has also been identified

Scheme 1 Synthesis of purines by polymerisation of cyanide to the HCN tetramer and formation of cyanoimidazole derivatives. The related

formation of glycine, formamidine and glycolonitrile was observed in ice–water experiments.

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as an organic precursor found in comets53 and prebiotic

chemistry laboratory simulations.54 A possible major mecha-

nism 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 the HCN

tetramer) to yield the carbamimidoyl cyanide derivative. This

molecule cyclises to 4-amino-2-cyanoimidazole-5-carbimidoyl-

cyanide. 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 eluci-

dation 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 identi-

fied 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 adenine.56

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 evaporation.57

One solution to this problem could be to consider alter-

natives to aqueous HCN chemistry. The formation of nucleo-

bases 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 polymer.59

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

years.60 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 experi-

ment, 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 products.62 Although the HCN pathway has been

extensively studied for the synthesis of purines, it has been

demonstrated that the polymerisation of cyanide could pro-

vide a pathway for the formation of the pyrimidines including

uracil, 5-hydroxyuracil and orotic acid.63 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 253 K and

195 K for 25 years. The analysis indicated the formation of

glycine and small amounts of alanine and aspartic acid.64 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 radia-

tion at 185 and 254 nm;67 the spectrum of this molecule has

been observed in the interstellar medium68 and by the Voyager

mission in Titan’s atmosphere,69 where crystalline condensates

of cyanoacetylene with acetylene may exist.70

The potential prebiotic relevance of cyanoacetylene in origin

of life studies was 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

ices.72

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

253 K to give cytosine and uracil in 0.005% and 0.02% yields,

respectively.73 In the same report, cyanoacetaldehyde reacted

with guanidine at 253 K 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 months.74 This reaction

may proceed through the cyanoacetaldehyde dimer, 4-(hydroxy-

methylene) pentenedinitrile, 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 solution is formed. At 262 K, 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.

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An unresolved issue with the cyanoacetylene pathway in the

synthesis of nucleobases is its reactivity to nucleophiles,75

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 uracil.76 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 a methane/nitrogen

based atmosphere was subjected to eutectic freezing77 at 253 K

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 a nitrogen/methane atmosphere

by spark discharges was performed in 2009.78 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 experi-

ments 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 of pyrimidines by oxidative alteration of

cytosine (UV irradiation, hydroxyl radical addition or other

free radical mechanisms and further oxidation to barbituric

acid) could explain the results observed.79 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 Bada.12

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 bio-

logical compounds, but they could mimic nucleobases

behaviour in nucleic acids and their potential prebiotic role

has been discussed.80 Their presence in meteorites remains

contentious.81

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, such 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 manner.82 Urea molecules in aqueous

fluids tend to form hydrogen bonds with neighbouring water

molecules at both the amino and the carbonyl groups.83

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 forma-

tion of dimers or clusters of urea molecules becomes dominant

at eutectic concentration.84 During freezing, the urea is segre-

gated from pure ice to accumulate in supercooled microfluid

inclusions of a supersaturated solution. This system is governed

by dehydration and association of solute molecules.85 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 sub-

jected 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 into ammonium

cyanate. Further decomposition of ammonium cyanate leads

to 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 observed.84

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. Another parallel pathway is

the formation of melamine by cyanamide polymerization.

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 and goes through a pentanedinitrile

intermediate.

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The melamine hydrolysis yields 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 460 K. 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 temperature;88 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 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 on the ice matrix,

together with the low availability of water vapour, could

explain the preferential synthesis of polycyclic aromatic

hydrocarbons (PAHs) by sparking a methane/nitrogen

atmosphere over an ice matrix.90 The model of PAH synthesis

is interesting because it could confirm the theoretical synthesis

of aromatics by acetylene insertion mechanisms proposed for

Titan’s atmosphere.91 In laboratory experiments at sub-zero

temperatures,65 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 such 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 a-dicarbonyl compounds such as

glyoxal.92 The reaction of glyoxal with urea under mild acidic

conditions yields hydantoin,93 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

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

methane/nitrogen atmosphere.

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generated in water solutions by photolysis or irradiation.79,94

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. As a con-

sequence, hydantoins could be the final products of alteration

of pyrimidines under 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 other alternative mechanisms as

the synthesis of uracil by reaction of urea with acetylene

dicarboxylic acid.95 This acid is the aqueous hydrolysis

product of dicyanoacetylene, which is an exotic product

of methane/nitrogen atmospheres observed in the Titan

atmosphere.96 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 tholins 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 interactions.97

The ice–water system in the origin of nucleic acids

The 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 primor-

dial 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-evaluated.99

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 life.100

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 possi-

bility was performed by Gryaznov and Letsinger in 1993.101 In

their experiment, the coupling of an a-bromoacyl-activated

oligonucleotide (bromoacetylamino-30-desoxythimidine in

the 30-terminus) with another oligonucleotide with a phos-

phorothioate group in the 50-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 concen-

tration of reactants in the fluid cavities in the ice matrix.

Scheme 4 Polycyclic aromatic hydrocarbons and acetylene polymers detected from sparking a methane/nitrogen atmosphere on the water–ice

matrix.

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The enhancing effect of the ice matrix on 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

50-monophosphorimidazolide solution at 255 K at a pH range

between 6 and 8 in the presence of magnesium and lead cations

(Scheme 5).102

The study of the ribonuclease A digestion products showed

that the oligomers obtained are mainly linear and that 30%

carry at least one 30–50 linkage. The fluorescence microscopy

observation of an ice layer under 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 Miller,103 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 interpre-

tation 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 pyrimidine

residues is obtained.105 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 50-overhang as a template.

Similarly, the non-enzymatic synthesis of polyadenosine in a

sea–ice matrix, directed by poly(U), was performed, using

adenosine-50-monophosphate (2-methyl) imidazolide asmonomer.

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 residues

(Scheme 6).107

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 form.108 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 matrix

The 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

agents,109 sulphide minerals,110 melting111 or hydrothermal

systems.112 Further studies suggest important roles of catalytic

surfaces, such as clays, or interfaces created by wet–dry cycling

of monomers on mineral surfaces.113

Based on this idea, Schwendinger and Rode found a parti-

cularly 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 catalyst.114 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 excess.115 However,

the so-called ‘proteinoids’ are mainly heteropolymers containing

only very small quantities of peptide bonds.116 The melting of a

mixture of urea and alanine yields the dipeptide Ala-Ala.81

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 agents.117

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

polymers.118 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 racemisation.119 This idea is connected

to a different approach to the problem of amino acid con-

densation 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 under 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?

Scheme 5 Scheme 6

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Liu and Orgel studied the oligomerisation of b-amino acids in

aqueous solutions under eutectic conditions using activation

by the water-soluble reagents EDAC (1-ethyl-3-(3-dimethyl-

aminopropyl)-carbodiimide) and carbonyl-diimidazole.120 The

oligomerisation of b-amino acids (L-aspartic acid, b-amino

adipic acid, b-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 were almost unaffected

by the solute concentration over a broad range of 0.1 to 100 mM

at 253 K. 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 a-amino

acids, the carboxyl group of the dipeptide can be activated and

then cyclise efficiently to give a diketopiperazine, thus inhibiting

oligomerisation.121 Cyclisation of an activated dimer of b-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

solvents.122 The data demonstrated that the coupling rates in

frozen dioxane at 254 K exceed by approximately one order of

magnitude of 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

solutions.123 However, no further investigation on these

possibilities has been performed.

Concluding remarks

Prebiotic chemistry in the range of stability of a liquid

water–ice interface (277 to 243 K under 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) is 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

the 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 performmore 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 reac-

tions 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.

Acknowledgements

We acknowledge the Centro de Astrobiologia (CSIC-INTA)

and the grants of the project AYA2009-13920-C02-01 from

the Ministerio de Ciencia e Innovacion (MICINN, Spain).

Notes and references

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