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Genetics first or metabolism first? The formamide cluew

Raffaele Saladino,*a Giorgia Botta,a Samanta Pino,b Giovanna Costanzoc and Ernesto Di Mauro*d

Received 6th March 2012 DOI: 10.1039/c2cs35066a

Life is made of the intimate interaction of metabolism and genetics, both built around the chemistry of the most common elements of the Universe (hydrogen, oxygen, nitrogen, and carbon). The transmissible interaction of metabolic and genetic cycles results in the hypercycles of organization and de-organization of chemical information, of living and non-living. The origin-of-life quest has long been split into several attitudes exemplified by the aphorisms ‘‘genetics-first’’ or ‘‘metabolism-first’’. Recently, the opposition between these approaches has been solved by more unitary theoretical and experimental frames taking into account energetic, evolutionary, proto-metabolic and environmental aspects. Nevertheless, a unitary and simple chemical frame is still needed that could afford both the precursors of the synthetic pathways eventually leading to RNA and to the key components of the central metabolic cycles, possibly connected with the synthesis of fatty acids. In order to approach the problem of the origin of life it is therefore reasonable to start from the assumption that both metabolism and genetics had a common origin, shared a common chemical frame, and were embedded under physical–chemical conditions favourable for the onset of both. The singleness of such a prebiotically productive chemical process would partake of Darwinian advantages over more complex fragmentary Downloaded by University of Oxford on 26 July 2012 chemical systems. The prebiotic chemistry of formamide affords in a single and simple

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A physical–chemical frame nucleic bases, acyclonucleosides, nucleotides, biogenic carboxylic acids, sugars, amino sugars, amino acids and condensing agents. Thus, we suggest the possibility that formamide could have jointly provided the main components for the onset of both (pre)genetic and (pre)metabolic processes. As a note of caution, we discuss the fact that these observations only indicate possible solutions at the level of organic substrates, not at the systemic chemical level.

1. Introduction the task is paramount. This endeavour necessarily passes through the untangling of the riddles specific to each type of It is a shared opinion that we will never know exactly how life biomolecules (nucleic acids, proteins, lipids, precursors of started on this planet but that at one point it will be possible to energy metabolisms) and to their initial, possibly cooperative reconstruct the likely chemical frames into which the different interactions. Before even considering the kick-starting events prebiotic processes organized themselves. Understanding the leading to the onset of cycles, the relevant prebiotically fertile key passages leading to their reciprocal interactions will follow. chemical frame(s) must be identified. Or was it rather a multifaceted single frame? The complexity of Building on the rationale illuminatingly defined by Victor Oparin,1 the last six decades have witnessed solid and con- a Dipartimento di Agrobiologia ed Agrochimica, tinuous progress, starting from the seminal Stanley Miller’s Universita` della Tuscia, Via San Camillo De Lellis, 01100 Viterbo, discoveries,2 reviewed in ref. 3. We will here examine ground- Italy. E-mail: [email protected] b Dipartimento di Biologia e Biotecnologie ‘‘Charles Darwin’’, breaking and recent observations related to the formation of ‘‘Sapienza’’ Universita` di Roma, P.leAldo Moro, 5, Rome 00185, the precursors of biomolecules, keeping in mind that an Italy. E-mail: [email protected] exhaustive analysis of this topic is impossible in a single article c Istituto di Biologia e e Patologia Molecolari, CNR, P.le Aldo Moro, 5, Rome 00185, Italy. E-mail: [email protected] and that restrictive choices need to be a priori made. Thus, we 4,5 d Istituto Pasteur ‘‘Fondazione Cenci-Bolognetti’’ c/o Dipartimento di reasoned following a Bayesian inference approach, consisting Biologia e Biotecnologie ‘‘Charles Darwin’’, ‘‘Sapienza’’ Universita` of changing our subjective degree of belief rationally in order di Roma, P.leAldo Moro, 5, Rome 00185, Italy. to account for evidence. When no decision is possible, we E-mail: [email protected]; Fax: +39 064440812; Tel: +39 0649912880 appealed to Occam’s lex parsimoniae, usually phrased: simpler w Part of the prebiotic chemistry themed issue. explanations are, other things being equal, generally better

5526 Chem. Soc. Rev., 2012, 41, 5526–5565 This journal is c The Royal Society of Chemistry 2012 View Online

than more complex ones. As restated by Bertrand Russel: applies to the possible initial cooperation between vesicles ‘‘whenever possible, substitute constructions out of known and RNA,7 and to the relationships among the key compo- entities for inferences to unknown entities’’.6 nents of metabolic cycles. As suggested by Vijayasarathy and The available evidence bayesianally leads to hydrogen Harold J. Morowitz,8 the first set of prebiotic processes cyanide (HCN) chemistry and, inside its frame of reference, reasonably consisted of a small group of organic reactions,

to formamide (NH2CHO) chemistry. The two frames are including the 11 key chemical transformations in the core of not in conflict (as will be discussed) but, rather, the second autotrophic anabolism: hydrolysis/dehydration, carboxylation/ is a chapter of the first. However, the specific attributes decarboxylation, oxidation/reduction, phosphorylation/ of stability, reactivity and the chemical properties of forma- dephosphorylation, isomerization, transamination and transfer mide make it a preferential starting point for prebiotic of functional groups. synthetic processes. We will present evidence pointing to Were these compounds present in the same ‘‘warm little the possible role of formamide-plus-minerals in the syntheses pond’’?9 A brief review of the principal models dealing with of a large part of the relevant starting components in pre- the onset of metabolisms will allow us to point to evidence biotic scenarios, namely nucleic acid components, amino related to a possible formamide-based unitary chemical frame acids, sugars, amino sugars and carboxylic acids. The connec- for metabolism and genetics. Thus, possible passages from the tion of formamide with all these compounds is presented in compounds afforded by formamide to higher complexity Fig. 1. organization levels will be indicated, particular attention being In Occamian terms, if polymerization of ribopolymers had devoted to the spontaneous generation of RNA, to the to occur, all the necessary components had to be present in the problem of the onset of metabolic cycles and to their possible same environment at the same time. A similar reasoning coexistence in the same chemical scenario.

Raffaele Saladino was born in Giorgia Botta was born in Rome in 1964. He obtained a Fredericton, Canada in 1979. Masters degree in Chemistry She received a Masters degree at University ‘‘Sapienza’’, Rome, in Medicinal Chemistry at the in 1989 and a PhD in Chemical University of Siena (Italy) in Sciences at the same University 2005 and a PhD in Medicinal in 1993. After post-doctoral Chemistry from the same

Downloaded by University of Oxford on 26 July 2012 training at the University University in 2008. Since of Montreal (Canada) and October 2010 she has been

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A teaching and research posi- working as a Postdoctoral tions at the University of Research Associate at the Tuscia, Viterbo, Italy he is University of Viterbo (Italy) now an Associate Professor of in collaboration with Prof. Organic & Bioorganic Chemistry Raffaele Saladino. Her Raffaele Saladino at the same University. He is Giorgia Botta research interests are in the involved in studies on prebiotic field of Organic, Analytical chemistry, green catalysis and oxidative transformations of and Medicinal Chemistry with particular interest in the Synthesis natural substances mainly focused on bioactive compounds. of novel Antiviral and Anticancer agents and in the field of Astrobiology with particular interest in the Origin of Life.

Samanta Pino was born in Giovanna Maria Costanzo Priverno, Italy in 1978. She was born in Rome, Italy, in studied Biology at the Roma 1962. She obtained her Degree Tre, University of Rome. She in Biological Sciences from received her PhD in Pasteurian ‘‘Sapienza’’ University of Sciences at the Department Rome, Italy, in 1987. In 1991 of Genetics and Molecular she joined the Dept. of Bio- Biology, University of Rome chemistry and Cell Biology ‘‘Sapienza’’ in 2009, working in at the University of New York the field of prebiotic chemistry. at Stony Brook (USA) as a Her primary research interests postdoctoral fellow. Since are currently focused on the 2001 she has worked as a study of the molecular researcher at CNR in Rome, mechanisms and of the physical– Italy. In the past years her Samanta Pino chemical environment allowing Giovanna Costanzo research interests were the formation of the first essentially on yeast transcription informational polymers on regulation and DNA chromatin structure. At present she is prebiotic Earth. working on the origin of the life.

This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 5526–5565 5527 View Online

Fig. 1 Syntheses from formamide.

2. Chemical precursors and prebiotic processes: the enzymatic systems and follow paths characterized by high formamide and hydrogen cyanide system. chemio-, regio- and stereoselectivity, leading to the formation General considerations of the glycosidic bond only in the b-configuration and, in the case of natural oligonucleotides, exclusively to the diphosphate Downloaded by University of Oxford on 26 July 2012 Nucleic acids are made out of three components, the purine bond at the 30- and 50-position of the sugar.

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A and pyrimidine nucleic bases (adenine, guanine, cytosine, and A plausibly prebiotic synthesis of nucleic acids should not thymine and uracil in DNA and RNA, respectively) and the only occur under experimental conditions coherent with those corresponding nucleosides and nucleotides. The level of structural that prevailed in primitive terrestrial or non-terrestrial environ- complexity increases gradually proceeding from nucleic bases ments, it should also allow the synthesis of nucleic bases, to nucleotides, with the initial formation of the glycosidic bond nucleosides and nucleotides in significant yields, possibly under between the nucleic base and the sugar (20-deoxy ribose or regioselectivity and stereochemical control. ribose), and the subsequent formation of phosphate derivatives. In the definition of the specific experimental conditions under The chemical processes that yield these compounds in extant which the origin of nucleic acids might have occurred, the first organisms are under the complete control of sophisticated step is the identification of chemical precursors that could have served as starting materials. In principle, the ideal chemical precursor should be a compound that is widely distributed in Ernesto Di Mauro was born the Universe, with a molecular weight lower than 300 Da and in Valmontone, Italy, in 1945. an elemental composition containing all the universal atomic In 1967 he obtained his components: H, C, N, and O. For the definition of universal Degree in Biological Sciences atomic components see ref. 10. An estimation of the possible from ‘‘Sapienza’’ University number of covalently bonded compounds with this elemental of Rome, Italy. In 1969 he composition and molecular weight yields millions of possible joined the Department of candidates.11 Among these, hydrogen cyanide (HCN)12 and 13,14 Genetics (Seattle), as a post- formamide (NH2CHO) have received increasing attention doctoral fellow. Appointed in as possible precursors of nucleic acid components. 1978 as an associate professor HCN and HCN polymers (for a recent review on HCN of Enzymology at the University polymers see ref. 15) have been detected in different parts of the of Rome, he has been a Universe including planets,16 satellites,17 dense stars,18 molecular professor of Molecular Biology clouds19 and in the gas phase of the interstellar medium as well 20 Ernesto Di Mauro since 1987. His research as in the comae of several comets. NH2CHO has been detected interests were centered on in interstellar dust associated with theicyphaseindensemolecular gene regulation, DNA and chromatin structure and topology clouds21 and in protostellar infrared sources,22 in comets such and, at present, on the various aspects of the origin of life. as C/1995 O1 (Hale–Bopp)23,24 and C/1996 B2 (Hyakutake),25

5528 Chem. Soc. Rev., 2012, 41, 5526–5565 This journal is c The Royal Society of Chemistry 2012 View Online

Scheme 1

26 in the interstellar medium, in the young stellar object the CN triple bond to give NH2CHO. These two competitive W33A27 and in the galactic sources Sagittarius SgrA and processes show a similar kinetics at relatively high HCN 28,29 SgrB2. Recent data suggest the presence of NH2CHO also concentration, in the range between 0.01 and 0.1 M, and at on Titan and Europa, where a stratosphere of liquid formamide alkaline pH (the optimal value being between pH 8 and 9). The 48 (pure or partially mixed with water) below the frozen surface of hydrolysis of NH2CHO prevails in more dilute solutions. the mantle has been hypothesized.30–32 A theoretical study on the possible concentration of HCN

Scheme 1 describes the basic chemistry of NH2CHO and on early Earth suggested that the steady state concentration of HCN considering selected examples of three main types of trans- HCN in the primitive ocean was 4 1012 M at pH 7 and 100 1C, formations: the preparative reactions, the degradation reactions with a slight increase of concentration at lower temperature and the reactions of conversion between the two compounds. (2 105 M at pH 7 and 0 1C).49 Similar results were obtained From the preparative point of view, HCN can be prepared from for NH2CHO. These concentration values appear to be too Downloaded by University of Oxford on 26 July 2012 gaseous mixtures of methane (CH4) and ammonia (NH3)inthe low for the formation of the appropriate amounts of bio- 33 34

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A presence of platinum or corona discharge, by ion-irradiation molecules suitable for the first living entities. On the other 35 and/or UV-photolysis of N2-rich ice containing CH4, from CH4 hand, alternative mechanisms exist for the solution of this and NH3 at high temperature in the presence of Al2O3 with a problem, as for instance the processes based on the colligative 36 suspension of Pt group metal and nitrides of Al, B, Ti, and Si or properties of the two compounds. HCN and NH2CHO might by yttria-stabilized zirconia (YSZ) cells with Pt electrodes.37 have accumulated through formation of eutectic phase with 49 NH2CHO may be formed from the same mixture of water at low temperature, as reported for HCN, or by evapora- reagents leading to the production of HCN. As an example, it tion processes, a phenomenon that affects mainly NH2CHO, can be prepared in appreciable yield from CH4 and NH3 which is characterized by a high boiling point (>200 1C) without through proton irradiation.38 Other synthetic pathways include azeotropic effects with water. For a comprehensive review on

the ultraviolet irradiation of icy mixtures of HCN, NH3 and NH2CHO properties see ref. 50. Further concentration processes 39 40 H2O, the photolysis of ices, or the pyrolysis of mixtures of might have been favored by the presence of minerals, considering 41 carbon monoxide (CO), NH3 and H2O. NH2CHO can also be as an example the high efficiency of absorption of NH2CHO in synthesized from organic compounds with higher structural clays of the montmorillonites family.51,52 In the latter case,

complexity, as in the case of the reaction between formic acid NH2CHO is included in the empty space between the layers of (HCOOH) and its esters HCOOR with NH3 over a wide range the mineral, mainly due to electrostatic interactions and hydrogen of experimental conditions.42,43 In the latter case various cata- bonds with the oxygen or metal atoms present in the crystal.53

lytic conditions have been identified for the optimization of the These interactions may increase the reactivity of NH2CHO by process, involving the use of alkoxides, ammonium methoxide44 increasing the electrophilic and nucleophilic properties of its and sodium methoxide,45 heterocyclic derivatives such as functional groups (the amide and amino moieties). thiamine hydrochloride,46 or polyols such as glycerine.47 In formamide chemistry, in addition to the direct synthesis

The synthesis of NH2CHO that probably has the major of precursors, degradation phenomena also have a significant significance in prebiotic scenarios is by the hydrolysis of HCN. role in the generation of other reactive species required for HCN is a gas at ambient pressure and temperature. Once different synthetic processes. absorbed in water it undergoes two possible processes depending As an example, in the presence of metal oxides at high

on pH and concentration: (a) direct condensation to biomolecules, temperature NH2CHO can be dehydrated to HCN, thus closing like adenine, or polymerization to poly(hydrogen cyanide) a cyclic transformation based on the hydrolysis–dehydration 42 derivatives that may be transformed further into biomolecules system. Other degradation reactions of NH2CHO potentially (see next section), or (b) the hydrolysis by addition of H2Oon relevant in the prebiotic context are the efficient generation of

This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 5526–5565 5529 View Online

CO and NH3 and other low molecular weight compounds via a characterized by freezing, such as the frozen regions of microwave-assisted thermal decomposition process,54 shock the primitive Earth, icy satellites or meteorites68 (Scheme 2, waves,55 mercury-(3P1)-photosensitized decomposition56 applied pathway B). A three-step synthesis of guanine was also reported as a tool for the synthesis of nitrogen-containing heterocycles.57,58 from frozen basic HCN solutions reacting to form DAMN,69 The generation of HNCO species by thermal decomposition followed by a two-photon photochemical rearrangement to 59 60 61,62 70 on Al2O3 or SiO2 and the pyrolysis to HCN polymers AICN. The process is completed by the reaction with the pre- 48 were also reported. Moreover, NH2CHO is hydrolysed under sumedly prebiotic reagent cyanogen or cyanogen bromide or by both acidic and basic conditions to ammonium formate reaction of AICA with guanidine or urea.71 Scheme 3 reports the + HCOO NH4 . For recent studies on the hydrolysis of proposed route of adenine and guanine formation from HCN. NH2CHO see ref. 63 and 64. This compound has been used For specific references on the proposed mechanism, see ref. 72–75. as a chemical precursor for the nucleic base adenine by Briefly, the HCN trimer, aminomalononitrile (AMN), is reaction at 100 1C with the HCN tetramer.65 obtained by sequential polymerization/addition of cyanide Irrespective of the processing route followed, NH2CHO and (CN ). The value of the rate constant for this addition at HCN were used as starting materials for the synthesis of the alkaline pH (pH 9.2) was 0.096 liter mole1 at 40 1C. Further components of the genetic machinery under experimental addition of CN yields DAMN. DAMN can react with forma- conditions compatible with those of the primitive Earth. In midine to provide the imidazole intermediate that by elimination the following sections these prebiotic syntheses are reported in of CN affords 4-aminoimidazole-5-carbonitrile (AICN). AICN detail and the specificity and selectivity of the reactions are was obtained from DAMN also by an alternative photochemical analyzed. pathway requiring the isomerization of the CQC double bond in the molecule and a ring closure reaction. For a detailed mass 3. Prebiotic synthesis of purine and pyrimidine spectrometry study on the structure of imidazole intermediates see nucleic acid bases ref. 76. In principle, under hydrolytic conditions AICN is in equilibrium with AICA. Both AICN and AICA can afford Since the fundamental Miller experiment2 on the prebiotic adenine and guanine. As an example, the reactions of AICN synthesis of amino acids, attention has been focused on HCN with formamidine or cyanogen afford adenine and guanine, as a potential chemical precursor for nucleic acids. respectively. Similarly, adenine can be obtained from AICA by reaction with HCN. An alternative reaction pathway was also 3.1. The syntheses from HCN proposed that suggests adenine 8-carbonitrile and adenine The first synthesis described in this context is the bona fide 8-carboxamide as key intermediates.77 Interestingly, adenine formation of adenine from a concentrated solution of ammonia can also be synthesized by the reaction between DAMN and Downloaded by University of Oxford on 26 July 2012 and HCN (1.0–11 M solutions of HCN) at reflux for several ammonium formate at 110 1C.78 In addition, the HCN dimer 66

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A days. In addition to adenine, purine precursors such as N-(methylimino)acetonitrile after treatment with AMN yields 4-aminoimidazole-5-carboxamide (AICA), 4-aminoimidazole- the HCN pentamer, a potential precursor of adenine.79 5-carboxamidine (AICAI), formamide, and formamidine were also detected (Scheme 2, pathway A). 3.1.1. The syntheses from HCN oligomers. In the prebiotic On the basis of these data, the authors suggested a reaction chemistry of HCN a key role is played by polymeric products, pathway involving the condensation of the HCN tetramer, usually referred to as HCN oligomers. HCN oligomers, also aminomalonodinitrile (DAMN), with formamidine to known as azulmic acids or azulmins, are heterogeneous 4-aminoimidazole-5-carboxamidine. This imidazole derivative compounds differently colored (from yellow/orange to can further react with formamidine to yield adenine.67 Guanine brown/black) depending on the degree of polymerization was also produced in the range of temperatures encompassed and/or cross-linking. Azulmins are widely diffused in the between 20 and 80 1C in a reaction lasting for long periods of Universe and are associated with dark matter in objects such time (months to years) and in yields ranging from 10 to 40 times as asteroids, moons, planets, comets (see, as examples, ref. 80 lower than those of adenine. This reaction requires high con- and 81) and with the reddish haze (tholins) present in the 82 centrations of NH4CN that can be obtained in environments atmosphere of Titan, the largest moon of Saturn.

Scheme 2

5530 Chem. Soc. Rev., 2012, 41, 5526–5565 This journal is c The Royal Society of Chemistry 2012 View Online

Scheme 3

Under terrestrial conditions HCN can spontaneously poly- The initial steps in the polymerization of HCN are well merize from relatively dilute solutions (>0.001 M HCN) at understood95 and require the formation of AMN and DAMN.

alkaline pH in the presence of bases such as NH3 and of free The mechanism of formation of higher order HCN oligomers radicals from ionizing radiations in a wide range of temperatures is much less clear. Radical species can be also detected in HCN and pressures.15 Other selected simulation experiments for the oligomers under spatial conditions.96 HCN oligomers are synthesis of HCN oligomers include spark discharge activation of precursors of nucleic acid bases and important biomolecules. 83 aqueous aerosols and liquid water in a CH4/N2/H2 atmosphere, As an example, boiling HCN oligomers in 6.0 N HCl yield Downloaded by University of Oxford on 26 July 2012 controlled decomposition of Prussian blue,84 g-irradiation85 and adenine, hypoxanthine, AICN, 3,4-dihydroxypyrimidine 1 86

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A low temperature processes, the first stages of the polymerization (an uracil isomer) and 5-hydroxyuracil 2 (Scheme 5, pathway A), process having been studied from a theoretical point of view while under basic conditions adenine, AICN and orotic acid 3 by computational approaches (see, for example, ref. 87 and 88). were recovered in appreciable yields.97 HCN oligomers can be also prepared by prolonged heating of Different purine derivatives [adenine, hypoxanthine, 2,4-diamino 89 NH2CHO at 185–220 1C, and their stability studied under purine 2,4-(DAMP), xanthine and guanine] were also obtained Earth orbit conditions.90 Several models trying to explain the by acidic or alkaline hydrolysis at high temperature of HCN structure of HCN oligomers have been proposed including oligomers produced in a frozen ammonium cyanide solution at Vo¨lker’s model (double-ladder rod-like structure), Matthew’s 78 1C for 27 years (Scheme 5, pathway B).98,99 model (heteropolypeptide pattern), Umemoto’s model Adenine, hypoxanthine, guanine, uracil, 2 and some carboxylic (mono ladder pattern) and Mamajanov and Herzfeld’s model acid derivatives were detected as adsorbed compounds on HCN (polyimidazole pattern) (Scheme 4).91–94 oligomers prepared from a solution of sodium cyanide and

Scheme 4

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Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A Scheme 5

ammonium chloride at 38 1C (Scheme 5, pathway C),100 while glycine, alanine, aspartic acid, non-protein amino acids and b-amino butyric acid were obtained by spark discharge experi- ments (Scheme 5, pathway D).101

3.1.2. Other synthetic pathways. Among pyrimidine nucleic bases, cytosine and thymine were never obtained in appreciable yields. On the other hand, the presence of relatively high amounts of unusual pyrimidine derivatives (such as compounds 1–3) suggested the possibility that pre-genetic materials could have existed allowing non-standard base-pairing with catalytic properties which were successively lost during molecular evolution.102 Cytosine may also be prepared from chemical precursors characterized by a structure more complex than HCN, as exemplified by the reaction between the cyano- acetylene 4 with cyanate (NCO–) at 30 1C,103 and of 4 with Scheme 6 urea at 100 1C,104 or by reaction between cyanoacetaldehyde 5 105 106 and urea or guanidine, also at 100 1C (Scheme 6). between the prebiotic chemistry of HCN (and HCN derivatives)

Irrespective of the procedure applied, cytosine easily lost and that of NH2CHO is in the role played by minerals. The ammonia to yield uracil. reactivity and selectivity of NH2CHO in the formation of nucleic acid bases can be finely tuned by the chemical and 3.2. The syntheses from H NCHO 2 physical properties of the minerals present in the reaction We have observed that all purine and pyrimidine nucleic medium, thus mimicking a genuinely primitive environment.

acid bases can be simultaneously synthesized from NH2CHO In these reactions the minerals act mainly as heterogeneous (ref. 13, 14 and references therein). The main difference catalysts through the reactivity of the metal atoms present

5532 Chem. Soc. Rev., 2012, 41, 5526–5565 This journal is c The Royal Society of Chemistry 2012 View Online

Scheme 7

in their structure. Simply, heating neat NH2CHO at 160 1C with different physical and chemical properties, a large panel of yields low amounts of purine, while adenine can be synthesized products, including purine, adenine, hypoxanthine, N9-formyl- in traces when the reaction is performed in the presence of purine 6, AICA, 5-formamidoimidazole-4-carboxamide 7 (fAICA), added HCN.107 The mechanism of formation of purine (and cytosine and uracil, were isolated in varying amounts depending 116 adenine) from NH2CHO is not completely understood and, in on the experimental conditions (Scheme 7). principle, both pyrimidine and imidazole derivatives are plausible Chemiomimesis. In accordance with what is known from intermediates for the condensation process. HCN chemistry, the imidazoles AICA and fAICA 7 are Nuclear magnetic resonance experiments performed on expected to be intermediates for the synthesis of both adenine reaction mixtures from labeled reagents showed the possibility and hypoxanthine. AICA ribonucleotide-50-monophosphate that three equivalents of HCN and two of NH CHO are 2 and fAICA ribonucleotide-50-monophosphate are intermediates embedded in the heterocyclic ring through a C–N bond fission in the biosynthesis of inosine-50-monophosphate (IMP), the main process.108 route to purine nucleotides in extant cells. The large similarity between the intermediates of this metabolic process and the 3.2.1. Catalysts: clays. The possibility of a prebiotic role of products observed during the condensation of NH CHO suggests NH CHO may be hypothesized based on thermal condensation 2 2 the possibility of ‘‘chemiomimesis’’, referring to defined bio- analyses performed in the presence of clays, minerals known to

Downloaded by University of Oxford on 26 July 2012 synthetic pathways considered as chemiomimetic of prebiotic have been largely diffused on the primitive Earth and present on chemistry. For a definition of chemiomimesis see ref. 117.

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A other celestial bodies. It has long been proposed that clays were Moving towards the evolution of molecular complexity, involved at different levels in prebiotic chemistry.109 Among clays, clays act as catalysts for the synthesis of the sugar moiety the minerals of the smectite family, such as montmorillonites and required in nucleoside derivatives. The aldol-like condensation kaolinite, have a layered structure composed of silica tetrahedral of formaldehyde (the formose reaction) is the main reaction sheets and alumina octahedral sheets in a ratio depending on invoked for the prebiotic synthesis of sugars (for critical the specific nature of the sample.110 Due to isomorphic sub- reviews see ref. 118 and 119). Noteworthily, the rate of the stitutions, a charge deficiency results that is counterbalanced reaction is increased by the presence of montmorillonites by the exchangeable metal ions (mainly Na+ and Ca+2 in under alkaline conditions,120,121 the sugars being mainly extant clays) held between the layers. Hydration of these metal formed in the interlamellar region of the clay affording stable ions causes expansion of the layers and the intercalation of intercalates.122 Adenine, cytosine and various sugars and organic molecules with appropriate charge and dimension.111 amino sugar derivatives 8–10 were synthesized from a mixture On the basis of these properties, it was suggested that clays of formaldehyde and formamide in the presence of montmor- could have concentrated prebiotic chemical precursors from illonite and dihydroxy acetone (DHA) (Scheme 8).123 In these the surrounding environment influencing the synthesis of reactions formamide provides the nitrogen atom of the amino biomolecules, as well as have protected them against chemical sugars on which nucleic acid bases can be assembled in and photochemical degradation.112 successive steps, while DHA acts as an electrophilic agent The formation of complexes between NH CHO and clays is 2 and an organocatalyst. For examples of conventional procedures usually a rapid and quantitative process, irrespective of the for the preparation of sugars see ref. 124 and 125. Clays were also elemental composition, morphology and crystallinity of the shown to be involved in the montmorillonite-catalyzed domino mineral.113 The guest NH CHO is arranged in the interlayer 2 one-pot [3+1+2] p coupling reactions between unprotected space in an ordered supramolecular structure based on a hexoses, amidines and natural amino acids to yield pyrimidine hydrogen bond network between the oxygen and nitrogen nucleoside analogues.126 atoms of the amide moiety and the adjacent silica and alumina sheets. For selected examples of interactions between clays and Clays as protective and organizing agents. In addition to the formamide see ref. 114 and 115. reported catalytic properties, clays probably played a crucial The noncovalent interactions and the ordered layout of the role in other prebiotic processes relevant for the origin of life. guest molecules can activate condensation processes. Thus, These minerals might have provided, in the formation of

when NH2CHO is heated in the presence of montmorillonites biopolymers, a protective environment against degradation,

This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 5526–5565 5533 View Online

Scheme 8

allowing their persistence and the possibility to self-replicate 3.2.2. Catalysts: metal oxides. Similarly to clays, simple

and evolve. In this scenario, clays can perform as an aggre- metal oxides, such as silica (SiO2), alumina (Al2O3) and gation nucleus to gather and organize key components of titanium dioxide (TiO2), are able to absorb biomolecules from the primitive cell. Inorganic cations and pH modulate the aqueous solutions promoting their polymerization. In this adsorption/binding process of nucleotides and nucleic acids on process, the formation of hydrogen bond networks between clays by reducing the electrostatic repulsion between absorbed monomers causes the condensation to proceed faster.144

negatively charged phosphates and the surface of the mineral. As for nucleic acid bases, both SiO2 and Al2O3 catalyze For examples of the role of cations and pH in the interaction the thermal condensation of NH2CHO to yield purine, between nucleic acids and clays, see ref. 127–130. adenine, cytosine and 4(3H)-pyrimidinone 11 (Scheme 10).145 Purines are adsorbed better than pyrimidines in the presence Compound 11 (Scheme 9) was observed as a minor pyrimidine of cations,128,131,132 and oligonucleotides are adsorbed on the derivative in the Murchison meteorite.146 Adenine was isolated

mineral surface with different selectivity depending on the in high yield in syntheses in the presence of SiO2, while Al2O3 nucleobase sequence and tertiary structure.133,134 Irrespective afforded mainly 11. The isomeric compound 2(1H)-pyrimidinone

of the mechanism of interaction between clays and nucleic 12 was synthesized in high yield from NH2CHO in the 147 acids (for a mechanism based on hydrogen bonding see presence of ZrO2 (Scheme 10). Compound 12 was used as Downloaded by University of Oxford on 26 July 2012 ref. 135, for coulombic attraction and van der Waals inter- starting material for the first successful instance of formation

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A actions see ref. 136), after the absorption nucleic acids were of the b-glycosidic bond between a pyrimidine derivative and a shown to retain their biological activity. Chromosomal DNA sugar under plausible prebiotic conditions.148

adsorbed on clay minerals could be amplified by Taq Titanium dioxide (TiO2) accounts for an appreciable part of polymerase,137 and both chromosomal and plasmid DNAs were Earth’s crustal rocks and is largely present in the Universe.

able to transform competent bacterial cells in the presence of Examples of detection of TiO2 in terrestrial and extraterrestrial 138 montmorillonite and kaolinite. Clay–nucleic acid complexes bodies are in ref. 149 and 150. NH2CHO treated at 160 1C enhance the stability of DNA and RNA towards agents of in the presence of TiO2 (in anatase phase) yields purine, biological origin (DNases, RNases and aspecific nucleases adenine, cytosine, thymine, 5-hydroxymethyl uracil (5-HMU), activities) and physical and chemical degrading factors (high N9-formylpurine 6, N9,N6-diformyladenine 13, and purine temperature, hydrolysis, UV and X-ray radiation).139,140 acyclonucleosides 14–16 (Scheme 10).151 Compounds 14–16 Clays also play a role in the supramolecular organization of have a sugar-like side chain bonded to the N(9)-atom in the a membrane-like boundary structure necessary for the onset of purine scaffold, and are currently referred to by the term cellular life. For a comprehensive review on chemistry and acyclonucleosides. Acyclonucleosides can in principle pair by physics of primitive membranes see ref. 141. In particular, the Watson–Crick interactions similarly to natural derivatives, surface of montmorillonite may bring together and organize and are possible components of pre-RNA and pre-DNA RNA molecules and fatty acids to afford complex systems that molecules.152,153 at the same time contain a catalytic site for prebiotic processes Since formaldehyde (HCOH) can be generated in situ from 154 (e.g. the polymerization of activated nucleotides) and informa- NH2CHO and TiO2, it probably played a key role in the tional biopolymers within a membrane. In this process, the formation of 14–16. HCOH was shown to be able to add as a formation of vesicles occurs in the proximity of the surface, finely tuned by the surface area and by the radius of curvature of the particles.142 Since the adhesion interactions of growing oligonucleotides with the surface increase by increasing the value of their molecular weight, the presence of vesicles can promote the release of newly synthesized biopolymers in the space between the mineral and the surface, thus enhancing their biological activity.143 This self-assembled abiotic system may represent an amazing self-replicating cell-like entity. Scheme 9

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Scheme 10

nucleophilic reagent on the formyl moiety of 6 and 13 in a acid bases was supposed for the synthesis of hypoxanthine by

formose-like condensation, growing the side chain through the UV irradiation of a mixture containing KCN, NH3 and 158 subsequent addition of a carbon and oxygen atom unit. H2O. Better results were obtained by UV irradiation of These data are of particular prebiotic relevance because of NH2CHO in the presence of a TiO2 (001) single crystal surface the known difficulty of building the b-glycosidic bond between under high vacuum and dry conditions.159 Under these experi- Downloaded by University of Oxford on 26 July 2012 nucleic acid bases and sugars.155 The nucleophilic addition of mental conditions adenine, cytosine, uracil, thymine, guanine

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A HCOH to the reactive C(5)-position of uracil is also a key step and HCN oligomers were obtained in very low yield for the previously unreported one-pot synthesis of thymine (Scheme 11). Due to the complexity of the mixture recovered and 5-hydroxymethyl uracil.156 after thermal desorption of the sample during the analytical procedures, the unambiguous identification of the products 159 Photocatalysis. Rutile and anatase TiO2 are photocatalysts was admittedly impossible. with a value of transition gap-bands energy of 3.0 and 3.2 eV, More recently, similar results were obtained by UV irradia-

respectively. Once the energy of UV photons is adsorbed by tion of NH2CHO in the presence of mineral phosphates during the surface of the mineral, interfacial electron transfer is heating. Relatively to the simple thermal condensation, a promoted between the excited semiconductor surface and 15-fold increase of the yield of adenine was obtained by adsorbed molecules. For a recent review see ref. 157. A possible photons irradiation. The observation that purine nucleobases

photocatalytic role of TiO2 in the prebiotic synthesis of nucleic are formed in UV-irradiated/heated NH2CHO solutions in

Scheme 11

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Scheme 12

the absence of inorganic catalysts (i.e. with ‘‘100% less salt’’) reactions in the parent bodies of carbonaceous meteorites could further relaxes the requirements for obtaining nucleic acid give rise to organic compounds engaged in both genetic and bases under prebiotic Earth conditions.160 metabolic apparatuses, introducing on the scene two key compo- nents of primitive life. The meteorite minerals had little or no 3.2.3. Catalysts: silicates and phosphates. Silicates and effect in promoting hydrolysis of RNA (see Section 8.2.1) at 80 1C phosphates also catalyze the synthesis of nucleic acid bases over a pH range from 4.2 to 9.3, the highest stability being 169 from NH2CHO. As an example, the thermal condensation of observed in the neutral pH range, with a half-life of 5 h. NH2CHO in the presence of silicates with different magnesium and Minerals can in principle perform at the same time as iron content, such as fayalite (FeSiO4), olivine (MgFeSiO4)and catalysts and reactants. This is the case of mineral phosphates. forsterite (MgSiO4), afforded cytosine, uracil, 4(3H)-pyrimidinone Most of the phosphorus in the early Earth would have been in 11 and 5,6-dihydrouracil 17, in addition to the simple purine the form of insoluble minerals. On the other hand, chemical and urea. The reaction showed the highest efficiency when and physical conditions exist generating reactive phosphorus performed in the presence of cosmic-dust analogues (CDAs) of from these insoluble materials. As an example, lightning olivines (Scheme 12).161 CDAs are ‘‘fluffy’’ grains of amorphous discharges in model prebiotic atmospheres reduce ortho- Downloaded by University of Oxford on 26 July 2012 silicates with variable proportions of silicium and carbonaceous phosphates to reactive phosphites.170 162 171 172

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A matrices, prepared by laser ablation techniques and repro- Soluble inorganic phosphates and phosphonic acids ducing the chemical composition and morphology of the silicate were used for the phosphorylation of nucleosides in water to dusts present in large amounts in space environments.163 yield mixtures of nucleotide derivatives. The efficiency of this As a general trend, the yield of nucleic acid bases increased reaction increased in the presence of condensing agents containing by increasing the amount of iron in the elemental composition activated multiple bonds (cyanamide and urea). A selection of of the mineral in the following order of reactivity: fayalite > relevant reference is in ref. 173. Further discussion on this olivine > forsterite, suggesting the possibility of a redox step topic is in Section 5. Reviews on phosphorylation, condensation as a crucial process for the formation of the pyrimidine scaffold. or polymerization of biomolecules with (poly)phosphates poten- These data show that CDAs are favorable microenvironments tially produced by volcanic activity have been reported.174–176

mainly for the synthesis of pyrimidine nucleic acid components, Simple inorganic phosphates (Na3PO4,Na4P2O7 and Na5P3O9) 164,165 2+ which are not easily obtained under terrestrial conditions. and mineral phosphates like hureaulite [Mn (PO3OH)2(PO4)2- 2+ 2+ CDAs analogues of silicates are also efficient catalysts for the (H2O)4], libethenite [Cu 2(PO4)(OH)], turquoise [Cu Al6- 2+ synthesis of simple organic compounds, such as hydrocarbons, (PO4)4(OH)8(H2O)4], childrenite [Mn (AlPO4(OH)2H2O], 2+ 2+ amine and nitrile derivatives, through Fisher–Tropsch- and vivianite [Fe 3(PO4)2(H2O)8] and vauxite [Fe Al2(PO4)2- 166 Haber–Bosch-like condensations at high temperature. In (OH)2(H2O)6] catalyze the thermal condensation of NH2CHO addition, silicates such as alkali feldspar [(Na, K)AlSi3O8]and to yield a large panel of nucleic acid precursors, including purine, clinopyroxene [(Ca, Mg, Fe)SiO3], present in rocks of the adenine, hypoxanthine, cytosine, uracil, 5,6-dihydrouracil 17,the Earth’s crust as well as on other planets of the Solar system, amino acid derivative N-formyl glycine 18 and low molecular are characterized by chiral crystal surfaces.167 weight compounds such as urea, parabanic acid 19 and Such natural chiral environments may provide stereoselective carbodiimide 20 (Scheme 13).177 functions and resolution of racemic mixtures in prebiotic processes. For a general recent review see ref. 168. Condensing agents. Even though the glycine derivative 18 Mineral components of the Murchison meteorite were was previously synthesized from mixtures of ammonia, carbon 178 investigated in terms of potential catalytic effects on NH2CHO dioxide and water by electric discharges, its formation from chemistry.169 Under thermal conditions the mineral surfaces formamide was unprecedented. Carbodiimide 20 is one of the

catalyzed condensation reactions of NH2CHO to form nucleic most powerful condensing agents in organic chemistry for the acid bases in addition to carboxylic acids, amino acids and sugar formation of amide or ester bonds.179 In the formation of

precursors. These results suggest that NH2CHO condensation peptide bonds this compound acts by initial addition of an

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Scheme 13

amino acid on its carbon atom with high electrophile character. The fundamental chemical step in this synthesis is the for- This process generates a good leaving group that can be mation of the b-glycosidic bond, which in extant molecules eliminated as an urea molecule during the formation of the allows the linkage between the nitrogen atom located on the Downloaded by University of Oxford on 26 July 2012 new bond between the two amino acid residues.180 Dehydration nucleic acid bases and the anomeric carbon of the sugar, the 181 0 Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A of urea yields back carbodiimide. ribose or the 2 -deoxyribose in RNA or DNA, respectively. In The presence of both urea and carbodiimide among the Nature, important limitations exist for the formation of this products of condensation of formamide suggests the existence bond. As for regiochemistry, the purine heterocyclic bases may of a carbodiimide–urea organocatalytic cycle, explaining the only use the nitrogen atom at the N(9)-position, while the presence of N-formylglycine 18 in the reaction mixture. In fact pyrimidines only the nitrogen atom at the N(1)-position. glycine, once formed in the presence of carbodiimide and of an As for stereochemistry, the glycosidic bond must be in the excess of formamide, can be easily formylated by a reaction b-configuration, i.e. the nucleic acid base must be facing mimicking the formation of a dipeptide. In principle, the upwards relative to the molecular plane of the sugar. In carbodiimide generated by degradation of formamide can principle, three possible routes can be followed for the synthesis catalyze the formation of both peptides and oligonucleotides, of nucleosides that meet these requirements. Assuming the the mechanism of formation of the ester bond being similar to contemporaneous prebiotic synthesis of the two reagents, the that of the amide bond. The possible role of the carbodiimide– nucleic acid base and the sugar, these molecules must directly urea cycle in the formation of N-formyl glycine is shown in the react with each other via a reaction that may be substantially lower part of Scheme 13. considered a dehydration process. Multisteps construction of

In conclusion, NH2COH affords all the nucleic bases the nucleic acid base on a sugar or the building of the sugar on present in extant nucleic acids. It does so in nonfastidious a nucleic acid base is possible alternatives. Examples of these reactions consisting in heating in the presence of one of a large three possibilities are briefly described below. class of simple and abundant catalysts. The instances of catalysts as metal oxides, clays, silicates and phosphates have 4.1. Dehydration procedures been here presented in some detail. Other catalysts (iron– sulfur minerals, borates, zirconium minerals and materials The easiest procedure to carry out a dehydration process from Murchison meteorite) afford, in addition to nucleic acid consists in reacting the nucleic acid base and the sugar in bases, also carboxylic acids and will be discussed in Section 10. anhydrous phase to favor the elimination of water during the reaction. However, this procedure does not allow for an 4. Prebiotic synthesis of nucleosides adequate regioselectivity, since the exocyclic nitrogen atoms (primary amino groups in both purines and pyrimidines) are The formation of purine and pyrimidine nucleosides is one of the more reactive as nucleophiles than the endocyclic N(9)- or least understood aspects of the prebiotic chemistry of nucleic acids. N(1)-atoms. As an example, when adenine and guanine were

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Scheme 14

treated with D-ribose by heating in dry phase, 6-ribosylamino syntheses of purine nucleosides under drying conditions adenine 21 and 2-ribosylamino guanine 22 were obtained as include the preparation of adenosine and deoxyadenosine the only recovered products. A slightly better regioselectivity from dry mixtures of adenine and sugars in the presence of 184 was observed when performing the reaction in the presence of feldspar, schist, TiO2, and ZnO, and the synthesis of magnesium salts and inorganic phosphates, in which case the deoxyadenosine in the presence of phosphates.185 More recently nucleosides riboadenosine and riboguanosine were detected it was discovered that the drying and heating of 2-pyrimidinone in low yield (4% and 9%, respectively) as a mixture of a and 12 with ribose produces a b-furanosyl ribonucleoside (named b-isomers (Scheme 14, pathway A).182 zebularine) 23 approximately with a 12% yield, in addition to Downloaded by University of Oxford on 26 July 2012 The increased regioselectivity of the reaction was probably other chemical isomers (Scheme 15). This work is the first

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A due to the occurrence of coordination processes involving the synthesis of a pyrimidine nucleoside derivative from a free nitrogen atoms of the nucleic acid bases. A detailed study on base and a non-activated sugar under drying conditions. the stereoselectivity of the reaction was performed using A comparison of 2-pyrimidinone with the purine bases that hypoxanthine, a purine nucleic acid base free of exocyclic amino have also been shown to form nucleosides in plausible pre- groups. The heating of hypoxanthine under solid-state conditions biotic reactions provides insights into which chemical features

in the presence of apatite, CaHPO4, MnSO4, (NH4)2SO4 and of the bases facilitate glycoside formation in drying–heating of a mixture of salts with a composition similar to the reactions.148 inorganic residue obtained after sea water evaporation yielded Detailed computational studies on the formation of 9-D-ribopyranosylhypoxanthine, a-inosine and b-inosine, zebularine at the density functional theory (DFT) level were the b-inosine being the main reaction product (Scheme 14, performed to explain the catalytic role of Mg2+ ions in the pathway B). reaction mechanism. The data showed that Mg2+ ions act as The maximum yield of b-inosine calculated on the basis of a Lewis acid, affording the necessary stabilization at the the initial amount of hypoxanthine was 20%. Similar results pyrimidinone derivative and promoting the elimination of a were obtained in the case of adenine, guanine and xanthine. water molecule during the glycoside bond formation. In addi- Natural pyrimidines failed to produce the corresponding nucleo- tion, predictions concerning the formation of nucleosides from sides under similar experimental conditions.183 Other prebiotic other pyrimidine bases were formulated, providing valuable

Scheme 15

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194 insights into the chemical features that might facilitate glyco- pyrimidine nucleic bases. As an example, D-ribose 5-phosphate 186 side formation in drying–heating reactions. 24, D-ribose 25, and D-arabinose 26 react with cyanamide in Organic Bro¨nsted acids,187 activated silyl reagents,188 the aqueous solutions to yield the corresponding aminooxazolines Vorbruggen glycosylation reaction and the silyl-Hilbert–Johnson 27–29, which further react with cyanoacetylene to yield reaction,189 glycosyl trifluoroacetimidates,190 Lewis acids in a-cytidylic acid 30, a-cytidine 31,andb-arabinosylcytosine 32, water191 have all been tested in the synthesis of nucleosides respectively (Scheme 16). Anomerization and epimerization of with varying degrees of efficiency. Their prebiotic valence has these and of related nucleosides and nucleotides occurred yet to be determined. upon irradiation in aqueous solutions (unfiltered 253 nm light source).195 4.2. Multi-steps construction of the nucleic acid base on a Similarly, pyrimidine nucleoside derivative analogues of sugar natural products have been synthesized, as in the case of An alternative route for the synthesis of nucleosides passes 6-methyluracil nucleoside 32 obtained by cyclocondensation 196 through the preparation of suitable heterocyclic sugar precursors between diketene and furanooxazoline 29 (Scheme 17). with one or more activated positions able to act as starting A significant improvement in the use of aminooxazolines in material for the construction of the nucleic acid base. Among all prebiotic chemistry has recently been described for the synth- 0 0 the heterocyclic precursors studied, aminooxazoline derivatives esis of b-ribocytidine-2 ,3 -cyclic phosphate. In this latter case, have certainly led to the most interesting results in relation to the furanooxazoline 29 was treated with cyanoacetylene to prebiotic chemistry. For a general review on the topic see ref. 192 give the anhydroarabinonucleoside 33, which subsequently and 193. The carbamimidic moiety in the aminooxazoline ring undergoes phosphorylation with rearrangement to afford 0 0 197 supports various condensation processes with formation of b-ribocytidine-2 ,3 -cyclic phosphate 34 (Scheme 18). Downloaded by University of Oxford on 26 July 2012 Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A

Scheme 16

Scheme 17

Scheme 18

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Scheme 19

4.3. Multi-steps construction of the sugar on the nucleic acid the sea bed as insoluble salts. The plausibility of polyphos- base phates or phosphate esters as a prebiotic reagent has been examined.175 Nucleoside derivatives can also be produced by the construc- In a series of ground-breaking studies appeared during a tion of the sugar on suitably modified nucleic acid bases. decade,212–215 from 1976 to 1988, Allen M. Schoffstall A simple example to illustrate this aspect is represented by reported the phosphorylation of nucleosides in reactions formyl purine derivatives. Formyl purines are bioactive com- occurring in water and in formamide, the source of phosphate pounds,198 sometimes used as reactive intermediates for the being provided by inorganic phosphates. Formamide proved synthesis of more complex nucleosides.199 These compounds its worth in this reaction, and appreciable yields of nucleotides may be of particular interest in prebiotic chemistry if involved obtained in a simple, prebiotically plausible reaction were for in processes of synthesis of sugars via condensation reactions the first time reported. As far as we know, these studies were of the ‘‘formose’’ type. For recent examples of formose-like not extended. Building on them, we tested a series of phos- synthesis of sugars see ref. 200–203. Formyl purines are in fact phate minerals, chosen as a representative of different classes, characterized by the presence in their structure of a masked in a simple reaction consisting in the heat treatment (130 1C, glycosidic bond corresponding to the electrophilic side-chain 72 h) of each phosphate mineral in formamide, followed by the carbon atom. As mentioned above, during thermal condensa- addition of a nucleoside, and reacted under various conditions Downloaded by University of Oxford on 26 July 2012 tion of NH CHO in the presence of TiO some purine 2 2 at temperatures encompassed between 60 and 90 1C.216,217 acyclonucleosides formed (compounds 14–16, see Scheme 10)

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A Two noticeable results were obtained: (i) upon treatment with in which ribose or deoxyribose is replaced by a non-cyclic formamide, numerous phosphate minerals acted as phosphate chain. Acyclonucleosides are bioactive compounds able to pair donors and phosphorylated nucleosides with an efficiency with nucleic acid bases by Watson–Crick interactions.204,205 comparable to that of the reference soluble KH PO . Phos- Their possible prebiotic role has been discussed from a theo- 2 4 phorylation occurred at any of the possible positions of the retical point of view,206 and experimentally evaluated in the sugar moiety: 20,30,50 and 20,30 or 30,50 cyclic. For differential case of glycerol-derived acyclonucleosides able to polymerize reactivity of the various positions, see ref. 216 and 217. after activation as phosphate derivatives.206 In the case of 14 Hydroxylapatite proved to be the most efficient phosphoryl- and 16, the HCOH produced in situ by TiO -catalyzed degra- 2 ation source, followed by several copper-containing minerals, dation of NH CHO can add to the electrophile side-chain 2 in the order: libethenite Cu2+ (PO )(OH), cornetite (Cu2+ - carbon atom, increasing step by step the complexity of the 2 4 3 PO (OH) ), ludjibaite (Cu2+ (PO ) (OH) ), reichenbachite alicyclic sugar side-chain (Scheme 19). In principle, the side 4 3 5 4 2 4 (Cu2+ (PO4) (OH) ). chain can grow up to 5 or 6 carbon atoms making intra- 5 2 4 The role of copper in phosphorylation is not unprecedented. molecular cyclization reactions possible. It was shown that copper ions can act as catalysts in phos- phorylation reactions: the phosphorylation of a variety of 5. Nucleoside phosphorylation sugars and of structurally related compounds is catalysed by copper salts,218 copper ions interacting with the phosphate The source of phosphate and the prebiotic routes for the moiety activating it towards nucleophilic additions.219 In transformation of nucleosides into nucleotides has been matter addition, copper ions are involved in the nucleoside phosphoryl- of early discoveries and debate,171,207–210 reviewed and for- ation activity of enzymes such as purine nucleoside phosphorylase, malized by Frank H. Westheimer.211 Additional discussion is a key enzyme in the purine-salvage pathway.220 by Yamagata et al.,174 highlighting both the problems and the Indications were obtained that on certain minerals the possible solutions. The problems essentially derive from the phosphorylation reaction occurred onto the mineral surface scarce solubility of the majority of phosphorus-containing (prominently, in the case of libethenite and hydroxylapatite).216 minerals (notably, of the most diffuse: hydroxylapatite). As Surface reaction studies were not extended. Nevertheless, the possible solution, it was reported that volcanic activity produces data reported allowed us to conclude that in these minerals

water soluble phosphates through partial hydrolysis of P4O10. surface interactions are a requirement of the reaction, as However, as pointed out,174 phosphates would precipitate to shown by the fact that phosphate solutes pre-released from

5540 Chem. Soc. Rev., 2012, 41, 5526–5565 This journal is c The Royal Society of Chemistry 2012 View Online

the minerals and separated from them were essentially inert, attempts to abiotic polymerization that cyclic nucleotides were and that the presence of the mineral was necessary for the a promising possibility. phosphorylation process to occur at high rate. Given that the 30–50-linked hexa-adenylic acid with a 20,30-cyclic phosphate phosphate component of the mineral was transferred to terminus couples on a polyuridylic acid template in the the recipient nucleoside (adenosine), these minerals cannot presence of ethylene diamine to form the dodecamer and be classified as catalysts. The catalyst function is exerted by octadecamer.243 The bond produced was largely that of the formamide. 20–50 isomer, but about 5% of 30–50 bonds also formed. The Under the reaction conditions analyzed, the life span of the same authors observed244 that upon annealing with a 30–50- 1 0 phosphorylated forms lasted several hundred hours (i.e. t2 of linked complementary polyU strand the stability of the 2–5 AMP at 90 1C in water is 4.5 102 h).216,221 Under these bond becomes about 900-fold lower than that of the 3–50 conditions the cyclic forms proved to be more stable, thus bond. This helical conformation-induced selective instability becoming the predominantly remaining compounds and a rapidly led to a majority of the ‘‘natural’’ 30–50 phosphoester potential source of abiotically activated nucleotides. bonds. A theoretical model on the early chemical evolution of nucleic acids based on these findings was presented.244 These 6. Polymerization studies support the prebiotic relevance of the formation of 20,30-cyclic AMP driven by soluble phosphate donors in The origin of genetic materials is not known. None of the formamide,216 and of the orders-of-magnitude larger stability fundamental questions has been definitively answered. The of this monophosphate.244 origin of the usage of the extant 4 bases has been a matter of Michael S. Verlander et al.245 reported in 1973 the self- long standing interest. Did the earliest nucleic acid start only polymerization of 20,30-cyclic adenosine monophosphate. By with purines, or only with a single purine–pyrimidine couple evaporating cyclic 20,30-phosphate from solution in the (possibly G:C),222 or with a variety of different purine– presence of simple catalysts such as aliphatic diamines at pyrimidine combinations derived from a complex pool that alkaline pH, maintaining the dry state at moderate temperatures was progressively simplified? An access to the specific litera- (25–85 1C), self-polymerization to give oligonucleotides of ture is in ref. 223–225. chain-lengths up to at least 6 nucleosides was observed. The Somewhat simplifying, and assuming that the genetic products contained an excess of 30–50-linkages over 20–50- materials originated using the same purine and pyrimidine linkages. After the analysis of the high molecular weight bases present in extant nucleic acids, the general questions material from the polymerization of 20,30-cyclic adenosine pertain to three different topics: the structure of the polymeric phosphate,245 the use of cyclic nucleotides as a source of chains, the abiotic mechanism leading to their formation, their naturally activated precursors was not further explored. Downloaded by University of Oxford on 26 July 2012 relative stability properties. In the polymerization process of nucleic acids, organisms

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A activate the monomers by converting them to phosphorylated 6.1. Precursors derivatives and then utilize the free energy of phosphate Was RNA preceded by other forms of polymeric organization hydrolysis to drive the reaction. Do extant polymerization of nucleic bases? Numerous nucleic bases-based polymers with processes mimic spontaneously occurring prebiotic reactions, totally different structures have been proposed, analyzed in thus representing a sort of biochemiomimesis117,246 descending detail and reviewed.226–234 The particularly interesting pre- from ancient pathways, or should it be considered a fully novel biotic properties of Peptide Nucleic Acid (PNA) have been cellular invention? reported.153,235–239 The properties and the advantages of the The need for activated precursors depends on the fact that phosphodiester chain as-we-know-it have been established none of the key polymers (polysaccharides, peptides, and since long, and reviewed.211,230 The reason why natural polynucleotides) may spontaneously form in an aqueous DNA is based on 20-deoxyribose with 30–50-phosphodiester solution from their monomers because of the Gibbs free- links was determined240,241 through the synthesis and the energy change, as critically reviewed.247,248 Thermodynamic analysis of the iso-DNA, based on 3-deoxyribose that makes considerations impose that amino acid polymerization or the 20–50 links instead of the normal 2-deoxyribose that makes formation of phosphodiester or glycosidic linkages will be 30–50 links. Iso-DNA makes a much weaker double helix with spontaneous only under highly dehydrating conditions. its conjugate or with the conjugate bases on normal DNA. Thus, either (i) life did not arise in aqueous environments, or Did RNA polymerization in prebiotic scenarios become (ii) pre-genetic polymerizations required activated monomers. possible only after peptide-based (proto)enzymatic activities The observation that cyclic nucleotides may form from nucleo- had reached sufficient complexity and diffuse availability? Or, sides and a phosphate source by heating in formamide216,217 in contrast, did RNA formation rely on the intrinsic chemical indicates that the two alternatives mentioned above are not properties of its precursors, its subsequent evolution being necessarily in contradiction: activated monomers can form made possible by its ribozymic242 abilities? under prebiotic conditions in a liquid, non-aqueous environ- Applying Occamian logics one might reformulate the ques- ment in the presence of phosphate minerals under conditions tion: which were the RNA precursors that met the criteria: that are compatible (see Section 6.2) with the thermodynamics nonfastidious abiotic formation, moderate reactivity (sufficient of polymerization. to allow polymerization when the right conditions are met, but A different approach to the activation of precursors for not too strong to prevent their accumulation because of RNA self-polymerization has met large success and has proven instability). It did not escape the attention of pioneering invaluable for studies of numerous aspects of polymerization

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in different systems. The ground-breaking finding goes back to A recent set of experiments showed that concentration can 1968, when Bernd J. Weimann et al.249 reported template- be easily obtained in solution by thermophoresis (or Soret directed synthesis with adenosine-50-phosphorimidazolides. effect), a phenomenon consisting in the movement of molecules This type of compounds is possibly difficult to reconcile with along a thermal gradient. If thermophoresis is perpendicular prebiotic scenarios because of the complexity of their prepara- to the convection flow and inside a long chamber, accumula- tion, decreasing the likelihood of their abundant presence in tion becomes very efficient. Thermophoresis was exploited to acellular environments. A critical evaluation of the require- obtain extreme accumulation of nucleotides in a simulated ments for a reaction to be considered as ‘‘prebiotic’’ was hydrothermal pore system,267 of DNA molecules, and to formulated by Lesley E. Orgel.250,251 mimic an abiotic replication system.268 Even considering Nevertheless, the possibility of prompt nonenzymatic poly- prebiotic environments different from hydrothermal pores, merization of nucleic acids has largely benefited the field, from thermophoresis is a physical phenomenon that is general nonenzymatic template-directed synthesis of RNA copolymers,252 enough to be applied to other scenarios. It provides the to the studies of the catalytic effects of minerals (notably proof-of-principle for the possibility to obtain elevated con- clays10,253–257), to molecular evolution studies.258,259 An inter- centrations of reactants from dilute solutions in the absence of esting alternative was presented by Kanaya and Yanagawa cellular structures. using cyanogen bromide for the template-directed polymeriza- Cyclic nucleotides provide an answer to the need for high- tion of oligoadenylates on polyU.260 energy precursors. The free energy for phosphodiester bond A detailed analysis of polymerization studies from highly formation was calculated to be 5.5 kcal mol1.269 The activated precursors is not within the aims of the present enthalpy of hydrolysis of cyclic nucleotides was calculated to review. Appropriate introduction to the related literature can be encompassed between 7.7 and 14.1 kcal mol1, depending be found in ref. 7, 143 and 261. on the type of bond (30,50 or 20,50)andthetypeofnucleoside.270 The reaction is a transesterification, does not involve hydrolysis and therefore is almost isoenergetic. This reaction requires no 6.2. The (inefficient) polymerization of cyclic purine enzyme and relies on the possible correct positioning of the nucleotides in solution reacting species. The low efficiency of a polymerization reaction releasing water The solubilization of 30,50-cyclic GMP (30,50-cGMP) and of to occur in water caused by the Gibbs free-energy change was 30,50-cyclic AMP (30,50-cAMP) in water,271 in formamide or in mentioned, and the biological solutions can be manifold. In N,N-dimethylformamide,272 followed by heating at moderate addition to the production of biotically synthesized high- temperatures resulted in the formation of oligomers up to a energy compounds (i.e. polyphosphorylated nucleotide deri- length of 30 units for 30,50-cGMP and to shorter oligomers Downloaded by University of Oxford on 26 July 2012 vatives) extant reactions usually take place in hydrophobic for 30,50-cAMP. Conditions for the polymerization of cyclic

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A pockets inside enzymes, and concentration is programmed and pyrimidines have not been found. The polymerization reaction often exploited as a regulatory mechanism. is not efficient, and the highest yield obtained was 1.3% of the The concentration of reactants for polymerization is a input monomer. This yield was obtained under the optimal problem difficult to deal within prebiotic terms. Did reactions conditions of time (1 h), temperature (80 1C), pH (9.0), presence of occur inside (potentially concentrating) vesicles,7,262,263 onto enhancers (like dimethylformamide or 1,8-diazabicycloundec-7-ene) potentially concentrating mineral surfaces,253–257 in brines of the base-dependent catalysis on which the reaction relies, concentrating by dehydration,254,264 in lipid environments by concentration (6 mM). A model of this reaction is shown in hydrating–dehydrating cycles,265,266 or even free in solution? Fig. 2; for detailed description of this click-like polymerization Each of these conditions entails an independent answer. process, see ref. 272.

Fig. 2 The mechanism of 30,50-cGMP polymerization generating RNA oligomers. For details see the text and ref. 272.

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In spite of the low efficiency of this reaction, the non- and provides the proof-of-principle of the possibility for a enzymatic polymerization of 30,50-cyclic purine nucleotides prebiotic sequence complementarity-based replication system. provides the proof-of-principle that RNA formation could The system allows for imprecise sequence matching, suggesting have occurred in the absence of enzymes and of pre-existing a mechanism for error-based sequence evolution.282 templates. Cyclic purine nucleotides undergo polymerization after pillaring 7.2. Ligation by stacking. Stacking of nucleic bases and of their derived forms in Ligation of fragments of genetic materials in extant organisms, 273–280 solution is a well characterized phenomenon. In particular, be it DNA or RNA, is a biologically exquisitely controlled the thermodynamic parameters for stacking of nucleic bases in process. Its analysis in biological systems is beyond the purport 274,278 aqueous solution were determined. The comparative analysis of this discussion. For an entry to the relevant literature the of the stacking free energy profiles for all 16 natural ribodinucleo- reader is referred to ref. 283. side monophosphates in aqueous solution275,277 has established that the stacked states have a 2–6 kcal mol1 lower free energy 7.2.1. Ligation of preactivated oligonucleotides. Nonenzy- than the unstacked states for purine–purine dimers and that A–A matic abiotic joining of oligoadenylates on a polyuridylic acid and G–G have the most favourable stacking. Stacking of purines template was reported as early as 1976,243 ligation being in the high dielectric aqueous solution is a very rapid phenomenon. performed via a20,30-cyclic phosphate terminus. Imidazole- In summary, the observed polymerization of cyclic nucleo- activated ligation reactions were reported,284,285 which were tides245,271,272 relies on favourable thermodynamics, on well also template-directed and highly regioselective for the formation established interaction and structural phenomena, on environment- of 30–50-phosphodiester bonds. These two latter studies led to determined nonfastidious concentration possibilities. It has the conclusion that RNA may correctly self-assemble as an been so far limited to purines, possibly because of the lower intrinsic consequence of its chemical structure, and that this stacking capacity of pyrimidines,275,276 for whom however no ability need not to be imposed by an external catalyst. The a priori reason exists precluding the possible identification of synthesis of oligoadenylates from the 50-phosphorimidazolide the appropriate physical–chemical conditions allowing non- of 20–50-diadenylate was carried out on a template made of enzymatic polymerization. decauridylate containing exclusively a 20–50-phosphodiester The interesting property of cyclic purines polymerization is bond.286 This synthesis was followed by joining of the products that it occurs among monomers free-floating in solution, held to yield long-chain oligoribonucleotides in a template-directed together by stacking. Is this reaction extendable to templated reaction.286 This approach was extended287 to nonenzymatic systems? And: how do stacking-driven and templated poly- ligation of short-chained 20–50-or30–50-linked oligoribo- merization compare? nucleotides on complementary templates, showing that a Downloaded by University of Oxford on 26 July 2012 homo-linkage system is preferable for the template-directed

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A 7. Abiotic RNA chain growth synthesis of RNA. With a chemically different approach, it was shown that The oligomerization of 30,50-cGMP in solution shows that oligonucleotide 50-polyphosphates are formed from oligo- abiotic polymerization of abiotically plausible monomers may nucleotide 50-phosphates and sodium trimetaphosphate.288 actually occur and that the size and the base composition of The oligonucleotides activated as polyphosphates underwent the products obtained are intrinsically limited. Thus, it also template-directed ligation. The possibility that these reactions sets a limit: the formation of a population of molecules complex could have produced longer oligonucleotide products from enough to kick-start a (pre)genetic system requires additional shorter substrates under prebiotic conditions was pointed mechanisms to foster the formation of longer polymers. Two out.288 such mechanisms were identified: RNA-templated polymeriza- tion and ligation. 7.2.2. Ligation of nonpreactivated oligonucleotides. The fact that RNA ligation may occur by phosphoester transfer 7.1. The templated polymerization of 30,50-cyclic guanosine was shown.289 The reaction required no external energy Exposure of preformed RNA oligonucleotides to 30,50-cyclic source, the energy for ligation being provided by the cleavage nucleotides resulted in the growth of RNA oligomers on the of the phosphodiester bond of a coupled reaction. The correct 30-end of the oligonucleotides. The system analyzed consisted of structure of the reacting groups was shown to play a pivotal oligoCs reacted with 30,50-cGMP, yielding molecules composed of role. the starting C-stretches on whose 30-end sequence-complementary These studies were extended in numerous directions. The oligonucleotides of various lengths polymerized.271,281,282 field that largely profited from this approach is that of the 0 0 290 291 More in detail, in the system analyzed (oligoC24 +3,5 - nonenzymatic spontaneous and template directed recombi- cGMP)271 the cyclic nucleotide actively reacted with the nation of RNAs, indicated as a possible mechanism for the preformed oligonucleotide, affording longer fragments. In formation of novel RNA sequences.292 The reactions were 293 particular, a group of molecules with number average (Navg) shown to be secondary structure-driven transesterifications. 42 formed in the presence of 30,50-cGMP, which grew up to an An additional simple approach to nonenzymatic ligation of observed length of >50 nucleotides in the presence of the RNA oligomers was more recently reported. Nontemplated non- highest concentrations of cyclic nucleotides tested.271 enzymatic ligation of preformed oligomers was observed,281,294,295 The observed 30-tail polymerization of 30,50-cGMP on which occurred according to two different mechanisms: complementary polyC sequences is an efficient reaction271,281 (i) ligation among oligomers held together in the reactive-prone

This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 5526–5565 5543 View Online Downloaded by University of Oxford on 26 July 2012 Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A

0 32 0 Fig. 3 Nonenzymatic terminal ligation of polyA oligonucleotides held by stacking. Panel A: gel electrophoretic analysis of untreated 5 - P-A24-3 0 32 0 0 0 (lane 1) and of the reaction products of the 5 - P-A24-3 oligomer reacted in water for 18 h at 60 1C in the presence of 10 mM 3 ,5 -cAMP (lane 2). The upper part of the panel is an overexposure (10) of the upper section of the image containing the dimers (48 nt) and the tetramers (96 nt), as indicated. Oligomers are idealized as rods whose 50 (red) and 30 (black) extremities are indicated throughout by this colour-code. During the permanence in water, the oligomers undergo hydrolysis, producing the ladder of decreasing length fragments, as indicated on the left side of lane 2. The identification of the fragments is shown on the right. Panel B: a model explaining for the observed directionality of the ligation events of polyA in water. Arrow A,50 to 50 ligation brings to the impossibility () of continuing the reaction to form the tetramer. Arrow B,30 to 30 ligation, the same. Arrow C, the antiparallel interaction of two parallel polyA double helices brings the reactive 50-phosphate and 30-hydroxyl group in the correct positioning. Data and models from ref. 294, with modifications.

conformation by stacking, and (ii) sequence complementarity- It was shown that the ligation is preceded by a first step of based ligation. pH-dependent interaction of polyA oligonucleotides based on stacking, followed by transphosphorylation at one extremity. Stacking-driven ligation. Homogeneous polyA oligomers Analysis of the orientation of the resulting ligation products were observed to undergo terminal ligation affording dimers, (which always formed as even-numbered multimers, dimers tetramers and more complex molecules.294 The reaction is and tetramers) provided a coherent mechanism explaining the driven by terminal transphosphorylation, similarly to what mechanics of the observed ligation, as described in Fig. 3. occurs in extant biological systems, with the difference that no The products of ligation reaction of a 24 nucleotide-long enzymatic system or high-energy cyclic phosphate bonds are polyA RNA adsorbed on mica were also analyzed by atomic required. This type of terminal ligation is favoured by low pH force microscopy.295 In that study the occurrence of oligo- to allow stacking among polyA oligomers. The net result is nucleotides at different degrees of polymerization was quantitatively multimerization of pre-existing oligomers.281,294,295 studied before and after the ligation reaction. The microscopy The nonenzymatic ligation in water of polyA sequences294 images at the nanoscale showed that nonenzymatic ligation of is a time-, pH- and temperature-dependent reaction. Liga- RNA monomers resulted in the formation of supramolecular tion efficiency depends on the oligonucleotide length and aggregates, with large prevalence of dimers and tetramers. sequence, and is strongly enhanced by adenine-based nucleo- Analytical conditions were defined allowing the identification, tide cofactors. the quantitative evaluation, and their distribution after ligation,

5544 Chem. Soc. Rev., 2012, 41, 5526–5565 This journal is c The Royal Society of Chemistry 2012 View Online

Fig. 4 The products of nonenzymatic stacking-driven terminal ligation observed by AFM. Panel A: AFM image on mica of polyA24 reacted with 30,50-cAMP (18 h, 60 1C). The bar marked in the lower-left part of the image corresponds to 100 nm. RNA concentration: 1 ng mL1 in a solution

Downloaded by University of Oxford on 26 July 2012 of 50% bidistilled water and 50% formamide. Height scale Dz = 1 nm. Panels B and C: linear profiles of differently sized RNA molecules, marked as 1, 2, and 3 in panel A. The objects 1, 2, and 3 correspond to a presumptive monomer, dimer, and tetramer, respectively. Panel D: 2D contour Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A plots and 3D image renderings of the sample described in Panel A; three typical objects assigned to the monomer (1), dimer (2), and tetramer (3) class. Right: linear profiles along the main orthogonal axes of the objects shown on the left; width (red lines), length (black lines). Inset: model interpretation of the oligomers: red, 50 extremity; black, 30 extremity. Data and models from ref. 295 with modifications.

also providing an estimate of the degree of hydration of the strand matching by stacking can be obtained with a number of objects. Such investigation (Fig. 4) provides the simplest yet sequence combinations and allows for internal sequence defects. model system for direct investigation of RNA reactions by As long as the two reactive extremities are matched in the correct advanced microscopy, potentially extendable to mineral surface reacting position (be it by stacking or by complementarity) the studies. strands have the possibility to evolve to higher complexity sequence combinations. Sequence complementarity-driven ligation. Analysis of a mechanistically similar terminal ligation reaction in sequence- 8. Stability complementary oligonucleotides confirmed the simplicity of ligation occurring by terminal transphosphorylation.281 The Once formed, polymers can evolve only if the physical–chemical experimental system analyzed consisted in reacting oligoC conditions of their environment and their intrinsic chemical with oligoG, one of the two reacting oligos bearing a phos- properties allow them to survive in polymeric form. It was phate group at 50. The oligos ligated promptly, the relative reported that the weak point of RNA (its 30-phosphoester position and orientation being determined by which of the two bond) is more stable when embedded in a ribopolymer than oligos bore the 50-ligating phosphate. when present in the monomer.221 This observation accounts on Ligation of short RNA fragments could have liberated one hand for the otherwise not easily understandable subsis- prebiotic polymerization systems from the thermodynamically tence of polymeric information in an abiotic environment, on demanding task of reaching a (pre)genetically meaningful size the other provides the background for the identification of pro- by stepwise additions of one precursor monomer at the time, life environments. The same analysis was performed in the and is a system fit for evolution. DNA system, where the weak bond is the 50-phosphoester.296 The requirement for the transmission of genetic informa- Given that the synthesis of nucleic bases, of acyclonucleosides tion is that the two strands replicate through a pairing and the phosphorylation of nucleosides by formamide require mechanism. Strand pairing in extant biological systems is a the presence of mineral catalysts, the effect of the same catalysts sequence complementarity-based phenomenon. Nevertheless, on the stability of RNA and DNA was determined.

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8.1. Stability of RNA and DNA phosphoester bonds in The fact that these conditions exist and that they are well monomers and in polymers under different physical–chemical within plausible prebiotic scenarios (as for instance mild conditions hydrothermal vents,267,311–313 or geothermal fields314) points to a possible mechanism leading to accumulation and survival The rapidity of the degradation of nucleic bases is well of macromolecular information. The stability of RNA is characterized86,98,99,297–299 and depolymerization studies of highly reduced in the presence of divalent cations, e.g. Zn2+, nucleic acids in H O date back to early studies by Robert 2 Fe2+/3+,Mg2+, etc. This fact has to be taken into account Shapiro and Erwin Chargaff,300 continued for decades.300–309 when considering the plausibility of prebiotic scenarios, and is The rate of hydrolysis of ribonucleotides was determined as discussed in the paragraphs that follow. Given that the core early as 1929.310 Direct comparison of the degradation rates of Darwinian property of a polymer is to maintain and transmit the various bonds when present in the nucleotides versus those its sequence information, these ‘‘relative stability’’ assays were present in the polymers was to our knowledge not reported, extended to different RNA sequences. The results315 revealed although from indirect comparison of the rate of cleavage of that a narrow pH range exists in which complex sequences b-glycosidic bonds of free deoxynucleosides302,303 relative to resist degradation markedly more than monotonous ones, that in single-stranded DNA304 one could infer the higher thus potentially favoring the evolution of sequence-based lability of the bond in the monomers. We have performed such genetic information. comparative analysis for the deoxyribose296 and the ribose221 The following section summarizes stability studies of DNA systems. The results revealed extensive differences depending and RNA in the presence of the minerals that were analyzed on the molecular context and on the physical–chemical for their synthetic ability in formamide/heat condensations. environment. For detailed information on these differences the reader is referred to ref. 221 and 296. 8.2. Stability of RNA and DNA in the presence of minerals The general indication obtained is that phosphoester bonds 8.2.1. RNA and minerals. The protection of RNA catalytic in monomers are not necessarily more stable than in polymers. activity by clays (namely, montmorillonites) was analyzed in Comparative measurements revealed that well defined condi- detail.129,130,133,140,316,317 The mechanistic aspects of RNA tions exist in which the opposite is actually true. protection rely on the large body of analyses centered on Let us focus here on RNA, also taking into consideration RNA degradation mechanisms301–309 that established that RNA parallel studies (when available) on DNA. The fact that DNA degradation follows the pathways summarized in Fig. 6, panel A. is probably not a prebiotic actor (presumably being a product of The cleavage of the phosphoester chain normally requires partici- cellular processes) does not decrease the interest of mechanistic pation of the 20-OH group as an internal nucleophile318 by two studies on the effects of physical–chemical conditions and of ‘‘nucleophilic cleavage’’ events: the transesterification and hydro- Downloaded by University of Oxford on 26 July 2012 mineral interaction analyses on nucleic acids stability in general. lysis reactions. During transesterification (pathway a), the 20-OH The stabilities of the bonds critical for the half-life of Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A nucleophile attacks the tetrahedral phosphorus to afford a ribonucleotides (the b-glycosidic and the 30-and50-phosphoester 20,30-cyclic monophosphate. This species is then hydrolyzed to bonds) were measured under a wide range of H O/NH CHO 2 2 a mixture of 30- and 20-phosphate monoesters (pathway b). ratios and temperatures.221 The stability of the 30-phosphoester Both steps are catalyzed by protons, hydroxide, nitrogen bond in RNA was also measured under the same conditions. derivatives and metal ions. The results obtained were coherent with previous reported Extending the studies that determined the protection by mechanistic studies on the stability of RNA308 and with the 309 clays, the stability of RNA in the presence of minerals was kinetics of RNA degradation by specific base catalysis. analyzed in numerous mineral classes: borates,319 iron–sulfur– The comparison revealed that the polymeric state of phos- copper minerals,320 phosphates,177,321 zirconium minerals147 phorylated nucleosides is thermodynamically favoured over and Murchison meteorite components.169 As reasonably expected, the monomeric one at high temperature both in pure water the results largely varied and strictly depended on the type of and in pure formamide and two defined thermodynamic niches mineral analyzed. No stringent unifying conclusion could be (Fig. 5) inside which polymers are favoured were identified. drawn and each class of minerals had its own behaviour. More so, Fig. 5 shows (panel A) the half-life of the indicated bonds in within each class different minerals exerted opposite behaviour, RNA and in monomers at three selected temperatures in so that the determination of the half-lives of RNA in the solutions varying in their H2O/NH2CHO relative composition. presence of the various minerals should be taken at its face 0 The shaded areas indicate higher stability of the 3 -bond in the value of preliminary rough-out screening to be used to orient polymer (redrawn from ref. 14) . Panel B presents the two areas in mechanistically focused further studies. Nevertheless, some the temperature (vertical) versus H2O/NH2CHO ratios (horizontal) conclusions are worth mentioning. plot in which such higher stability behaviour was observed. This plot summarizes a series of measurements consisting of the analysis Borates319,322. A typical assay is shown in Fig. 6, panel B. 0 of 42 temperatures vs. H2O/NH2CHO combinations (redrawn The assay consisted of the treatment of a 5 terminally labelled from Fig. 5 of ref. 296). Similar analyses were performed for RNA for the indicated time in water in the absence or in the 296 3+ DNA, keeping in mind its lower prebiotic relevance. The presence of the borate mineral ludwigite (Mg2Fe BO5). The weakest bond in DNA is the 50-phosphoester (see below). protective effect of the mineral towards hydrolytic degradation Comparative analyses showed that the DNA favourable thermo- at longer times is evident.322 Interestingly, out of 20 different dynamic niche, in the purport given above, is only present at low borates analyzed (representative of the most common classes of

H2O/NH2CHO ratios and at high temperature. this type of minerals), only ludwigite, hambergite (Be2(BO3)OH)

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Fig. 5 Differential stability of phosphoester bonds in a ribo-monomer (AMP) and in the polymer (RNA) in aqueous formamide solutions. Panel A: the differential stability of P bonds in RNA. The half-life of the 30-phosphoester bond in RNA is compared with that of the same bond in 30-AMP and with that of the 50-phosphoester bond in 50-AMP as a function of formamide concentration in water and temperature. Data are taken from ref. 221. Grey shades indicate the areas highlighting the conditions in which bond stability is higher in the polymer than in the monomer. Panel B: thermodynamic niches for RNA. Cartoon of the higher stability of the 30 bond in the polymer, derived summarizing 42 measurements of 1 0 differential t2 values (see Section 8.1. and ref. 221 and 296). The 5 bond in the deoxy system is more stable in DNA than in the monomer only at the highest temperatures and formamide concentrations, as indicated (data from ref. 296). The background is made of scanning profiles of electrophoretic analyses of degradation of DNA oligonucleotides, observed at the indicated combination of temperature/formamide:water (%) (from ref. 296).

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and jeremejevite (Al6B5O15F2.5(OH)0.5) had a protective effect, observed versus the degradation in formamide was exerted by 322 2+ 2+ and only at low concentration. In water all the other borates libethenite (Cu 2(PO4)(OH)), cornetite (Cu 3(PO4)(OH)3), and 2+ had a marked degradative effect, which was somewhat malachite (Cu 5(PO4)2(OH)4) (all copper containing minerals) unexpected due to the well established protection exerted by presumably because free phosphate is released into solution by borates on ribose.200 these minerals by formamide.216 Coherently, soluble phosphates do protect RNA versus hydrolytic degradation. Phosphates177,321. Phosphate minerals are, as a general We describe here as an example (Fig. 6, panel C) the effect of

trend, essentially inert relative to the RNA stability, both in three forms of soluble phosphates (NaH2PO4,Na4P2O7 and 0 water and in formamide. The representative phosphates tested: Na5P3O9) on the stability of a 5 labelled short RNA (RNA P1, pyromorphite (Pb5(PO4)3Cl), wardite (NaAl3(OH)4(PO4)2(H2O)2), containing in 21 nucleotides 14 of the 16 possible different base 2+ vivianite (Fe 3(PO4)2(H2O)8), herderite (Ca[BePO4F]), turquoise steps), as compared with the half-life of the same RNA in their 2+ (Cu Al6(PO4)4(OH)8(H2O)4) and hydroxylapatite Ca5(PO4)3OH absence. The protective effect exerted by di- and triphosphates exhibited no effect (data not published). The only protection is clear. The detailed quantitative analysis is in ref. 177 and 323. Downloaded by University of Oxford on 26 July 2012 Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A

Fig. 6 RNA stability in the presence of minerals. Panel A: mechanism of RNA degradation. For a description, see Section 8.2. Panel B: RNA 3+ 322 0 32 protection in water by ludwigite (Mg2Fe BO5), performed as described. Briefly, a 5 - P-labelled polyA24 oligomer was treated for the period of time indicated on top of each lane (h = hours) in water at 80 1C and analyzed in 16% acrylamide gel electrophoresis. The decrease of autoradiographic signal is proportional to the first-hit kinetics degradation of the 50-terminally labelled RNA molecules. For a detailed description of the method see Saladino et al.177 and Ciciriello et al.315 The % of the remaining full-length molecules in the presence (K) or in the absence (J) of ludwigite was determined and plotted. U = untreated. Panel C: soluble phosphates protect the phosphodiester bonds of RNA.177 Mixed

sequence P1 RNA was treated at 90 1C in water for the time periods indicated on top, in the presence of 1.3 mM NaH2PO4,Na4P2O7,Na5P3O9 or in the absence of added salt, as indicated. The autoradiograms show the increasingly (top to bottom) protective effects exerted by the phosphate salts. Panel D: iron–sulfur minerals modify the degradative reaction of RNA in formamide. (top) Degradation profiles of the 50-labeled 50-AAAAAAAAAAAAAAU-30 ribooligomer in the absence (leftmost panel) or in the presence of a mineral (as indicated above each profile). The reactions were performed as described in panel B and as detailed.320 The size of the resulting fragments is indicated (in nucleotides) to the right of the first profile. (bottom) Kinetics of degradation of the oligomer in the absence or presence of the indicated minerals, reported as the percentage of the full-sized molecules (ordinate) as a function of the reaction time (abscissa).

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This type of assays provides the basis for the analysis of the Phosphates act as catalysts for the synthesis of nucleic effect of the various minerals on the half-life of RNA and on precursors (Section 3), as substrates for the phosphorylation which aspect of the degradative reaction is affected. RNA of nucleosides (Section 5) and as stabilizing agents for the stability was analyzed in the presence of Fe–S–Cu minerals,320 eventually formed polymers (this section). Thus, the formamide– of zirconium minerals147 and of minerals from the Murchison phosphate system appears particularly worth considering in a meteorite.169 All the tested Fe–S–Cu and zirconium minerals and, prebiotic perspective. to lower extent, Murchison materials increased RNA degradation (as exemplified in Fig. 6, panel D, for Fe–S–Cu minerals). 8.2.2. DNA and minerals. Degradation of DNA oligo- One of the relevant aspects of these analyses in a prebiotic nucleotides occurs through two different mechanisms in aqueous perspective relates to the fact that in order to avoid an formamide solutions. The first mechanism – dubbed hydrolysis otherwise futile cycle of polymerizations/depolymerizations, the following nucleobases degradation (HND) – occurs more survival of informational polymers required a thermodynamic set efficiently in the absence of water at temperatures of B100 1C of conditions which could provide them with sufficient stability. or more. It is initiated by the nucleophilic addition of forma- These pro-polymer thermodynamic niches were identified, as mide at position C-8 of purines, and positions C-4 and C-6 of described in the previous section. However, the role of minerals pyrimidines.324,325 This results in the elimination of both the in the possible extension of these niches was not previously 30- and the 50-phosphoester bonds followed by chain cleavage. analyzed. In this perspective it is interesting that several The second mechanism – dubbed hydrolysis following nucleo- among the minerals tested (i.e. borates, zirconium minerals base substitution (HNS) – involves the hydrolytic degradation and Murchison) do no protect RNA but favour its degrada- of oligonucleotides in water, which occurs after the cleavage of tion, both in water and in formamide. Soluble di- and the b-glycosidic bond. The two mechanisms296 have different triphosphates177,323 and basalt320 are the only actively protective reaction rates; HNS is faster than HND, both preferentially agents. cleaving the 30-phosphoester bond (Fig. 7, panel A). Downloaded by University of Oxford on 26 July 2012 Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A

Fig. 7 DNA stability in the presence of minerals. Panel A: mechanisms of DNA degradation. Pathway A: hydrolysis by Nucleophilic Degradation (HND), consisting of formamide or hydroxyl nucleophilic addition at the C-8 position of a purine (adenine) ring followed by nucleobase degradation/elimination and two b-elimination reactions with C(30)–O and C(50)–O bond cleavages. Pathway B: hydrolysis by Nucleophilic Substitution (HNS) consisting of direct hydroxide nucleophilic substitution of adenine followed by two b-elimination reactions with C(30)–O and C(50)–O bond cleavages (modified from Saladino et al.296). See Section 8.2. Panel B: the effects of phosphate minerals on the instability of DNA molecules in water. General assay conditions as for RNA (Fig. 6). Reproduced from ref. 322 with modifications. Treatment in 1 H2O in the absence or in the presence of the indicated amounts (mg mL ) of the phosphate mineral indicated on top of each panel. Similar assays were performed with the other 22 minerals whose results are summarized in Fig. 8.

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The protection of DNA oligonucleotides was analyzed protective effect against the DNA-cleavage reactions, and for metal oxides,145 cosmic dust analogues of olivines,161 changed the 50 versus 30 cleavage specificities, as shown by 116 151 177,321 321 montmorillonite clays, TiO2 and phosphates. The the modification of the cleavage patterns. As observed for type of assay was similar to that for RNA and, as for RNA, RNA, the active and variegate behaviour of phosphate minerals different types of results were obtained with different minerals. appears particularly interesting in a prebiotic perspective. For these, the reader is addressed to the specific articles In summary, different classes of minerals exert different indicated. As a general trend, the results obtained for DNA actions on RNA and DNA stability, RNA degradation being differ from those obtained for RNA, mostly because of a more in general enhanced by all the minerals tested except those 151 diffuse protection for pyrimidines (i.e. in the TiO2 and releasing soluble phosphates. Discussion of chemical rationals montmorillonite116 systems). In the olivine-like class of minerals,161 and of more specific and mechanistic aspects of this topic is in

fayalite (Fe2SiO4) showed a particularly strong protective the specific reference and in ref. 14 and 156. behaviour, while the oxides analyzed SiO, SiO2, FeO, Al2O3, CuO, CaO, MgO showed differing results, as detailed in 9. Continuity ref. 145. The behaviour of phosphate minerals is noteworthy. In The data described allow us to hypothesize a continuous contrast to what observed for RNA, numerous phosphates thread from formamide to complex (pre)genetic polymers. exerted protection. Fig. 7, panel B shows examples of Fig. 9 presents this possibility. Formamide is derived from a (i) no interference by the considered mineral in the half- pool of a few alternative simple reactions which may essentially

life of an oligo DNA in water. The instance of laueite be boiled down to the central H2O + HCN = H2NCHO. 2+ 3+ (Mn [Fe 2(PO4)2(OH)2(H2O)2](H2O)4(H2O)2) is reported; Formamide condenses into pools of more stable chemical (ii) protection, the instance of lazulite (Mg[Al(PO4)(OH)]2) structures, whose composition mostly depends on the catalyst is reported; present, the physical–chemical conditions essentially and ideally (iii) enhanced degradation, as exemplified by variscite consisting of temperatures moderately higher than 100 1C,

(Al(PO4)(H2O)2). nothing else being required. In the case of temperatures The effect of the whole set of 25 phosphate minerals o100 1C, the synthetic reactions allow the presence of water. analyzed, both in water and in formamide, is reported in The fact that purine is synthesized in the absence of catalysts (and Fig. 8. In addition, soluble phosphates exerted a strong that it is always present as a product, whatever catalyst is used) Downloaded by University of Oxford on 26 July 2012 Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A

Fig. 8 The effect of phosphate minerals on DNA in water and in formamide.

5550 Chem. Soc. Rev., 2012, 41, 5526–5565 This journal is c The Royal Society of Chemistry 2012 View Online

Fig. 9 The possible continuous line from universal elements to complex (pre)genetic chemical information. The prebiotic plausibility of each step is discussed in the text in the indicated sections. The difficulties presented by ribonucleosides are discussed in Section 4. Formamide chemistry is effective up to the RNA polymerization step included.

tells that formamide acts as a substrate and a catalyst at the metabolic machineries were much simpler, and the network of same time, quite similarly to the role of formaldehyde in the proto-metabolic reactions was less orderly and selective than autocatalytic process established for the formose reaction extant metabolisms. In these elementary systems the molecules leading to sugars326 and to the role hypothesized for HCN that collectively were the best catalysts of the synthesis of their by Albert Eschenmoser.327 own constituents may have been selected with higher probability We see formamide chemistry in its whole as an autocatalytic than less organized communities, the self-replication process process that may be differently oriented and fine-tuned by becoming a property of the metabolic networks rather than a specific additional catalysts. The relevant point in a biological property of individual molecules.328 perspective is the long way that the series of successive reactions In the absence of enzymes, minerals might have been critical

Downloaded by University of Oxford on 26 July 2012 may cover. The scheme in Fig. 9 summarizes the possible for the control of the selectivity and specificity of chemical continuity from the simplest compounds to (pre)genetic polymers, transformations in otherwise very complex networks, as well

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A referring to the specific sections in which the data were presented. as for accelerating the reaction rates. Possibly because of this The complexities and alternatives concerning the major gap, the initial and important function, metal clusters329 and cofactors330 synthesis of nucleosides, are described in Section 4. are still ubiquitous in the majority of extant enzymes. Multimeric The data gathered so far mostly refer to the nucleic acids systems, in which primitive amino acids, nucleotides and cofactors domain, the search for other compounds, such as amino acids, were linked in random assemblages, may have also played an sugars, amino sugars, components of condensation processes important role in the selection processes of key molecular such as carbodiimide and urea, still being at its initial stage. pathways (for a discussion on multimeric systems see ref. 331). Nevertheless, as described, instances of all these compounds Among all the chemical transformations characterizing have been encountered. Formamide chemistry is in force up to extant metabolism, the transformations which are able to 0 0 the RNA polymerization step. Polymerization of 3 ,5 -cyclic produce biomolecules from carbon dioxide (CO2) under reducing purine nucleotides (30,50-cGMP271,272,281,282 and 30,50-cAMP)271 conditions appear to be the most consistent with the environ- occurs in water,271,281,282 and both in formamide and in mental conditions presumably existing on the primitive Earth.332 dimethylformamide.272 In dimethylformamide polymerization These transformations include autotrophic pathways333 such is actually more efficient than in water both in terms of yield as the reductive pentose phosphate pathway,334 the reductive and of polymer length, due to the base catalysis mechanism acetyl-CoA pathway,335 the 3-hydroxypropionate cycle,336 the onto which it relies. The ligation reaction has not been tested in 4-hydroxy butyrate cycle,337 and the reductive tricarboxylic solvents different than water. acids (rTCA) cycle.338

10. Synthesis of precursors of metabolic cycles 10.2. The reductive tricarboxylic acids cycle The rTCA pathway has recently received particular attention 10.1. About primitive metabolisms as a possible prebiotic ‘‘core of the core’’ of primitive pre- Cellular genetic apparatuses require for their functions the metabolism. This reductive cycle runs in the opposite direction presence of externally provided molecules and the use of relative to the Krebs cycle, the oxidative citric acid cycle,339 chemical energy made available by metabolism. The metabolic based on the intervention of 11 carboxylic acid derivatives machinery is today a complex network of highly controlled which are transformed into each other through a series of chemical transformations whose specificity and selectivity depend hydrolysis/dehydration, oxidation/reduction, group transfer on the catalytic activity of enzymes. Reasonably, the initial and isomerization reactions (Scheme 20).

This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 5526–5565 5551 View Online Downloaded by University of Oxford on 26 July 2012 Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A

Scheme 20

In modern photoautotrophs and oxidizing heterotrophs the Photo- and radiation chemistry. Some redox steps of the sequence of reactions in the cycle runs oxidatively, while in cycle can be photochemically run starting from preformed several chemolithoautotrophs it runs reductively, appearing as acids in the presence of sphalerite ZnS particles, prepared by 343 a topological network of autocatalytic transformations and a mixing ZnSO4 with Na2S. Under these experimental condi- platform for more advanced self-replicating biotic systems.340 tions, oxaloacetate was converted to malate and fumarate to Intermediates in the cycle are key starting compounds for the succinate, while the transformation of ketoglutarate to oxalo- biosynthesis of amino acids, nucleic acid bases, sugars and succinate occurred only in low yield, and that of succinate to lipids, suggesting the concept of the ‘‘universality in inter- ketoglutarate and oxalosuccinate to isocitrate did not occur at mediary metabolism’’.341 all (Scheme 21). As a class of compounds, a-ketocarboxylic Citric acid, pyruvic acid, and other possible rTCA inter- acids have high free energies of formation, which disfavors mediates have been detected in carbonaceous meteorites.342 their production under simple experimental conditions. On the other hand, in the ZnS photochemistry framework pyruvate is 10.2.1. Syntheses of rTCA components. Several experi- synthesized from lactate and ketoglutarate from pyruvate. In ments have been performed to test the possibility of the particular, pyruvate is produced in 70% yield and a quantum synthesis of rTCA intermediates under plausible prebiotic efficiency of 0.009 at 15 1C across the wavelength range of conditions, analyzing predominantly photochemical and thermal 200–400 nm. Pyruvate further proceeds by photooxidation to reactions. ketoglutarate with a 50% yield and a quantum efficiency of 0.0036.

5552 Chem. Soc. Rev., 2012, 41, 5526–5565 This journal is c The Royal Society of Chemistry 2012 View Online Downloaded by University of Oxford on 26 July 2012 Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A

Scheme 21

Noteworthily, isocitrate, glutarate, succinate, and acetate are arc-discharge experiments starting from elemental carbon351 or also produced in low amounts as side-products (Scheme 21).344 from HCN oligomers (tholins) in saline aqueous aerosols.352 In addition, the reaction of glyoxylate with carbon dioxide to produce lactate is promoted when ZnS is irradiated by Temperature. Other intermediates of the cycle can be synthe- ultraviolet light.345 Furthermore, the enzyme-like reversible sized in the presence of metal sulfides under thermal condi- amination of ketoglutarate to glutamate, and of other three tions. In the context of the ‘‘iron–sulfur-world theory’’,353,354 intermediates pertinent to rTCA, can be also performed by thioacetic acid (an activated form of acetate and possible 346 photoirradiation of NH3 on ZnS (Scheme 21). precursor of acetyl-CoA) was prepared from CH3SH and Intermediates of the rTCA cycle are also produced by CO at 100 1C in the presence of (Fe,Ni)S, NiS, Ni(OH)2 irradiation of an aqueous solution of acetic acid AcOH with and CoS,355 pyruvate was obtained by CO fixation at 2000 g-rays.347 The latter procedure was extended to test the effect bar and 250 1C with the FeS–RSH system,356 and a-amino of clays of the family of montmorillonites. The results showed acids were synthesized from ketoacids through an amination/

a decrease in the yield of carboxylic acids due to the increase of reduction procedure catalyzed by FeS and/or Fe(OH)2 at 357,358 degradative processes with predominant formation of CO2 100 1C (Scheme 22). 348 and CH4. The X-rays irradiation of frozen and liquid HCN CO is considered as a possible alternative to CO2 as a solutions at the dose range from 0 to 419 kGy produced carbon source359 and is omnipresent in volcanic gases.360 carboxylic acids as well as some amino acids and urea. Organic Interestingly, thioacetic acid can act as an acylating agent compounds were synthesized even at low temperature and low for different amino acids under oxidative conditions catalyzing radiation doses.349 Complex mixtures of carboxylic acids can the formation of amide linkages.361 It has been proposed that 350 be also formed from CH4 and H2O gaseous mixtures, and by hydrothermal systems may have provided an appropriate

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Scheme 22

environment for the abiotic formation and accumulation of n-alkan-2-ones, n-alkanes, n-alkenes, methyl alkanoates, organic matter on the primitive Earth, thus supplying bio- methylalkanes and phenylalkanes, it could have played a molecules necessary for the evolution of life.362–364 Theoretical significant role in the prebiotic formation of lipidic amphiphilic calculations suggested that synthetic processes in hydrothermal derivatives, potential precursors of membrane constituents. The vents are energetically possible in metastable equilibrium states in formation of these lipid constituents is inferred to proceed by the presence of mineral clusters at relatively high temperatures. insertion of a CO group at the terminal end of a carboxylic acid These computational studies point out that carboxylic acids may functionality to form a-oxoalkanoic acids, followed by reduction be the dominant organic compounds produced in these systems, to n-alkanoic acids, to n-alkanals, then to n-alkanols.371,372 Downloaded by University of Oxford on 26 July 2012 in addition to lower concentrations of alcohols, aldehydes, and The synthesis of amino acids (Strecker-type) under hydro- 365–367

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A ketones. thermal conditions has been also reported for reactions per- The abiotic formation of organic compounds under hydro- formed in the presence of a nitrogen source to yield glycine, thermal conditions proceeds by reduction/polymerization of alanine, aspartic acid, serines, glutamic acid, sarcosine and 373 the CO–CO2 system with H2 in the presence of H2O and 2-aminobutyric acid. The experiments performed in the mineral clusters as heterogeneous catalysts. Noteworthily, the presence of pyrite, pyrrhotite and magnetite in buffer pH organic compounds synthesized by thermocatalytic reactions yielded glycine, racemic alanine, aspartic acid and glutamic are usually characterized by homologous series with no carbon acid.374 Oligomerization of amino acids and nucleotide monomers number preferences.368,369 was also reported under various hydrothermal constraints.375,376 The conversion and synthesis of rTCA components is also operative under submarine hydrothermal vent conditions. 10.3. Formamide and carboxylic acids When ketoglutarate, succinate and fumarate were treated in Numerous intermediates of the rTCA cycle can be prepared

a flow-reactor system simulating the hydrothermal environ- starting from NH2CHO in both thermal and photochemical ment, malate was detected as a reaction product. Similarly, processes. citrate was prepared from pyruvate and from all the other rTCA components. The synthesis of citrate decreased signifi- 10.3.1. Iron–sulfur minerals. The first evidence of the possi-

cantly in the absence of pyruvate in the initial reaction mixture bility of synthesizing carboxylic acids from NH2CHO was (Scheme 23).370 obtained in a study of its reactivity in the presence of different Under these experimental conditions the presence of metals iron–sulfur minerals,320 previously suggested as key redox can increase the efficiency and selectivity of the reactions. components for the energetics of the Wa¨chtersha¨user model.

Thus, when a mixture of reagents known to be present in In particular, pyrite (FeS2) interacts efficiently with different the vicinity of submarine hydrothermal vents (e.g. CO, H2S) organic species, including NH2CHO. The reactivity of the was treated in the presence of NiS, carboxylic acids and related mineral surface during this process is tuned by the defect thioesters containing up to four carbon atoms were obtained, density value,377 the concentration of Fe+2 ions and the pH of in addition to thiols and alkyl mono- and disulfides. In these the solution. In fact, an increase in the iron activity increases the reactions the Ni(0) species appear to be the key catalysts abundance of iron groups on the surface providing more sites probably by hydroformylation-like processes. Since this pro- suitable for the interaction with negatively charged species that are cess can lead to organic compounds of any chain length, such thus activated for further transformations.378 Purine, adenosine, as n-alkanols, n-alkanoic acids, alkyl formates, n-alkanals, adenosine monophosphate and sugars interact with pyrite.379

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Scheme 23 Downloaded by University of Oxford on 26 July 2012 Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A

Scheme 24

Iron–sulfur minerals, such as FeS2 and pyrrothine [Fe(1 x)S], in the hydrogen bonding interactions with natural nucleo- and iron–sulfur–copper minerals chalcopyrite (FeCuS2), bornite bases. Oxalic acid 36 has been detected in various organisms, (FeCu5S4), tetrahedrite [(Fe, Cu, Sb)S], and covellite (CuS) including animals, plants and fungi, and its biosynthesis is 320 382 catalyze the thermal condensation of NH2CHO to yield mainly related to photosynthesis and carbohydrate metabolism. purine, adenine, 2(1H)-pyrimidinone12, urea, carbodiimide 20, isocytosine 35 and oxalic acid 36 (Scheme 24). The con- 10.3.2. Borates. Better results, in relation to the synthesis densation was operative also in mixed formamide–water solu- of intermediates of the cycle rTCA, were obtained by

tions (up to 30 wt% water). thermal condensation of NH2CHO in the presence of mineral Isocytosine [2-aminopyrimidin-4(3H)one] is a structural borates. Boric acid (H3BO3) and mineral borates such as isomer of cytosine. Nucleoside derivatives of isocytosine are ulexite, kernite and colemanite catalyze the formation and useful probes for the study of the formation of a DNA stabilization of ribose under formose-like conditions by triplex380 and for mechanistic studies of RNA catalysis.381 In formation of complexes with the vicinal 1,2-diol moiety in addition, isocytosine could perform as bioisostere of guanine key intermediates.200,383,384

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The chemical relationship between formamide and borate and glyoxylic acid, and two N-formylated amino acids, glycine minerals is in the ‘‘water paradox’’ stating that polymerization and alanine. Pyruvate is directly involved in the rTCA cycle, of biomolecules requiring release of water is, in water, thermo- while glyoxylate is involved in the glyoxylate cycle, a variation dynamically disfavoured. In fact, during the dry–wet cycles of the TCA cycle, an anabolic metabolic pathway occurring in expected on the early Earth, two processes can occur: (i) the plants, bacteria, protists, fungi and several microorganisms, formation of borate evaporites, mineral deposits that emerge such as E. coli and yeast (Scheme 25). In addition, glycolate from evaporating water (as discussed in ref. 385), and (ii) the is involved in the photosynthetic oxidation of glycolate to 386 concentration of NH2CHO, which is characterized by a boiling glyoxylate. Carboxylic acids can be produced through a point of 210 1C in the absence of eutectics with water. common reaction pathway involving a reactive HCN-dimer and/or 327 The heating of NH2CHO at 160 1C in the presence of DAMN. The hydrolysis of the nitrile moiety in DAMN mineral borates (hydrates of borates containing hydroxyl or affords hydroxyoxaloacetic acid tautomers (2,3-dihydroxymaleic, halogen moieties, anhydrous borates containing hydroxyl or 2,3-dihydroxyfumaric, and 2-hydroxyoxaloacetic acid, respec- halogen moieties, borosilicates, borocarbonates and multiple tively) that are easily converted to oxaloacetic acid (not detected) oxides) afforded319 purine, adenine, cytosine, isocytosine, uracil, and its decarboxylation product pyruvic acid via astepwise 2(1H)-pyrimidinone 12 and carbodiimide 20, different carboxylic addition of over two electrons and two protons.117,387 Further acids: oxalic acid 36, lactic acid, pyruvic acid, glycolic acid reduction of pyruvic acid yields lactic acid. Similarly, glyoxylic acid Downloaded by University of Oxford on 26 July 2012 Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A

Scheme 25

5556 Chem. Soc. Rev., 2012, 41, 5526–5565 This journal is c The Royal Society of Chemistry 2012 View Online

can be formed by hydrolysis of the HCN-dimer, and glycolic Murchison minerals are a complex mixture of microscopic acid by a second two electron–two proton reductive step of the silicate particles ranging from sub-micron single crystals to formyl moiety in glyoxylic acid. aggregates tens of microns in size. Very little metallic iron is It is noteworthy that under these experimental conditions present (0.03% by weight). The mineral components are

NH2CHO behaves as a chemical precursor for the contem- primarily classified as pyroxene and olivine. Trace amounts poraneous synthesis of intermediates of the genetic apparatus of iron and nickel oxides and sulfides have been also and of metabolism. This property does not appear to be detected.391

limited to the catalysis by mineral borates. The heating of NH2CHO at 160 1C in the presence of dust from the Murchison meteorite afforded purine, uracil, iso- 10.3.3. Zirconium minerals. Similar results were obtained cytosine, 4(3H)-pyrimidinone 11, 3-hydroxypyrimidine 37, with minerals of zirconium. Zirconium compounds occur isocyanate 38, parabanic acid 19, dihydroxyacetone (DHA) almost wherever ancient sediments are present.388 They rise 39, N-formylglycine and some carboxylic acid derivatives, through the crust in igneous rocks and are eroded into the including oxalic acid, oxaloacetic acid, malic acid and lactic stream system, washed out to sea, and laid down in the acid. Again, oxaloacetic acid and malic acid are directly sediment beds where they become part of the next cycle of involved in the rTCA cycle (Scheme 27). DHA is a chemical sandstone, remaining totally unaffected.389 Zirconium minerals precursor for sugar. that exceed 4 billion years in age have been found in a quartz conglomerate.390 10.3.5. TiO . Finally, the UV irradiation of NH CHO in Geological processes of the Earth’s surface over billions of 2 2 the presence of TiO anatase with synchrotron light (3.2 eV; years have probably modified the properties of the minerals 2 ca. 387.5 nm corresponding to the band gap energy of anatase) that catalyzed prebiotic syntheses of biomolecules. In this at 25 1C afforded392 6 of the 11 carboxylic acid intermediates scenario, zirconium minerals can provide a mineral probe that of rTCA, including pyruvic acid, succinic acid, malic acid, simulates ancient Earth. The role of zirconium minerals in the oxaloacetic acid, a-ketoglutaric acid and fumaric acid synthesis of biomolecules as well as in the stability of RNA has (Scheme 28). Other carboxylic acid derivatives, glycolic acid, been recently reported.147 Zircon (zirconium silicate, ZrSiO ), 4 lactic acid, ethanimidic acid, propanoic acid, 2,3-dihydroxy- baddeleyite (zirconium dioxide, ZrO ) and different metal 2 propanoic acid, 2,4-dihydroxybutanoic acid, 3,4-dihydroxy- zirconates catalyzed the condensation of formamide to yield butanoic acid, oxalic acid and malonic acid (not shown) were purine, adenine, isocytosine 35, 2(1H)-pyrimidinone 12, urea, also detected in the reaction mixture. Under these experi- carbodiimide 20, and carboxylic acid derivatives oxalic acid, mental conditions, nucleic acid bases were not detected. The succinic acid, fumaric acid and maleic acid (Scheme 26). UV irradiation of neat formamide in the absence of TiO only Downloaded by University of Oxford on 26 July 2012 Fumaric acid and succinic acid are intermediates of the 2 afforded traces of oxalic acid diamide, triazine and urea, in rTCA cycle, while lactic acid is a precursor of pyruvic acid. 393 Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A accordance with data previously reported. 10.3.4. Murchison meteorite. Also non-terrestrial minerals About the mechanism of formation of carboxylic acids, catalyze the synthesis of nucleic acid bases and carboxylic pyruvic acid and lactic acid may be formed with a process

acids from NH2CHO, as observed in the case of material similar to that previously described for mineral borates by sampled from the Murchison meteorite.169 conversion of a DAMN intermediate. Succinic acid, malic acid

Scheme 26

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Scheme 27

and fumaric acid can be derived from oxaloacetic acid by a systems look promising for an experimental approach to these

Downloaded by University of Oxford on 26 July 2012 two electron–two proton reduction step (to yield malic acid) hypotheses.

concomitant with, or followed by, a b-elimination of H2O Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A (to yield fumaric acid), and reductive dehydroxylation step (to yield succinic acid), while a-ketoglutaric acid is a product 11. Concluding remarks of photochemical transformation of succinic acid in which We have started from the consideration that formamide is the TiO acts as a catalyst instead of zinc sulfide. Finally, long 2 simplest possible amide and that it contains within its diverse chain carboxylic acids can be obtained394 by successive addi- chemistry the functional groups and chemical bonds of the tion of a one-carbon unit through a known process of induced central biomolecules. We have considered the sources of carbon–carbon bond formation via TiO photocatalysis. 2 formamide (meteorites, comets, interstellar dusts) and we have discussed how formamide can be at the same time solvent and 10.4. Towards a unified scheme for the origin of primitive reactant. Having observed the products obtained simply by metabolism warming formamide in the presence of one out a large number On the basis of the data described above, two main processes of different catalysts, we adhere to the conclusion that for- appear to be involved in the origin of primitive metabolism: the mamide chemistry is quite versatile and nonfastidiously yields chemical reactions necessary for the synthesis of metabolism in rich and complex combinations nucleobases, carboxylic intermediates, and the chemical (or photochemical) transforma- acids, amino acids, sugars, amino sugars and condensing tions needed for the interconversion of the intermediates of the agents. These reactions occur in the presence of necessary catalytic cycles. catalysts, as discussed in the specific sections. If the cradle of Some reagents and minerals may contribute to both these life contained formamide, the walls of the cradle were made of

processes, as observed in NH2CHO–minerals systems where one out of many different possible minerals, presumably the thermal pathway predominantly leans towards syntheses, of combinations thereof. As noted, phosphate minerals were while photochemistry favors interconversions. a likely ingredient. In other systems, as observed for ZnS photochemically- We have considered the first steps towards (pre)genetic based transformations, the interconversions pathway prevails. materials, from nucleosides to their phosphorylation to Reasonably, the primitive environment in which these trans- nucleotides, and have discussed the available evidence leading formations occurred was quite complex as for mineral compo- towards the formation of complex RNA molecules, passing sition, ensuring the contemporaneous presence of compounds through polymerization and ligation. Attention has been able to catalyze syntheses and to provide the energy necessary devoted to the problem of the stability of nucleic polymers. to run the cycles. The formamide chemistry–mixed minerals In the origin-of-life perspective, the system would be blocked if

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Scheme 28 Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A

nucleic syntheses could not somehow couple with proto- A simple and unitary chemistry might support the possibility metabolic processes. Far from suggesting any detailed that on this planet life might have emerged more than once396 solution, we note that precursors of genetics and metabolism and at different geological sites.397,398 form under the same conditions and with the same catalysts. If origin of life has been a matter of interacting systems, its key 11.1. The limits of the formamide scenario requisite is that the necessary components had to be present in the same place at the same time. Its origin is expected to have The contribution that HCN/formamide chemistry provides to met conditions of contemporaneity and availability. the general picture of the origins is limited to the proof-of- This natural consideration is often overlooked. Given for principle that a unifying chemistry is at least conceivable. The granted with respect to thermodynamics and the control of scenario is far from being fully and satisfactorily sketched. channelled energy flows, the kinetic optimization of all the Riddles remain. reactions, and a programmed and orderly avoidance of clogging The first riddle is the concentration problem. We have these reactions by accumulation of secondary or underused mentioned in Section 2 that the steady state concentration of products, the basic need remains, in the origins, for an actual HCN in the primitive ocean was evaluated to be 4 1012 M

source of nucleic bases, amino acids, carboxylic acids. at 100 1C, that similar values were reported for NH2CHO and In order to have a set of metabolic reactions in place and to that even at lower temperatures concentrations were too low evolve the specific molecules of inheritance, spatial compart- to foster biomolecular syntheses in solution. Concentration mentalization was at some point needed. However, that was processes of formamide by eutectics, by absorption onto or probably not before that ‘‘simple chemistry in diverse environ- into appropriate minerals such as clays, and by evaporation

ments on the early Earth led to the emergence of ever more (the boiling point of NH2CHO being 204 1C), have been complex chemistry’’.395 studied (see Sections 2 and 3.2.1). Noteworthily, the stability

We have not been dealing here with the emergence of of NH2CHO towards hydrolysis increases proportionally to its 399 complex chemistry. Nevertheless, the non-fastidious single- concentration, and efficient prebiotic syntheses from NH2CHO chemistry scenario set by formamide points to the possibility are operative also in 30% water (v/v).320 Another possible concen- that the diverse environments imagined for the various com- tration means as thermophoresis have not yet been sufficiently ponents did not need, all considered, to be that diverse. explored experimentally to indicate novel possible solutions.

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The fact that hydrolysis of HCN to formamide is optimal low pH (1–2), and devoid of compartmentalization are not under alkaline conditions and that many of the reported particularly suited for the prebiotic synthesis of biomolecules.413,414 reactions are favoured by alkaline conditions point to alkaline On the other hand, the alkaline hydrothermal vents produced hydrothermal vents311,312,400–404 as a favourable setting. In by serpentinisation (the hydroxylation of olivine to serpentine) vents, thermophoresis and mineral nanopore and/or chemical show environmental conditions more compatible with synthetic absorption would be the preferred concentrating means pro- processes, the vent being warm, alkaline and loaded with dis- viding an interesting prebiotic scenario. This scenario needs to solved hydrogen.312 Under these experimental conditions, some consider formamide stability. organic compounds such as hydrocarbons, formic acid and Thus, the second riddle is the stability problem. The activa- acetate can be synthesized in appreciable yields,414,415 the process 1 tion free energy value of 31.0 kcal mol suggests that the energy for these transformations being the reduction of CO2 by 405 neutral hydrolysis of formamide does not take place at all. H2 catalyzed by minerals, able to produce primitive analogues of The mechanism of the base-catalyzed hydrolysis of formamide the ‘‘acetyl CoA pathway’’400 and ATP.416 The reducing power 417 has been extensively studied both experimentally and theore- of hydrothermal vents is sufficient to fix N2 as ammonia, a tically as a key model for peptides stability.406–409 The reaction reagent useful for the synthesis of nitrogen containing bio- is first order in hydroxide concentration, with a free energy of molecules such as amino acids418 and formamide.419 In this activation of 21.5 kcal mol1 and rate constants of 3.2 s1 (398 K) environment formamide can be also directly produced from 1 399 419–422 and 0.032 s (329 K). These values are of the same order of CO2 and H2 in the presence of a nitrogen source. magnitude as those measured for the corresponding amino acid The experimental exploration of formamide chemistry in the derivatives, as in the case of glycine amide,410 confirming the alkaline hydrothermal vents environment looks particularly similar stability of the amide bond in formamide and in peptides. promising. This identifies a third riddle. Which was the optimal temperature? We have mentioned in the Introduction that complex The synthetic reactions affording the various combinations of problems are best confronted by a Bayesian approach, by

biomolecules from NH2CHO + minerals were typically optimal successive runs of experience-driven approximations, each at 140 1C, efficient syntheses occurring at temperature as low as time possibly selecting the simplest alternative following 100 1C. The effect of UV irradiation160 as a source of energy Occam’s approach, best if in its updated Russel’s version. may be a marginal observation in terrestrial scenarios and It seems to us that formamide chemistry matches these criteria does not detract from the central fact that on planet Earth heat of simplicity and that in the quest for a common general frame and one out of a large ensemble of common mineral catalysts it might meet successive runs of validation. are sufficient conditions. The fourth riddle is systemic. The data gathered so far

Downloaded by University of Oxford on 26 July 2012 Acknowledgements indicate that NH2CHO provides a single and fertile chemical

Published on 08 June 2012 http://pubs.rsc.org | doi:10.1039/C2CS35066A frame only at the level of organic products. At systems level Thanks to Silvia Lopizzo for helpful contributions. the kinetics and the conditions necessary for the optimization of a series of connected reactions have not been explored. The References data gathered so far only provide the proof-of-principle that 1 A. I. Oparin, in Vozniknovenie Zhisni na Zemle (The Origin of the reactions involved do actually occur starting from a single Life on Earth), Izdatelstvo Akademii Nauk SSSR, Moscow, substrate and that nucleic bases and carboxylic acids may form 3rd edn, 1957, [Translated in English (New York Academic Press together. The limit of this type of observations was lucidly Inc), 1957]. defined by Lesley Orgel,411 stating the implausibility of meta- 2 S. L. Miller, Science, 1953, 117, 528–529. 3 L. Delaye and A. Lazcano, Phys. Life Rev., 2005, 2, 47–64. bolic cycles on the early Earth. The reasons indicated were the 4 T. Rev. Bayes, Philos. Trans. R. Soc. London, 1736, 53, 370–418. difficult optimization of the various catalyses, the clogging of 5 G. P. Tocchini Valentini and M. A. Tocchini Valentini, FASEB the reactions by plethoras of secondary products, the difficulty J., 2012, 36, 5–8. 6 B. Russel, in The Philosophy of Logical Atomism, ed. D. F. Pears, in obtaining the right assembly of catalytic minerals, coherent La Salle, Open Court edn., 1918, pp. 35–155. chemical plausibilities and the right combination of efficiencies 7 S. S. Mansy, J. P. Schrum, M. Krishnamurthy, S. Tobe´, and specificities. The cycle allowing the conversion of formaldehyde D. A. Treco and J. W. Szostak, Nature, 2008, 454, 122–125. into a mixture of monosaccharides by an aldol-like reaction412 8 V. Vijayasarathy and H. J. Morowitz, Biol. Bull., 2009, 217, 222–232. remains a prebiotically interesting exception. 9 C. Darwin, The life and letters of Charles Darwin, 1888, vol. 3, The same problems can be invoked in the formulation of a p. 18, Letter to Joseph Hooker. John Murray, London. precise scenario envisaging the onset of metabolic cycles, their 10 G. Wald, in Horizons in Biochemistry, (Life in the Second and cooperation, their eventual interaction with (pre)biotic systems. Third Periods; or, Why Phosphorus and Sulfur for High-Energy Bonds?), ed. M. Kasha and B. Pullman, Marcel Academic Press, The fact that NH2CHO yields rich panels of biomolecules in a New York, 1962, pp. 127–142. large set of conditions and increases the likelihood of combina- 11 T. Fink, H. Bruggesser and J. L. Reymond, Angew. Chem., Int. Ed., tory events shows the versatility and elasticity of its chemistry. In 2005, 44, 1504–1508. 12 Cellular Origin and Life in Extreme Habitats and Astrobiology, this respect we deem as particularly promising the concurrence (The HCN world: Establishing protein-nucleic acid life via hydrogen 311,312,400–404 of alkaline hydrothermal vent chemistry based on cyanidepolymers),ed.C.N.Mattews,Origins,Genesis,Evoluation CO2, for whose production robust and abundant mechanisms and Diversity of Life, vol. 6, 2004, pp. 121–135. have been identified. 13 R. Saladino, C. Crestini, G. Costanzo and E. Di Mauro, Top. Curr. Chem., 2005, 259, 29–68. Deep submarine hydrothermal vents (‘‘black smokers’’) of 14 R. Saladino, C. Crestini, F. Ciciriello, G. Costanzo and E. Di Mauro, volcanic origin, characterized by high temperature (350 1C), Chem. Biodiversity, 2007, 4, 694–720.

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