At some stage in the origin of life, an informational polymer must have arisen by purely chemical means. According to one version of the 'RNA world' hypothesis this polymer was RNA, but attempts to provide experimental support for this have failed. In particular, although there has been some success demonstrating that 'activated' ribonucleotides can polymerize to form RNA, it is far from obvious how such ribonucleotides could have formed from their constituent parts (ribose and nucleobases). Ribose is difficult to form selectively, and the addition of nucleobases to ribose is inefficient in the case of purines and does not occur at all in the case of the canonical pyrimidines. Here we show that activated pyrimidine ribonucleotides can be formed in a short sequence that bypasses free ribose and the nucleobases, and instead proceeds through arabinose amino-oxazoline and anhydronucleoside intermediates. The starting materials for the synthesis-cyanamide, cyanoacetylene, glycolaldehyde, glyceraldehyde and inorganic phosphate-are plausible prebiotic feedstock molecules, and the conditions of the synthesis are consistent with potential early-Earth geochemical models. Although inorganic phosphate is only incorporated into the nucleotides at a late stage of the sequence, its presence from the start is essential as it controls three reactions in the earlier stages by acting as a general acid/base catalyst, a nucleophilic catalyst, a pH buffer and a chemical buffer. For prebiotic reaction sequences, our results highlight the importance of working with mixed chemical systems in which reactants for a particular reaction step can also control other steps.

Amide bond formation is one of the most important reactions in both chemistry and biology 1-4 , but there is currently no chemical method to achieve α-peptide ligation in water that tolerates all twenty proteinogenic amino acids at the peptide ligation site. The universal genetic code establishes the biological role of peptides predates Life's last universal common ancestor and that peptides played an essential role in the origins of Life 5-9 . The essential role of sulfur in the citric acid cycle, non-ribosomal peptide synthesis and polyketide biosynthesis points towards thioester-dependent peptide ligations preceding RNA-dependent protein synthesis during the evolution of Life 5,9-13 . However, a robust mechanism for aminoacyl thioester formation has never been demonstrated 13 . Here, we report a chemoselective, high yielding a-aminonitrile ligation that exploits only prebiotically plausible molecules-hydrogen sulfide, thioacetate 12,14 and ferricyanide 12,14-17 or cyanoacetylene 8,14 -to yield apeptides in water. The ligation is extremely selective for a-aminonitrile coupling and tolerates all 20 proteinogenic amino acid residues. Two essential features enable the peptide ligation in water: 1) the reactivity and pKaH of a-aminonitriles makes them compatible with ligation at neutral pH, and 2) Nacylation stabilises the peptide product and activates the peptide precursor to (biomimetic) N®C peptide ligation. Our model unites prebiotic aminonitrile synthesis and biological a-peptides, suggesting short N-acyl peptide nitriles were plausible substrates during early evolution.To improve the efficiency and selectivity of peptide ligation in water we sought to develop a novel mechanism for non-enzymatic peptide synthesis, which would operate via biomimetic N®C ligation in near-neutral pH water, and we suspected that a combination of sulfur and nitrile chemistry would be required ( Fig. 1a) 8,9,14,[18][19][20][21] . Proteinogenic a-aminonitriles (AA-CN) are readily synthesised 8,18 , and their direct ligation would provide the simplest prebiotic pathway to peptides. Unfortunately, incubation of AA-CN in water results in extremely ineffective peptide synthesis 22 . a-Amino acids (AA) are widely assumed to be prebiotic precursors of peptides, but the harsh conditions (typically strongly acidic or alkaline solutions) required for AA formation from AA-CN are incompatible with the integrity of both peptides and electrophilic activating agents. Therefore, we sought a more congruent and direct pathway from a-aminonitriles to a-peptides, and although the conversion of AA-CN to AA-SH has never been reported 23 , harnessing the AA-CN nitrile moiety for thioacid synthesis seemed more prudent than dissipating its activation through exhaustive hydrolysis.Orgel has previously suggested that a-aminothioacids (AA-SH) 16 might offer an interesting alternative to biological thioesters 10,11 . AA-SH unite excellent aqueous stability with highly selective (electrophilic or oxidative) activation 12,14,16,24 , but their prebiotic synthesis...

A plausible process for non-enzymatic RNA replication would greatly simplify models of the transition from prebiotic chemistry to simple biology. However, all known conditions for the chemical copying of an RNA template result in the synthesis of a complementary strand containing a mixture of 2′-5′ and 3′-5′ linkages, rather than the selective synthesis of only 3′-5′ linkages as found in contemporary RNA. Here we show that such backbone heterogeneity is compatible with RNA folding into defined three-dimensional structures that retain molecular recognition and catalytic properties and, therefore, would not prevent the evolution of functional RNAs such as ribozymes. Moreover, the same backbone heterogeneity lowers the melting temperature of RNA duplexes that would otherwise be too stable for thermal strand separation. By allowing copied strands to dissociate, this heterogeneity may have been one of the essential features that allowed RNA to emerge as the first biopolymer.The ability of RNA molecules to fold into defined three-dimensional structures with exquisitely specific molecular recognition and catalytic properties is the conceptual basis of the RNA World hypothesis, an early stage in the evolution of life in which RNA served not only as the polymer of inheritance, but as the central functional polymer of biochemistry [1][2][3] . This model is most strikingly supported by the observation that all modern proteins are synthesized by the peptidyl transferase ribozyme at the heart of the ribosome 4,5 . With the RNA World hypothesis so strongly supported by this and other evidence 1 , the central question in the origin of life field concerns the pathway from the prebiotic chemistry of the early Earth to the emergence of simple forms of cellular life containing RNA genomes coding for RNA enzymes. While there has been considerable recent progress towards the elucidation of potentially prebiotic pathways for ribonucleotide synthesis 6-8 and the assembly of activated nucleotides into oligonucleotides 9,10 , the non-enzymatic replication of RNA oligonucleotides remains problematic. A series of seemingly intractable difficulties * Correspondence to: szostak@molbio.mgh.harvard.edu. † Current Address: Department of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street, London, WC1H 0AJ, UK Author ContributionsAll authors contributed to the design of the experiments and to writing the paper. Experiments were conducted by A.E.E. and M.W.P. Competing Financial Interests StatementThe authors declare no competing financial interests.Published as: Nat Chem. 2013 May ; 5(5): 390-394. HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscriptcontinues to make a robust system for the chemical replication of RNA elusive [11][12][13][14] . These problems include the slow rate, poor fidelity and low regioselectivity of non-enzymatic RNA template copying; in addition, activated substrates typically hydrolyze on the same timescale as polymerization. The importance of the latter point ...

UK 2A central problem for prebiotic synthesis of the biological amino acids and nucleotides is avoiding the concomitant synthesis of undesired or irrelevant byproducts. Additionally, multistep pathways require mechanisms that enable the sequential addition of reactants and purification of intermediates that are consistent with reasonable geochemical scenarios. Here, we show that 2-aminothiazole reacts selectively with two-and three-carbon sugars (glycolaldehyde and glyceraldehyde, respectively), which results in their accumulation and purification as stable crystalline aminals. This permits ribonucleotide synthesis, even from complex sugar mixtures. Remarkably, aminal formation also overcomes the thermodynamically favoured isomerisation of glyceraldehyde to dihydroxyacetone because only the aminal of glyceraldehyde separates from the equilibrating mixture. Finally, we show that aminal formation provides a novel pathway to amino acids that avoids synthesis of the non-proteinogenic α,α-disubstituted analogues. The common physicochemical mechanism that controls proteinogenic amino acid and ribonucleotide assembly from prebiotic mixtures suggests these essential classes of metabolite had a unified chemical origin. 3The conservation of the genetic code, amino acids, and nucleotides in biology suggests a single origin of life on Earth. [1][2][3][4][5][6][7][8][9][10][11][12][13] Proteins are built from a highly restricted set of about 20 amino acids according to a universal triplet code of four ribonucleotides. Therefore, it is essential to learn how this specific small constellation of molecules became irrevocably linked at the advent of life. In contrast to the narrow distribution of universal metabolites observed in biology, typical prebiotic reactions are notorious for their complex product distributions. Accordingly, it has been recognised that "the chief obstacle to understanding the origin of RNA-based life is identifying a plausible mechanism for overcoming the clutter wrought by prebiotic chemistry". [4][5][6][7] For example, the most-efficient and specific proposed prebiotic pathway to the pyrimidine ribonucleotides requires synthesis of the key intermediate pentose aminooxazoline (1) (Fig. 1a). [2][3][4][5][6][22][23][24] However, the plausibility of this proposed prebiotic synthesis of pentose aminooxazoline (1) has been questioned because it is contingent upon the strictly controlled sequential delivery of pure glycolaldehyde (2a) to cyanamide (3) to yield 2-aminooxazole (4), followed by pure glyceraldehyde (2b) to 2-aminooxazole (4) to yield the desired product (Fig. 1a). This is a serious problem because both of these reactions lack the intrinsic selectivity required to exclusively yield their respective products (2-aminooxazole (4) and pentose aminooxazoline (1)) from mixtures of glycolaldehyde (2a) and glyceraldehyde (2b). The problem becomes increasingly worse in the presence of other sugars. Without a separate and sequential delivery of glycolaldehyde (2a) and glyceraldehyde (2b), a complex mixture...

Peptide biosynthesis is performed by ribosomes and several other classes of enzymes, but a simple chemical synthesis may have created the first peptides at the origins of life. α-Aminonitriles—prebiotic α–amino acid precursors—are generally produced by Strecker reactions. However, cysteine’s aminothiol is incompatible with nitriles. Consequently, cysteine nitrile is not stable, and cysteine has been proposed to be a product of evolution, not prebiotic chemistry. We now report a high-yielding, prebiotic synthesis of cysteine peptides. Our biomimetic pathway converts serine to cysteine by nitrile-activated dehydroalanine synthesis. We also demonstrate that N-acylcysteines catalyze peptide ligation, directly coupling kinetically stable—but energy-rich—α-amidonitriles to proteinogenic amines. This rare example of selective and efficient organocatalysis in water implicates cysteine as both catalyst and precursor in prebiotic peptide synthesis.

SUMMARYLiving organisms are the most complex chemical system known to exist, yet exploit only a small constellation of universally conserved metabolites to support indefinite evolution. The conserved chemical simplicity belying biological diversity strongly indicates a unified origin of life. Thus, the chemical relationship between metabolites suggests that a simple set of predisposed chemical reactions predicated the appearance of life on Earth. Conversely, if prebiotic chemistry produces highly complex mixtures, this then implies that the feasibility of elucidating life's origins is an insurmountable task. Prebiotic systems chemistry, however, has recently been exploiting the chemical links between different metabolites to provide unprecedented scope for exploration of the origins of life, and an exciting new perspective on a 4 billion-year-old problem. At the heart of the systems approach is an understanding that individual classes of metabolites cannot be considered in isolation. This review highlights several recent advances suggesting that the canonical nucleotides and proteinogenic amino acids are predisposed chemical structures. KEYWORDSOrigins of life, prebiotic chemistry, systems chemistry, predisposed chemistry, RNA, nucleotides, amino acids, sugars, metabolism, crystallization. BIGGER PICTUREAdvancing our understanding of the spontaneous emergence of life requires innovation across scientific disciplines as broad as astrophysics to phylogenetics, yet the primacy of chemistry cannot be overestimated. Cellular life is a chemical system of awe-inspiring complexity yet, perhaps surprisingly, life exploits only a 2 small constellation of universally conserved metabolites working in concert to support indefinite evolution.The conserved chemical simplicity that belies biodiversity is a strong indication that a simple set of predisposed reactions predicated the sudden appearance of life on Earth. The wonder of nature's greatest feat of invention-the self-assembly of living cells-positions the origins of life as one of the greatest challenges in chemistry. Building chemical systems that can self-assemble, process information, control the transport and accumulation of chemicals, orchestrate reaction pathways, and ultimately self-replicate will no doubt have a major impact on evolving technology, but nature has had a 4 billion-year head start in implementing controlled chemical evolution, and the lessons to be learnt from its prior art merely await discovery. eTOCPrebiotic systems chemistry is providing unprecedented scope for exploring the origins of life and an exciting new perspective on a 4 billion-year-old problem. At the heart of this new systems approach is an understanding that individual classes of metabolites cannot be considered in isolation if the chemical origin of life on Earth is to be successfully elucidated. This review aims to highlight several recent advances that suggest the canonical nucleotides and proteinogenic amino acids are predisposed chemical structures.

Non-equilibrium conditions must have been crucial for the assembly of the first informational polymers of early life-by supporting their formation and continuous enrichment in a long-lasting environment. Here we explored how gas bubbles in water subjected to a thermal gradient, a likely scenario within crustal mafic rocks on the early Earth, drive a complex, continuous enrichment of prebiotic molecules. RNA precursors, monomers, active ribozymes, oligonucleotides, and lipids are shown to (1) cycle between dry and wet states, enabling the central step of RNA phosphorylation, (2) accumulate at the gas-water interface to drastically increase ribozymatic activity, (3) condense into hydrogels, (4) form pure crystals, and (5) encapsulate into protecting vesicle aggregates that subsequently undergo fission. These effects occurred within less than 30 minutes. The findings unite physical conditions in one location which were crucial for the chemical emergence of biopolymers.They suggest that heated microbubbles could have hosted the first cycles of molecular evolution.Life is a non-equilibrium system. By evolution, modern life has created a complex protein machinery to maintain the nonequilibrium of crowded molecules inside dividing vesicles. Based on entropy arguments, equilibrium conditions were unlikely to trigger the evolutionary processes during the origin of life 1 . External non-equilibria had to be provided for the accumulation, encapsulation, and replication of the first informational molecules. They can locally reduce entropy, give rise to patterns 2 , and lean the system towards a continuous, dynamic self-organization 3 . Non-equilibrium dynamics can be found in many fluid systems, including gravity-driven instabilities in the atmosphere 4 , the accumulation of particles in nonlinear flow 5,6 , and shear-dependent platelet activation in blood 7 . Our experiments discuss whether gas-water interfaces in a thermal gradient could have provided such a nonequilibrium setting for the emergence of life on early Earth.Non-equilibrium systems in the form of heat flows were a very common and simplistic setting, found ubiquitously on the planet 8 . Hydrothermal activity is considered abundant on early Earth and intimately linked to volcanic activity 9 . Water is thereby circulating through the pore space of the volcanic rocks, which is formed by magmatic vesiculation (primary origin) and fractures (secondary origin). These systems have been studied as non-equilibrium driving forces for biological molecules in a variety of processes 10-17 .Gases originating from degassing of deeper magma bodies percolate through these water-filled pore networks. At shallow depths bubbles are formed by gases dissolved in water and formation of vapor where sufficient heat is supplied by the hydrothermal system. The bubbles create gas-water interfaces, which previously have been discussed in connection with atmospheric bubble-aerosol-droplet cycles 18 , the adsorption of lipid monolayers and DNA to the interface 19,20 , or the formation of pep...

The recent synthesis of pyrimidine ribonucleoside-2',3'-cyclic phosphates under prebiotically plausible conditions has strengthened the case for the involvement of ribonucleic acid (RNA) at an early stage in the origin of life. However, a prebiotic conversion of these weakly activated monomers, and their purine counterparts, to the 3',5'-linked RNA polymers of extant biochemistry has been lacking (previous attempts led only to short oligomers with mixed linkages). Here we show that the 2'-hydroxyl group of oligoribonucleotide-3'-phosphates can be chemoselectively acetylated in water under prebiotically credible conditions, which allows rapid and efficient template-directed ligation. The 2'-O-acetyl group at the ligation junction of the product RNA strand can be removed under conditions that leave the internucleotide bonds intact. Remarkably, acetylation of mixed oligomers that possess either 2'- or 3'-terminal phosphates is selective for the 2'-hydroxyl group of the latter. This newly discovered chemistry thus suggests a prebiotic route from ribonucleoside-2',3'-cyclic phosphates to predominantly 3',5'-linked RNA via partially 2'-O-acetylated RNA.