Author Correction: Peptide ligation by chemoselective aminonitrile coupling in water.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Peptide ligation by chemoselective aminonitrile coupling in water.

Amide bond formation is one of the most important reactions in both chemistry and biology1-4, but there is currently no chemical method of achieving α-peptide ligation in water that tolerates all of the 20 proteinogenic amino acids at the peptide ligation site. The universal genetic code establishes that the biological role of peptides predates life's last universal common ancestor and that peptides played an essential part in the origins of life5-9. The essential role of sulfur in the citric acid cycle, non-ribosomal peptide synthesis and polyketide biosynthesis point towards thioester-dependent peptide ligations preceding RNA-dependent protein synthesis during the evolution of life5,9-13. However, a robust mechanism for aminoacyl thioester formation has not been demonstrated13. Here we report a chemoselective, high-yielding α-aminonitrile ligation that exploits only prebiotically plausible molecules-hydrogen sulfide, thioacetate12,14 and ferricyanide12,14-17 or cyanoacetylene8,14-to yield α-peptides in water. The ligation is extremely selective for α-aminonitrile coupling and tolerates all of the 20 proteinogenic amino acid residues. Two essential features enable peptide ligation in water: the reactivity and pKaH of α-aminonitriles makes them compatible with ligation at neutral pH and N-acylation stabilizes the peptide product and activates the peptide precursor to (biomimetic) N-to-C peptide ligation. Our model unites prebiotic aminonitrile synthesis and biological α-peptides, suggesting that short N-acyl peptide nitriles were plausible substrates during early evolution.

Selective Synthesis of Lysine Peptides and the Prebiotically Plausible Synthesis of Catalytically Active Diaminopropionic Acid Peptide Nitriles in Water.

Why life encodes specific proteinogenic amino acids remains an unsolved problem, but a non-enzymatic synthesis that recapitulates biology's universal strategy of stepwise N-to-C terminal peptide growth may hold the key to this selection. Lysine is an important proteinogenic amino acid that, despite its essential structural, catalytic, and functional roles in biochemistry, has widely been assumed to be a late addition to the genetic code. Here, we demonstrate that lysine thioacids undergo coupling with aminonitriles in neutral water to afford peptides in near-quantitative yield, whereas non-proteinogenic lysine homologues, ornithine, and diaminobutyric acid cannot form peptides due to rapid and quantitative cyclization that irreversibly blocks peptide synthesis. We demonstrate for the first time that ornithine lactamization provides an absolute differentiation of lysine and ornithine during (non-enzymatic) N-to-C-terminal peptide ligation. We additionally demonstrate that the shortest lysine homologue, diaminopropionic acid, undergoes effective peptide ligation. This prompted us to discover a high-yielding prebiotically plausible synthesis of the diaminopropionic acid residue, by peptide nitrile modification, through the addition of ammonia to a dehydroalanine nitrile. With this synthesis in hand, we then discovered that the low basicity of diaminopropionyl residues promotes effective, biomimetic, imine catalysis in neutral water. Our results suggest diaminopropionic acid, synthesized by peptide nitrile modification, can replace or augment lysine residues during early evolution but that lysine's electronically isolated sidechain amine likely provides an evolutionary advantage for coupling and coding as a preformed monomer in monomer-by-monomer peptide translation.

Prebiotic Catalytic Peptide Ligation Yields Proteinogenic Peptides by Intramolecular Amide Catalyzed Hydrolysis Facilitating Regioselective Lysine Ligation in Neutral Water.

The prebiotic origin of catalyst-controlled peptide synthesis is fundamental to understanding the emergence of life. Building on our recent discovery that thiols catalyze the ligation of amino acids, amides, and peptides with amidonitriles in neutral water, we demonstrate the outcome of ligation depends on pH and that high pKa primary thiols are the ideal catalysts. While the most rapid thiol catalyzed peptide ligation occurs at pH 8.5-9, the most selective peptide ligation, that tolerates all proteinogenic side chains, occurs at pH 7. We have also identified the highly selective mechanism by which the intermediate peptidyl amidines undergo hydrolysis to α-peptides while demonstrating that the hydrolysis of amidines with nonproteinogenic structures, such as ß- and γ-peptides, displays poor selectivity. Notably, this discovery enables the highly α-selective protecting-group-free ligation of lysine peptides at neutral pH while leaving the functional ε-amine side chain intact.

Photochemistry of 2-thiooxazole: a plausible prebiotic precursor to RNA nucleotides.

Potentially prebiotic chemical reactions leading to RNA nucleotides involve periods of UV irradiation, which are necessary to promote selectivity and destroy biologially irrelevant side products. Nevertheless, UV light has only been applied to promote specific stages of prebiotic reactions and its effect on complete prebiotic reaction sequences has not been extensively studied. Here, we report on an experimental and computational investigation of the photostability of 2-thiooxazole (2-TO), a potential precursor of pyrimidine and 8-oxopurine nucleotides on early Earth. Our UV-irradiation experiments resulted in rapid decomposition of 2-TO into unidentified small molecule photoproducts. We further clarify the underlying photochemistry by means of accurate ab initio calculations and surface hopping molecular dynamics simulations. Overall, the computational results show efficient rupture of the aromatic ring upon the photoexcitation of 2-TO via breaking of the C-O bond. Consequently, the initial stage of the divergent prebiotic synthesis of pyrimidine and 8-oxopurine nucleotides would require periodic shielding from UV light either with sun screening chromophores or through a planetary scenario that would protect 2-TO until it is transformed into a more stable intermediate compound, e.g. oxazolidinone thione.

Selective Prebiotic Synthesis of α-Threofuranosyl Cytidine by Photochemical Anomerization.

The structure of life's first genetic polymer is a question of intense ongoing debate. The "RNA world theory" suggests RNA was life's first nucleic acid. However, ribonucleotides are complex chemical structures, and simpler nucleic acids, such as threose nucleic acid (TNA), can carry genetic information. In principle, nucleic acids like TNA could have played a vital role in the origins of life. The advent of any genetic polymer in life requires synthesis of its monomers. Here we demonstrate a high-yielding, stereo-, regio- and furanosyl-selective prebiotic synthesis of threo-cytidine 3, an essential component of TNA. Our synthesis uses key intermediates and reactions previously exploited in the prebiotic synthesis of the canonical pyrimidine ribonucleoside cytidine 1. Furthermore, we demonstrate that erythro-specific 2',3'-cyclic phosphate synthesis provides a mechanism to photochemically select TNA cytidine. These results suggest that TNA may have coexisted with RNA during the emergence of life.

One-step protecting-group-free synthesis of azepinomycin in water.

We report an efficient, atom economical general acid-base catalyzed one-step multi-gram synthesis of azepinomycin from commercially available compounds in water. We propose that the described pH-dependent Amadori rearrangement, which couples an amino-imidazole and simple sugar, is of importance as a potential step toward predisposed purine nucleotide synthesis at the origins of life.

Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions.

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.

Spontaneous Peptide Ligation Mediated by Cysteamine.

The fundamental and universal nature of life's exploitation of peptides suggests they must have played a vital role during the onset of life, but their spontaneous chemoselective synthesis in water remains unknown. Aminonitriles (1) are widely accepted as prebiotic precursors of both amino acids and peptides, but they do not spontaneously polymerize in water to yield peptides. Here, we demonstrate that the simple prebiotically plausible aminothiol, cysteamine (5), participates in Strecker chemistry to furnish ß-mercaptoethyl-α-aminonitriles (8) and ß-mercaptoethyl-amino acids (16), which are predisposed to spontaneously form peptides in water. Intramolecular thiol catalyzed ligation is faster, higher-yielding, and more α-selective than previously reported prebiotic peptide ligation chemistries, enabling, for example, the highly regioselective α-ligation of Asp- and Glu-dinitriles in quantitative yields. Our findings suggest that cysteamine (5), the thiol bearing moiety of the universal thiol cofactor coenzyme A, may have played an important role in the selective chemical synthesis of prebiotic α-peptides.

Prebiotically plausible chemoselective pantetheine synthesis in water.

Coenzyme A (CoA) is essential to all life on Earth, and its functional subunit, pantetheine, is important in many origin-of-life scenarios, but how pantetheine emerged on the early Earth remains a mystery. Earlier attempts to selectively synthesize pantetheine failed, leading to suggestions that "simpler" thiols must have preceded pantetheine at the origin of life. In this work, we report high-yielding and selective prebiotic syntheses of pantetheine in water. Chemoselective multicomponent aldol, iminolactone, and aminonitrile reactions delivered spontaneous differentiation of pantoic acid and proteinogenic amino acid syntheses, as well as the dihydroxyl, gem-dimethyl, and ß-alanine-amide moieties of pantetheine in dilute water. Our results are consistent with a role for canonical pantetheine at the outset of life on Earth.

Detection of potential TNA and RNA nucleoside precursors in a prebiotic mixture by pure shift diffusion-ordered NMR spectroscopy.

In the context of prebiotic chemistry, one of the characteristics of mixed nitrogenous-oxygenous chemistry is its propensity to give rise to highly complex reaction mixtures. There is therefore an urgent need to develop improved spectroscopic techniques if onerous chromatographic separations are to be avoided. One potential avenue is the combination of pure shift methodology, in which NMR spectra are measured with greatly improved resolution by suppressing multiplet structure, with diffusion-ordered spectroscopy, in which NMR signals from different species are distinguished through their different rates of diffusion. Such a combination has the added advantage of working with intact mixtures, allowing analyses to be carried out without perturbing mixtures in which chemical entities are part of a network of reactions in equilibrium. As part of a systems chemistry approach towards investigating the self-assembly of potentially prebiotic small molecules, we have analysed the complex mixture arising from mixing glycolaldehyde and cyanamide, in a first application of pure shift DOSY NMR to the characterisation of a partially unknown reaction composition. The work presented illustrates the potential of pure shift DOSY to be applied to chemistries that give rise to mixtures of compounds in which the NMR signal resolution is poor. The direct formation of potential RNA and TNA nucleoside precursors, amongst other adducts, was observed. These preliminary observations may have implications for the potentially prebiotic assembly chemistry of pyrimidine threonucleotides, and therefore of TNA, by using recently reported chemistries that yield the activated pyridimidine ribonucleotides.

Multicomponent assembly of proposed DNA precursors in water.

We propose a novel pathway for the prebiotic synthesis of 2'-deoxynucleotides. Consideration of the constitutional chemical relationships between glycolaldehyde and ß-mercapto-acetaldehyde, and the corresponding proteinogenic amino acids, serine and cysteine, led us to explore the consequences of the corresponding sulfur substitution for our previously proposed pathways leading to the canonical ribonucleotides. We demonstrate that just as 2-aminooxazole-an important prebiotic ribonucleotide precursor-is readily formed from glycolaldehyde and cyanamide, so is 2-aminothiazole formed from ß-mercapto-acetaldehyde and cyanamide in water at neutral pH. Indeed, both the oxazole and the thiazole can be formed together in a one-pot reaction, and can be co-purified by crystallization or sublimation. We then show that 2-aminothiazole can take part in a 3-component carbon-carbon bond-forming reaction in water that leads to the diastereoselective synthesis of masked 2'-thiosugars regiospecifically tethered to purine precursors, which would lead to 2'-deoxynucleotides upon desulfurization. The possibility of an abiotic route to the 2'-deoxynucleotides provides a new perspective on the evolutionary origins of DNA. We also show that 2-aminothiazole is able to sequester, through reversible aminal formation, the important nucleotide precursors glycolaldehyde and glyceraldehyde in a stable, crystalline form.

Prebiotic synthesis and triphosphorylation of 3'-amino-TNA nucleosides.

Nucleosides are essential to the emergence of life, and so their synthesis is a key challenge for prebiotic chemistry. Although amino-nucleosides have enhanced reactivity in water compared with ribonucleosides, they are assumed to be prebiotically irrelevant due to perceived difficulties with their selective formation. Here we demonstrate that 3'-amino-TNA nucleosides (TNA, threose nucleic acid) are formed diastereoselectively and regiospecifically from prebiotic feedstocks in four high-yielding steps. Phosphate provides an unexpected resolution, leading to spontaneous purification of the genetically relevant threo-isomer. Furthermore, 3'-amino-TNA nucleosides are shown to be phosphorylated directly in water, under mild conditions with cyclic trimetaphosphate, forming a nucleoside triphosphate (NTP) in a manner not feasible for canonical nucleosides. Our results suggest 3'-amino-TNA nucleosides may have been present on the early Earth, and the ease with which these NTPs form, alongside the inherent selectivity for the Watson-Crick base-pairing threo-monomer, warrants further study of the role they could play during the emergence of life.

Prebiotic triose glycolysis promoted by co-catalytic proline and phosphate in neutral water.

Proline and phosphate promote a near-quantitative aldol reaction between glycolaldehyde phosphate and formaldehyde at neutral pH in water. Our results demonstrate the important role of general acid-base catalysis in water and underscore the essential role that amino acid catalysis may have played in early evolution of life's core metabolic pathways.

Chemoselective multicomponent one-pot assembly of purine precursors in water.

The recent development of a sequential, high-yielding route to activated pyrimidine nucleotides, under conditions thought to be prebiotic, is an encouraging step toward the greater goal of a plausible prebiotic pathway to RNA and the potential for an RNA world. However, this synthesis has led to a disparity in the methodology available for stepwise construction of the canonical pyrimidine and purine nucleotides. To address this problem, and further explore prebiotically accessible chemical systems, we have developed a high-yielding, aqueous, one-pot, multicomponent reaction that tethers masked-sugar moieties to prebiotically plausible purine precursors. A pH-dependent three-component reaction system has been discovered that utilizes key nucleotide synthons 2-aminooxazole and 5-aminoimidazoles, which allows the first divergent purine/pyrimidine synthesis to be proposed. Due to regiospecific aminoimidazole tethering, the pathway allows N9 purination only, thus suggesting the first prebiotically plausible mechanism for regiospecific N9 purination.

Prebiotic synthesis of cysteine peptides that catalyze peptide ligation in neutral water.

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.

Analyses of Aliphatic Aldehydes and Ketones in Carbonaceous Chondrites.

Aliphatic aldehydes and ketones are essential building blocks for the synthesis of more complex organic compounds. Despite their potentially key role as precursors of astrobiologically important molecules, such as amino acids and carboxylic acids, this family of compounds has scarcely been evaluated in carbonaceous chondrites. The paucity of such analyses likely derives from the low concentration of aldehydes and ketones in the meteorites and from the currently used chromatographic methodologies that have not been optimized for meteorite analysis. In this work, we report the development of a novel analytical method to quantify the molecular distribution and compound-specific isotopic analysis of 29 aliphatic aldehydes and ketones. Using this method, we have investigated the molecular distribution and 13C-isotopic composition of aldehydes and ketones in 10 carbonaceous chondrites from the CI, CM, CR, and CV groups. The total concentration of carbonyl compounds ranged from 130 to 1000 nmol g-1 of meteorite with formaldehyde, acetaldehyde, and acetone being the most abundant species in all investigated samples. The 13C-isotopic values ranged from -67 to +64‰ and we did not observe clear relationships between 13C-content and molecular weight. Accurately measuring the relative abundances, determining the molecular distribution, and isotopic composition of chondritic organic compounds is central in assessing both their formation chemistry and synthetic relationships.

Heated gas bubbles enrich, crystallize, dry, phosphorylate and encapsulate prebiotic molecules.

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 explore 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 occur within less than 30 min. The findings unite, in one location, the physical conditions that were crucial for the chemical emergence of biopolymers. They suggest that heated microbubbles could have hosted the first cycles of molecular evolution.

Prebiotic nucleic acids need space to grow.

What were the conditions on early Earth when nucleotides were formed, and what are the most plausible nucleoside candidates? Answering these questions will require mechanistic chemistry and planetary science to work together, enhancing not limiting each other's scope of investigation.

Selective aqueous acetylation controls the photoanomerization of α-cytidine-5'-phosphate.

Nucleic acids are central to information transfer and replication in living systems, providing the molecular foundations of Darwinian evolution. Here we report that prebiotic acetylation of the non-natural, but prebiotically plausible, ribonucleotide α-cytidine-5'-phosphate, selectively protects the vicinal diol moiety. Vicinal diol acetylation blocks oxazolidinone formation and prevents C2'-epimerization upon irradiation with UV-light. Consequently, acetylation enhances (4-fold) the photoanomerization of α-cytidine-5'-phosphate to produce the natural ß-pyrimidine ribonucleotide-5'-phosphates required for RNA synthesis.