Prebiotic Nucleoside Synthesis: The Selectivity of Simplicity

Ever since the discovery of nucleic acids 150 years ago,^[1] major achievements have been made in understanding and decrypting the fascinating scientific questions of the genetic code.^[2] However, the most fundamental question about the origin and the evolution of the genetic code remains a mystery. How did nature manage to build up such intriguingly complex molecules able to encode structure and function from simple building blocks? What conditions were required? How could the precursors survive the unhostile environment of early Earth? Over the past decades, promising synthetic concepts were proposed providing clarity in the field of prebiotic nucleic acid research. In this Minireview, we show the current status and various approaches to answer these fascinating questions.     

Darwin's Warm Little Pond: A One‐Pot Reaction for Prebiotic Phosphorylation and the Mobilization of Phosphate from Minerals in a Urea‐Based Solvent

The poor reactivity of insoluble phosphates, such as apatite‐group minerals, has been a long‐appreciated obstacle for proposed models of prebiotic organophosphate formation. This obstacle presents a significant challenge to the nascent development of an RNA world and other models for the origins of life on Earth. Herein, we demonstrate that a scenario based on the formation of a urea/ammonium formate/water (UAFW) eutectic solution leads to an increase in phosphorylation when compared to urea alone for phosphate sources of varying solubility. In addition, under evaporative conditions and in the presence of MgSO₄, the UAFW eutectic mobilizes the phosphate sequestered in water‐insoluble hydroxyapatite, giving rise to a marked increase in phosphorylation. These results suggest that the prebiotic concentrations of urea in a geologically plausible evaporitic environment could solve the problem of organic phosphorylation on a prebiotic Earth.     

Noncanonical RNA Nucleosides as Molecular Fossils of an Early Earth—Generation by Prebiotic Methylations and Carbamoylations

The RNA‐world hypothesis assumes that life on Earth started with small RNA molecules that catalyzed their own formation. Vital to this hypothesis is the need for prebiotic routes towards RNA. Contemporary RNA, however, is not only constructed from the four canonical nucleobases (A, C, G, and U), it also contains many chemically modified (noncanonical) bases. A still open question is whether these noncanonical bases were formed in parallel to the canonical bases (chemical origin) or later, when life demanded higher functional diversity (biological origin). Here we show that isocyanates in combination with sodium nitrite establish methylating and carbamoylating reactivity compatible with early Earth conditions. These reactions lead to the formation of methylated and amino acid modified nucleosides that are still extant. Our data provide a plausible scenario for the chemical origin of certain noncanonical bases, which suggests that they are fossils of an early Earth.     

Synthesis of Pyrimidines and Triazines in Ice: Implications for the Prebiotic Chemistry of Nucleobases

From urea to nucleobases : Freeze–thaw cycles in urea ( 1 ) solutions under methane/nitrogen atmospheres lead, with application of an energy source, to the synthesis of pyrimidines (mainly cytosine ( 2 ) and uracil ( 3 )), triazines (such as cyanuric acid ( 4 )), and adenine. This synthesis appears to be dependent on the atmosphere and the freezing conditions. At room temperature, hydantoin ( 5 ) is obtained. However, a freezing urea/water system subjected to an energy source under an inert atmosphere generates s‐triazines.

     

Prebiotic Route to Thymine from Formamide—A Combined Experimental–Theoretical Study

Synthesis of RNA nucleobases from formamide is one of the recurring topics of prebiotic chemistry research. Earlier reports suggest that thymine, the substitute for uracil in DNA, may also be synthesized from formamide in the presence of catalysts enabling conversion of formamide to formaldehyde. In the current paper, we show that to a lesser extent conversion of uracil to thymine may occur even in the absence of catalysts. This is enabled by the presence of formic acid in the reaction mixture that forms as the hydrolysis product of formamide. Under the reaction conditions of our study, the disproportionation of formic acid may produce formaldehyde that hydroxymethylates uracil in the first step of the conversion process. The experiments are supplemented by quantum chemical modeling of the reaction pathway, supporting the plausibility of the mechanism suggested by Saladino and coworkers.     

Synthesis and Incorporation of k2U into RNA

Lysidine (k²C) is one of the most modified pyrimidine RNA bases. It is a cytidine nucleoside, in which the 2-oxo functionality of the heterocycle is replaced by the ϵ-amino group of the amino acid lysine. As such, lysidine is an amino acid-containing RNA nucleoside that combines directly genotype (C-base) with phenotype (lysine amino acid). This makes the compound particularly important in the context of theories about the origin of life and here especially for theories that target the origin of translation. Here, we report the total synthesis of the U-derivative of lysidine (k²U), which should have the same base pairing characteristics as k²C if it exists in the isoC-like tautomeric form. To investigate this question, we developed a phosphoramidite building block for k²U, which allows its incorporation into RNA strands. Within RNA, k²U can base pair with the counter base U and isoG, confirming that k²U prefers an isoC-like tautomeric structure that is also known to dominate for k²C. The successful synthesis of a k²U phosphoramidite and its use for RNA synthesis now paves the way for the preparation of a k²C phosphoramidite and RNA strands containing k²C.     

Simple Ion–Gas Mixtures as a Source of Key Molecules Relevant to Prebiotic Chemistry

Very simple chemistry can result in the rapid and high-yield production of key prebiotic inorganic molecules. The two reactions investigated here involve such simple systems, (a) carbon disulfide (CS₂) and acetate (CH₃COO¯) and (b) sulfur dioxide (SO₂) and formate (HCOO¯). They have been carried out under non-aqueous conditions, either in an organic solvent or with a powdered salt exposed to the requisite gas. Under such dry conditions the first reaction generated the thioacetate anion [CH₃COS]¯ while the second produced the radical [SO₂^·]¯anion. Anhydrous conditions are not rare and may have arisen on the early earth at sites where an interface between different phases (liquid/gas or solid/gas) could be generated. This is one way to rationalize the formation of molecules and ions (such as we have produced) necessary in the prebiotic world. Interpretation of our results provides insight into scenarios consistent with the more prominent theories of abiogenesis.     

Plasma Modeling and Prebiotic Chemistry: A Review of the State-of-the-Art and Perspectives

We review the recent progress in the modeling of plasmas or ionized gases, with compositions compatible with that of primordial atmospheres. The plasma kinetics involves elementary processes by which free electrons ultimately activate weakly reactive molecules, such as carbon dioxide or methane, thereby potentially starting prebiotic reaction chains. These processes include electron–molecule reactions and energy exchanges between molecules. They are basic processes, for example, in the famous Miller-Urey experiment, and become relevant in any prebiotic scenario where the primordial atmosphere is significantly ionized by electrical activity, photoionization or meteor phenomena. The kinetics of plasma displays remarkable complexity due to the non-equilibrium features of the energy distributions involved. In particular, we argue that two concepts developed by the plasma modeling community, the electron velocity distribution function and the vibrational distribution function, may unlock much new information and provide insight into prebiotic processes initiated by electron–molecule collisions.     

Microfluidic Production of Pyrophosphate Catalyzed by Mineral Membranes with Steep pH Gradients

Pyrophosphate might have functioned as an energy storage/currency molecule on early Earth, essential for the emergence of life. Here we synthesized mineral membranes involving iron(II), iron(III), and other divalent metal cations (calcium, manganese, cobalt, copper, zinc, and nickel) and tested their ability to catalyze the formation of pyrophosphate from phosphate and acetyl phosphate across steep pH gradients in microfluidic devices. We studied the chemical compositions of the precipitate membranes (which included vivianite, goethite, and green rust) using in situ and ex situ micro-Raman spectroscopy. The yields of pyrophosphate were determined by aqueous ³¹P NMR spectroscopy. We found that Fe²⁺ and Ca²⁺ were the best catalysts for pyrophosphate synthesis among investigated ions; Fe³⁺ and mixed-valence iron membranes were also able to promote pyrophosphate formation. In addition, the pH gradients across the membranes affected the pyrophosphate yields and the smallest pH gradient resulted in the highest yield. These results suggest a possible route of substrate phosphorylation in prebiotic hydrothermal systems.     

Template-Directed Copying of RNA by Non-enzymatic Ligation

The non-enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA-based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template-directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3′-amino group at the 3′-end of each oligonucleotide, in combination with an N-alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution.     

Template‐Directed Copying of RNA by Non‐enzymatic Ligation

The non‐enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA‐based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template‐directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3′‐amino group at the 3′‐end of each oligonucleotide, in combination with an N‐alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution.     

Binding of nucleosides with the cytotoxic plant alkaloid sanguinarine: Spectroscopic and thermodynamic studies

In spite of a large number of studies of the interaction of the cytotoxic plant alkaloid sanguinarine(SAN) with nucleic acids,the anticancer mechanism of SAN is still not clear.In contrast to the large number of studies of the interaction mechanism of SAN with DNA,there have been relatively few studies of the interaction of SAN with nucleosides.In this work,the interaction of SAN with three nucleosides-thymidine(T),uridine(U),and adenosine(A)-was investigated using a combination of conventional fluorescence and UV-vis spectroscopic techniques;thermodynamic calculations were also carried out at physiological pH 7.2.The binding processes of SAN with the different nucleosides were characterized by hypochromic and bathochromic effects in the absorption spectra of SAN and by the quenching of the fluorescence intensity of SAN.The measurements of fluorescence lifetime,the variations of the absorption spectra of the fluorophore,and the dependence of the quenching on the temperature indicated that the fluorescence quenching is static.The Stern-Volmer plot is nonlinear and approximately quadratic showing that,in this process,one SAN molecule can bind with two nucleoside molecules.These studies,together with our earlier studies of the binding of SAN with cytidine(C) and guanosine(G),showed that the binding constants of SAN with the five nucleosides at T = 308.15,318.15,and 328.15 K decreased in the order C > G > T > U > A and at T = 298.15 K decreased in the order G > C > T > U > A,and that the binding of SAN with the various nucleosides is not only slightly exothermic but also entropy-driven.All these results together with fluorescence quenching experiments advance good evidence concerning the interaction of SAN with various nucleosides.Such studies of the interaction mechanism of alkaloids with DNA may promote the development of new drugs.     

Spectroscopic Characterization of 3-Aminoisoxazole,a Prebiotic Precursor of Ribonucleotides

The processes and reactions that led to the formation of the first biomolecules on Earth play a key role in the highly debated theme of the origin of life. Whether the first chemical building blocks were generated on Earth (endogenous synthesis) or brought from space (exogenous delivery) is still unanswered. The detection of complex organic molecules in the interstellar medium provides valuable support to the latter hypothesis. To gather more insight, here we provide the astronomers with accurate rotational frequencies to guide the interstellar search of 3-aminoisoxazole, which has been recently envisaged as a key reactive species in the scenario of the so-called RNA-world hypothesis. Relying on an accurate computational characterization, we were able to register and analyze the rotational spectrum of 3-aminoisoxazole in the 6–24 GHz and 80–320 GHz frequency ranges for the first time, exploiting a Fourier-transform microwave spectrometer and a frequency-modulated millimeter/sub-millimeter spectrometer, respectively. Due to the inversion motion of the −NH₂ group, two states arise, and both of them were characterized, with more than 1300 lines being assigned. Although the fit statistics were affected by an evident Coriolis interaction, we were able to produce accurate line catalogs for astronomical observations of 3-aminoisoxazole.     

Amino Acid Modified RNA Bases as Building Blocks of an Early Earth RNA-Peptide World

Fossils of extinct species allow us to reconstruct the process of Darwinian evolution that led to the species diversity we see on Earth today. The origin of the first functional molecules able to undergo molecular evolution and thus eventually able to create life, are largely unknown. The most prominent idea in the field posits that biology was preceded by an era of molecular evolution, in which RNA molecules encoded information and catalysed their own replication. This RNA world concept stands against other hypotheses, that argue for example that life may have begun with catalytic peptides and primitive metabolic cycles. The question whether RNA or peptides were first is addressed by the RNA-peptide world concept, which postulates a parallel existence of both molecular species. A plausible experimental model of how such an RNA-peptide world may have looked like, however, is absent. Here we report the synthesis and physicochemical evaluation of amino acid containing adenosine bases, which are closely related to molecules that are found today in the anticodon stem-loop of tRNAs from all three kingdoms of life. We show that these adenosines lose their base pairing properties, which allow them to equip RNA with amino acids independent of the sequence context. As such we may consider them to be living molecular fossils of an extinct molecular RNA-peptide world.     

On the Prebiotic Synthesis of D‐Sugars Catalyzed by L‐Peptides: Assessments from First‐Principles Calculations

What accounts for a particular chiral selection in the case of a few sugars of prebiotic relevance, thereby mirroring the asymmetry observed in nature? By using first‐principles calculations, the generation of pentoses from glycolaldehyde (the initial product of the autocatalytic formose reaction), which has been detected in outer space), has been modeled by using L ‐Val‐L ‐Val as a primeval catalyst. Our theoretical study provides insight into the mechanism of this reaction and satisfactorily explains a few key molecular events. Our rationale agrees with the reported experimental data and shows that the D ‐configuration is only favored for ribose. L ‐pentoses are usually favored in the presence of L ‐configured dipeptides, as observed experimentally, although no chiral selection could be observed in the case of xylose. These results confirm that a prebiotic sugar soup could be fine‐tuned in the presence of shorter peptides as catalysts and that D ‐ribose would have also resulted in an advantageous imbalance for further amplification and chemical evolution.