A possible prebiotic synthesis of purine, adenine, cytosine, and 4(3H)-pyrimidinone from formamide: implications for the origin of life

The synthesis of prebiotic molecules is a major problem in chemical evolution as well as in any origin-of-life theory. We report here a plausible new prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide under catalytic conditions. In the presence of CaCO(3) and different inorganic oxides, namely silica, alumine, kaolin, and zeolite (Y type), neat formamide undergoes the formation of purine, adenine, cytosine, and 4(3H)-pyrimidinone, from acceptable to good yields. The role of catalysts showed to be not limited to the improvement of the yield but it is also relevant in providing a high selectivity in the products distribution.     

Preparation of guanine and diaminopurine from biuret. Part III

Because of their potential prebiotic origin and relative chemical stability, urea, biuret, formic acid, and glycine amide might have played a role in the assembly process of purine bases. In this paper, we describe a short reaction path to purine nucleobases from these acyclic precursors. The formation of different purines was verified by UV and NMR spectroscopy, as well as by mass spectrometry.     

The Role of Self-Assembled Monolayers of the Purine and Pyrimidine Bases in the Emergence of Life

The experimental evidence for the spontaneous formation and structure determination of two-dimensional monolayers of the purine and pyrimidine bases is examined. The plausibility of such structures forming spontaneously at the solid-liquid interface following their prebiotic synthesis suggests a functional role for them in the emergence of life. It is proposed that prebiotic interactions of enantiomorphic monolayers of mixed base composition with racemic amino acids might be implicated in a simultaneous origin of a primitive genetic coding mechanism and biomolecular homochirality. The interactions of these monolayers with carbohydrates and other derivatives is also discussed.     

Purine Biosynthetic Intermediate-Containing Ribose-Phosphate Polymers as Evolutionary Precursors to RNA

The RNA world hypothesis proposes that RNA once functioned as the principal genetic material and biological catalyst. However, RNA is a complex molecule made up of phosphate, ribose, and nucleobase moieties, and its evolution is unclear. Yakhnin has proposed a period of prebiotic chemical evolution prior to the advent of replication and Darwinian evolution, in which macromolecules containing polyols joined by phosphodiester linkages underwent spontaneous transesterification reactions with selection for stability. Although he proposes that the nucleobases were obtained during this stage from less stable macromolecules, the ultimate source of the nucleobases is not addressed. We propose that the purine nucleobases arose in situ from simpler precursors attached to a ribose-phosphate backbone, and that the weaker and less specific intra- and interstrand interactions between these precursors were the forerunners to the base pairing and base stacking interactions of the modern RNA nucleobases. Further, in line with Granick’s hypothesis of biosynthetic pathways recapitulating evolution, we propose that these simpler precursors were the same or similar to intermediates of the modern de novo purine biosynthetic pathway. We propose that successive nucleobase precursors formed progressively stronger interactions that stabilized the ribose-phosphate polymer, and that the increased stability of the parent polymer drove the selection and further chemical evolution of the purine nucleobases. Such interactions may have included hydrogen bonding between ribose hydroxyls, hydrogen bonding between carbonyl oxygens and protonated amine side groups, the intra- and interstrand coordination of metal cations, and the stacking of imidazole rings. Five of the eleven steps of the modern de novo purine biosynthetic pathway have previously been shown to have alternative nonenzymatic syntheses, while a sixth step has also been proposed to occur nonenzymatically, supporting a prebiotic origin for the pathway.     

Conversion of Biosynthetic Precursors of RNA to Those of DNA by Photoredox Chemistry

Soon after the origin of RNA-based life, depletion of prebiotically synthesised ribonucleotides would have driven the evolution of a biosynthetic pathway to these key building blocks. Ribozyme-catalysed nucleosidation—the key biosynthetic step—requires that ribose and the nucleobases are produced by abiotic chemistry and are relatively stable to the conditions of their synthesis. The most plausible prebiotic synthesis of sugars involves photoreduction of cyanohydrins by hydrogen sulphide in the presence of copper(I) cyanide, and we therefore subjected ribose to these conditions whereupon it was partially converted to 2-deoxyribose. Furthermore, a derivative of uracil is reduced under similar conditions to thymine. Thus, DNA biosynthetic precursors can be formed abiotically from those of RNA allowing for an early evolutionary transition to life based on RNA and DNA.     

Structural Phylogenomics Reveals Gradual Evolutionary Replacement of Abiotic Chemistries by Protein Enzymes in Purine Metabolism

The origin of metabolism has been linked to abiotic chemistries that existed in our planet at the beginning of life. While plausible chemical pathways have been proposed, including the synthesis of nucleobases, ribose and ribonucleotides, the cooption of these reactions by modern enzymes remains shrouded in mystery. Here we study the emergence of purine metabolism. The ages of protein domains derived from a census of fold family structure in hundreds of genomes were mapped onto enzymes in metabolic diagrams. We find that the origin of the nucleotide interconversion pathway benefited most parsimoniously from the prebiotic formation of adenine nucleosides. In turn, pathways of nucleotide biosynthesis, catabolism and salvage originated ∼300 million years later by concerted enzymatic recruitments and gradual replacement of abiotic chemistries. Remarkably, this process led to the emergence of the fully enzymatic biosynthetic pathway ∼3 billion years ago, concurrently with the appearance of a functional ribosome. The simultaneous appearance of purine biosynthesis and the ribosome probably fulfilled the expanding matter-energy and processing needs of genomic information.     

Characterization of novel Na+-dependent nucleobase transport systems at the blood-testis barrier

In the testis, nucleosides and nucleobases are important substrates of the salvage pathway for nucleotide biosynthesis, and one of the roles of Sertoli cells is to provide nutrients and metabolic precursors to spermatogenic cells located within the blood-testis barrier (BTB). We have already shown that concentrative and equilibrative nucleoside transporters are expressed and are functional in primary-cultured rat Sertoli cells as a BTB model, but little is known about nucleobase transport at the BTB or about the genes encoding specific nucleobase transporters in mammalian cells. In the present study, we examined the uptake of purine ([3H]guanine) and pyrimidine ([3H]uracil) nucleobases by primary-cultured rat Sertoli cells. The uptake of both nucleobases was time and concentration dependent. Kinetic analysis showed the involvement of three different transport systems in guanine uptake. In contrast, uracil uptake was mediated by a single Na+-dependent high-affinity transport system. Guanine uptake was inhibited by other purine nucleobases but not by pyrimidine nucleobases, whereas uracil uptake was inhibited only by pyrimidine nucleobases. In conclusion, it was suggested that there might be purine- or pyrimidine-selective nucleobase transporters in rat Sertoli cells.     

SYNTHESIS OF RACEMIC AND ENANTIOMERIC 3-PYRROLIDINYL DERIVATIVES OF PURINE AND PYRIMIDINE NUCLEOBASES

The present work relates to the synthesis of pyrrolidine nucleoside analogs. Starting from malic acid, we have elaborated a high-yield synthesis of racemic and enantiomeric N-protected 3-pyrrolidinols and their O-mesyl derivatives as key compounds for alkylations of purine and pyrimidine nucleobases. On varying base and solvent, we have found conditions providing both satisfactory N-/O-regioisomeric ratio and acceptable yield for pyrimidine compounds.     

Synthesis of racemic and enantiomeric 3-pyrrolidinyl derivatives of purine and pyrimidine nucleobases

The present work relates to the synthesis of pyrrolidine nucleoside analogs. Starting from malic acid, we have elaborated a high-yield synthesis of racemic and enantiomeric N-protected 3-pyrrolidinols and their O-mesyl derivatives as key compounds for alkylations of purine and pyrimidine nucleobases. On varying base and solvent, we have found conditions providing both satisfactory N-/O-regioisomeric ratio and acceptable yield for pyrimidine compounds.     

The role of spin in biological processes: O2, NO,nucleobases, nucleosides,nucleotides and Watson–Crick base pairs

The experimental electron affinities of adenine, guanine, cytosine, thymine and uracil have been determined from reduction potentials and negative ion photoelectron spectra. Updated values for purine, pyrimidine and other nitrogen heterocyclics, which have not been measured in the gas phase, are presented. The electron affinity of Watson–Crick guanine–cytosine is estimated empirically. The experimental values are consistent with quantum mechanical semi-empirical multiconfiguration configuration interaction calculations. The bulk hydration energies of the nucleobase anions, 2.34 eV, determined from the experimental data and sequential anion hydration energy difference of about 0.20(5) eV suggest that 10–15 water molecules complete the hydration shell. The electron affinities for the formation of doublet and quartet anions of the nucleobases, nucleosides, nucleotides and Watson–Crick base pairs are calculated. We postulate that low-lying quartet anion states and their spin distribution can and will participate in electron conduction, radiation damage, oxidation damage and repair, strand breakage and protein synthesis.     

From earth's primitive atmosphere to chiral peptides--the origin of precursors for life

To re-enact the long way to the origin of life with today's chemical methods, many steps have to be investigated in the light of a primordial scenario deduced from geochemical research. After the formation of our planet and its atmosphere, prebiotic chemical evolution started its course with the formation of the first building blocks for the formation of biomolecules. In the case of proteins, those building blocks were amino acids that had to be formed in the primitive atmosphere, and then had to react to peptides and proteins as the main pillar of first life. In this paper, we describe the processes in the primordial atmosphere according to contemporary geochemical knowledge leading to the synthesis of amino acids until the formation of homochiral peptides, and, thus, show a plausible pathway towards the origin of life.     

Formamide chemistry and the origin of informational polymers

Formamide (HCONH2) provides a chemical frame potentially affording all the monomeric components necessary for the formation of nucleic polymers. In the presence of the appropriate catalysts, and by moderate heating, formamide yields a complete set of nucleic bases, acyclonucleosides, and favors both phosphorylations and transphosphorylations. Physico-chemical conditions exist in which formamide favors the stability of the phosphoester bonds in nucleic polymers more than that of the same bonds in monomers. This property establishes 'thermodynamic niches' in which the polymeric forms are favored. The hypothesis that these specific attributes of formamide allowed the onset of prebiotic chemical equilibria capable of Darwinian evolution is discussed.     

Hydrogen bonding versus stacking stabilization by modified nucleobases incorporated in PNA·DNA duplexes

The effects of incorporation of the modified nucleobases, 2,6-diaminopurine (D) (substituting for adenine) and 7-chloro-1,8-naphthyridin-2-(1H)-one (bicyclic thymine, bT) (substituting for thymine), that stabilize PNA·DNA duplex formation by increasing hydrogen bonding and/or base pair stacking interactions have been studied by thermal denaturation in terms of thermodynamics. Although the stabilizing effect of the bT base (in contrast to that of D base) is abolished upon addition of dimethyl formamide, thereby indicating that the stabilization is predominantly due to hydrophobic stacking forces, duplex stabilization was found to be enthalpic for both nucleobases. Increased stabilization (although not fully linearly) was observed with increasing numbers of modified bases, and single base sequence discrimination was only slightly compromised, but showed significant dependence on the sequence context.     

Mineral interface in extreme habitats: a niche for primitive molecular evolution for the appearance of different forms of life on earth

Innumerable primitive membrane and protocell models in latter stages of chemical evolution are based on the properties of minerals' interfaces with primitive seawater. The ordering mechanism induced by mineral interfaces has been the basis of several prebiotic models of molecular complexification and compartmentalization towards the appearance and evolution of different forms of life. Since mineral-aqueous media interfaces have been considered as initial stages of prebiotic models dealing with the formation of energy-transducing systems, the interface formed by pyrite in the presence of artificial primitive seawater was chosen to show the functional richness of this special niche. Interfaces--especially sulphide interfaces--were proposed as suitable niches for a two-carbon extant metabolism, synthesis and polymerization of nucleotides--to form ancient RNA strands--and assembly of amino acids synthesized in its vicinity. Accumulation of precursors at sulphide interfaces could have avoided their dilution into the Hadean seas and provided a suitable geochemical environment for a variety of molecular interactions. In this essay, we present a short review of the proposed roles of mineral interfaces in chemical evolution towards the appearance of primitive membranes, which might have been relevant for the advent of cellular life before its divergent evolution and differentiation. This survey covers several previous studies on the early cycles of energy conservation and of the formation of molecules carrying genetic information.     

Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro.

Thymine glycols were produced in M13 DNA in a concentration dependent manner by treating the DNA with osmium tetroxide (OsO4). For the formation of urea-containing M13 DNA, OsO4-oxidized DNA was hydrolyzed in alkali (pH 12) to convert the thymine glycols to urea residues. With both thymine glycol- and urea-containing M13 DNA, DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment was decreased in proportion to the number of damages present in the template DNA. Sequencing gel analysis of the products synthesized by E. coli DNA polymerase I and T4 DNA polymerase showed that DNA synthesis terminated opposite the putative thymine glycol site and at one nucleotide before the putative urea site. Substitution of manganese for magnesium in the reaction mix resulted in increased processivity of DNA synthesis so that a base was incorporated opposite urea. With thymine glycol-containing DNA, processivity in the presence of manganese was strongly dependent on the presence of a pyrimidine 5' to the thymine glycol in the template.     

Trypanosoma brucei: a survey of pyrimidine transport activities

Purine uptake has been studied in many protozoan parasites in the last few years, and several of the purine transporters have been cloned. In contrast, very little is known about the salvage of preformed pyrimidines by protozoa, and no pyrimidine transporters have been cloned, yet chemotherapy based on pyrimidine nucleobases and nucleosides has been as effective as purine antimetabolites in the treatment of infectious and neoplastic disease. Here, we surveyed the presence of pyrimidine transporters in Trypanosoma brucei brucei. We could not detect any mediated uptake of thymine, thymidine or cytidine, but identified a very high-affinity transporter for cytosine, designated C1, with a K(m) value of 0.048+/-0.009 microM. We also confirmed the presence of the previously reported U1 uracil transporter and found it capable of mediating uridine uptake as well, with a K(m) of 33+/-5 microM. A higher-affinity U2 uridine transporter (K(m)=4.1+/-2.1 microM) was also identified, but efficiency of the C1 and U2-mediated transport was low. Pyrimidine antimetabolites were tested as potential trypanocidal agents and only 5-fluorouracil was found to be effective. This drug was efficiently taken up by bloodstream forms of T. b. brucei.     

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.     

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.     

Exploratory studies to investigate a linked prebiotic origin of RNA and coded peptides. 3rd communication. Behaviour of 5-amino-1H-imidazole-4-carbonitrile derivatives

The potentially prebiotic synthesis of pyrimidine ribonucleotides by stepwise nucleobase assembly on arabinose-3-phosphate derivatives has been demonstrated in previous work. The generation of xylose-2-phosphate derivatives by aldolisation, and the behaviour of these compounds under the conditions of pyrimidine nucleobase assembly have also been described. In this paper, the scope for generation of purine nucleotides via 3,3'-anhydro-xylo-nucleotides is investigated. In neutral D2O solution, the potential intermediate 47 (Schemes 6 and 8) undergoes H-C2 --> D-C2 exchange, but no appreciable reaction with cyanide or cyanamide occurs. The exchange chemistry expands options for purine nucleobase assembly on sugar phosphate scaffolds.     

Enhancement of Electrochemical Differentiation Ability of Nucleobases in Phosphate Buffer Solution at pH 7.4

The electrochemical behavior of nucleobases has been studied in 0.1 M phosphate buffer solution at pH 7.4, using a bare graphite electrode. Guanine and adenine produced well-defined oxidation peaks at about +0.63 and +0.91 V at 100 mV/s, respectively. Nucleobases exhibit an irreversible and hybrid-controlled electrochemical process, including adsorption and diffusion. The nucleobase oxidation peaks shift due to the selective interactions of nucleobases with each other. The oxidation peaks for three different pyrimidine bases, uracil, cytosine, and thymine, can be clearly identified at +1.26, +1.41, and +1.32 V, respectively. These differences in the electrochemical behavior among nucleobases can be attributed to their different chemical structures.