John D. Sutherland

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.

Common origins of RNA, protein and lipid precursors in a cyanosulfidic protometabolism

A minimal cell can be thought of as comprising informational, compartment-forming and metabolic subsystems. To imagine the abiotic assembly of such an overall system, however, places great demands on hypothetical prebiotic chemistry. The perceived differences and incompatibilities between these subsystems have led to the widely held assumption that one or other subsystem must have preceded the others. Here we experimentally investigate the validity of this assumption by examining the assembly of various biomolecular building blocks from prebiotically plausible intermediates and one-carbon feedstock molecules. We show that precursors of ribonucleotides, amino acids and lipids can all be derived by the reductive homologation of hydrogen cyanide and some of its derivatives, and thus that all the cellular subsystems could have arisen simultaneously through common chemistry. The key reaction steps are driven by ultraviolet light, use hydrogen sulfide as the reductant and can be accelerated by Cu(I)-Cu(II) photoredox cycling.

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 simple sugars by photoredox systems chemistry

A recent synthesis of activated pyrimidine ribonucleotides under prebiotically plausible conditions relied on mixed oxygenous and nitrogenous systems chemistry. As it stands, this synthesis provides support for the involvement of RNA in the origin of life, but such support would be considerably strengthened if the sugar building blocks for the synthesis--glycolaldehyde and glyceraldehyde--could be shown to derive from one carbon feedstock molecules using similarly mixed oxygenous and nitrogenous systems chemistry. Here, we show that these sugars can be formed from hydrogen cyanide by ultraviolet irradiation in the presence of cyanometallates in a remarkable systems chemistry process. Using copper cyanide complexes, the process operates catalytically to disproportionate hydrogen cyanide, first generating the sugars and then sequestering them as simple derivatives.

Prebiotic Chemistry: A Bioorganic Perspective

1. Scope 2. The Primordial Earth 3. Prebiotic Feedstock Molecules 4. Synthesis of Amino Acids 5. Synthesis of Purines and Chemistry of HCN 6. Synthesis of Pyrimidines 7. Synthesis of Sugars 8. Synthesis of Sugar Phosphates 9. Synthesis of Riboflavin 10. Theories 11. The R N A W o r l d 12. In vitro Evolution 13. Pyranosyl RNA and Hexose Nucleic Acids 14. Peptide Nucleic Acid 15. Iron Pyrites 16. Clay Crystals 17. Prebiogenesis of the Natural Nucleic Acids 18. Alternative Backbone Nucleic Acids 19. An Alternative RNA Disconnection 20. On the Interconnection between RNA and DNA 21. Refinements to Theory and Future Directions

Prebiotic chemistry: a new modus operandi.

A variety of macromolecules and small molecules—(oligo)nucleotides, proteins, lipids and metabolites—are collectively considered essential to early life. However, previous schemes for the origin of life—e.g. the ‘RNA world’ hypothesis—have tended to assume the initial emergence of life based on one such molecular class followed by the sequential addition of the others, rather than the emergence of life based on a mixture of all the classes of molecules. This view is in part due to the perceived implausibility of multi-component reaction chemistry producing such a mixture. The concept of systems chemistry challenges such preconceptions by suggesting the possibility of molecular synergism in complex mixtures. If a systems chemistry method to make mixtures of all the classes of molecules considered essential for early life were to be discovered, the significant conceptual difficulties associated with pure RNA, protein, lipid or metabolism ‘worlds’ would be alleviated. Knowledge of the geochemical conditions conducive to the chemical origins of life is crucial, but cannot be inferred from a planetary sciences approach alone. Instead, insights from the organic reactivity of analytically accessible chemical subsystems can inform the search for the relevant geochemical conditions. If the common set of conditions under which these subsystems work productively, and compatibly, matches plausible geochemistry, an origins of life scenario can be inferred. Using chemical clues from multiple subsystems in this way is akin to triangulation, and constitutes a novel approach to discover the circumstances surrounding the transition from chemistry to biology. Here, we exemplify this strategy by finding common conditions under which chemical subsystems generate nucleotides and lipids in a compatible and potentially synergistic way. The conditions hint at a post-meteoritic impact origin of life scenario.

Engineering of Penicillium chrysogenum for fermentative production of a novel carbamoylated cephem antibiotic precursor

Penicillium chrysogenum was successfully engineered to produce a novel carbamoylated cephalosporin that can be used as a synthon for semi-synthetic cephalosporins. To this end, genes for Acremonium chrysogenum expandase/hydroxylase and Streptomyces clavuligerus carbamoyltransferase were expressed in a penicillinG high-producing strain of P.chrysogenum. Growth of the engineered strain in the presence of adipic acid resulted in production of adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA) and of several adipoylated pathway intermediates. A combinatorial chemostat-based transcriptome study, in which the ad7-ACCCA-producing strain and a strain lacking key genes in beta-lactam synthesis were grown in the presence and absence of adipic acid, enabled the dissection of transcriptional responses to adipic acid per se and to ad7-ACCCA production. Transcriptome analysis revealed that adipate catabolism in P.chrysogenum occurs via beta-oxidation and enabled the identification of putative genes for enzymes involved in mitochondrial and peroxisomal beta-oxidation pathways. Several of the genes that showed a specifically altered transcript level in ad7-ACCCA-producing cultures were previously implicated in oxidative stress responses.

Evolutionary optimisation of enzymes

Aside from the demonstration that individual molecular traits of enzymes can be evolutionarily optimised, the discovery that several traits can be simultaneously optimised is a major advance. The first observations of the effects of evolutionary optimisation at the structural level, through X-ray crystallography, reinforce the view that enzymes are best optimised by evolution and not by design.

Phosphate‐Mediated Interconversion of Ribo‐ and Arabino‐Configured Prebiotic Nucleotide Intermediates

Compound 2 is produced alongside ribose aminooxazoline 3 and its lyxo and xylo diastereoisomers when 2-aminooxazole 4 adds to glyceraldehyde 5. The synthesis of 4 from glycolaldehyde 6 and cyanamide 7 only takes place efficiently at neutral pH when phosphate was included as a general acid– base catalyst. Furthermore, the subsequent reaction of 2 with cyanoacetylene 8, giving the anhydronucleoside 9, is also controlled by phosphate at pH 6.5, where it acts as a pH buffer, thereby preventing hydrolysis of 9 to arabinocytidine. Heating 9 with phosphate in formamide or in the dry state then results in the formation of 1 (base = Cyt). In a final step, 1 (base = Cyt) is partly converted into 1 (base = Ura) by UV irradiation in aqueous solution. Enantiomerically pure monomers are considered essential for the abiogenesis of RNA (racemic or scalemic monomers potentially giving rise to enormously complex diastereomeric mixtures of polymers), but as it stands, the synthesis does not yield enantiomerically pure ribonucleotides 1 unless the starting glyceraldehyde 5 is enantiomerically pure. Whilst enantioselectivity in the formation of glyceraldehyde 5 by aldolization of glycolaldehyde 6 and formaldehyde is easy to imagine if a suitable chiral catalyst could be found, formation of enantiopure 5 seems unlikely. Fractional crystallization of the ribonucleotides 1 or chiral intermediates in the synthesis offers a potential means of further enantioenrichment. The aminooxazolines 2 and 3 are less polar than 1 or the anydronucleosides such as 9, and so offer the best hope of such a crystallization. However, the arabino-configured 2 needed for ribonucleotide synthesis is more water-soluble than the ribo-configured 3 along with which it is produced. Indeed, upon cooling or concentration of a solution of all four aminooxazolines, 3 selectively crystallizes. Furthermore, scalemic 3 is enantioenriched by crystallization such that enantiopure 3 can be obtained from 5 with an enantiomeric excess (ee) of 60 %. We have therefore sought a means whereby 3, purified and enriched to enantiomeric purity in such a way, might subsequently be partly converted into 2. Such a conversion would maintain the enantiopurity established by crystallization but transfer it from the ribo series to the arabino series and from there onwards to the ribo nucleotides 1. Phosphate has proved to be a key player in the systems chemistry aspects of the synthesis, so we took the requirement for phosphate to be present in the subsequent conversion of 2 into 9 as a clue that phosphate might also catalyze the conversion of 3 into 2. Furthermore, we envisaged a potential mechanism for the interconversion of 2 and 3 that appeared to require general acid–base catalysis (Scheme 2), and we had previously found phosphate to be an ideal mediator of such catalysis. When exploring the effects of pH buffering by phosphate on the reactions of aminooxazolines 2 and 3 with cyanoacetylene 8, we had not seen any interconversion of 2 and 3. Scheme 1. Potentially prebiotic synthesis of activated pyrimidine nucleotides 1. Pi = inorganic phosphate.

Synthesis of cytidine ribonucleotides by stepwise assembly of the heterocycle on a sugar phosphate.

Although various syntheses of the nucleic acid bases exist and ribose is a product of the formose reaction, no prebiotically plausible methods for attaching pyrimidine bases to ribose to give nucleosides have been described. Kinetic and thermodynamic factors are thought to mitigate against such condensation reactions in aqueous solution. This inability to produce pyrimidine nucleosides and hence nucleotides is a major stumbling block of the “RNA World” hypothesis and has led to suggestions of alternative nucleic acids as evolutionary precursors to RNA. Here, we show that a process in which the base is assembled in stages on a sugar phosphate can produce cytidine nucleotides. The sequential action of cyanamide and cyanoacetylene on arabinose‐3‐phosphate produces cytidine‐2′,3′‐cyclophosphate and arabinocytidine‐3′‐phosphate.