Urea-acetylene dicarboxylic acid reaction: A likely pathway for prebiotic uracil formation

A number of routes have been suggested for the prebiotic synthesis of uracil involving the reaction of urea with malic acid, propiolic acid, cyanoacetylene and others. Cyanoacetylene has been detected in the interstellar medium as well as simulated prebiotic experiments. It is therefore plausible that dicyanoacetylene and its hydrolytic product acetylene dicarboxylic acid, (ADCA) may have played a role in chemical evolution. This aspect has been examined in the present work for the synthesis of uracil from ADCA and urea reaction.It was found that when ADCA reacted with urea, uracil was formed only in the presence of phosphoric acid and phosphates. Ammonium phosphates gave higher yields of uracil than other phosphates. In the absence of phosphoric acid or phosphates no uracil formation took place. This type of synthesis could have taken place in prebiotic oceans which contained ammonium phosphates and other salts.     

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.     

Attempted nonenzymatic template-directed oligomerizations on a polyadenylic acid template: Implications for the nature of the first genetic material

Summary Previous attempts to produce nonenzymatic template-directed oligomerizations of activated pyrimidines on polypurine templates have been unsuccessful. The only efficient reactions are those where the template is composed primarily of pyrimidines, especially cytosine. Because molecular evolution requires that a synthesized daughter polynucleotide be capable of acting as a template for the synthesis of the original polynucleotide, the one-way replication achieved thus far is inadequate to initiate an evolving system.Several uracil analogs were used in this investigation in order to search for possible replacements for uracil. The monomers used in this investigation were the imidazolides of UMP, xanthosine 5-monophosphate, the bis-monophosphates of the acyclic nucleosides of uracil, and 2,4-quinazolinedione. The concentrations of various salts, buffers, pH, and temperature were among the different variables investigated in attempts to find conditions that would permit template-directed oligomerizations. Although the different monomers in this study demonstrated varying abilities to form very short oligomers, we were unable to detect any enhancement of this oligomerization that could be attributed to the poly(A) template.Although special conditions might be found that would allow purine-rich templates to work, these reactions cannot be considered robust. The results of our experiments suggest that pyrimidines were not part of the original replicating system on the primitive Earth. It has already been shown that ribose is an unlikely component of the first replicating systems, and we now suggest that phosphate was absent as well. This is due to the low solubility of phosphate in the present ocean (3×10^–6 M), as well as the difficulty of prebiotic activation of phosphates.     

Alternative bases in the RNA world: The prebiotic synthesis of urazole and its ribosides

Urazole is five-membered heterocyclic compound which is isosteric with uracil's hydrogen-bonding segment. Urazole reacts spontaneously with ribose (and other aldoses) to give mixture of four ribosides: and pyranosides and furanosides. This reaction occurs in aqueous solution at mild temperatures. Thermodynamic and kinetic parameters for the reaction of urazole with ribose were determined. In contrast, uracil is completely unreactive with ribose under these conditions. Urazole's unusual reactivity is ascribed to the hydrazine portion of the molecule. Urazole can be synthesized from biuret and hydrazine under prebiotic conditions. The prebiotic synthesis of guanazole, which is isosteric in part to diaminopyrimidine and cytosine, is accomplished from dicyandiamide and hydrazine. Kinetic parameters for both prebiotic reactions were measured. Urazole and guanazole are transparent in the UV, which would be favorable property in the absence of an ozone layer on the early Earth. Urazole makes hydrogen bonds with adenine in DMSO similar to those of uracil, as established by IH NMR. All of these properties make urazole an attractive potential precursor to uracil and guanazole a potential precursor to cytosine in the RNA or pre-RNA world.Correspondence to: S.L. Miller 0805Presented at the ISSOL Meeting in Barcelona, 5 July 1993 V.M. Kolb is on sabbatical leave from the University of Wisconsin-Parkside, Kenosha, WI 53141     

Studies on the Lead-Catalyzed Synthesis of Aldopentoses

The object of this work was to find an efficient means of synthesizing ribose in a manner that could be considered prebiotic. The starting point for synthesis was an aqueous solution of formaldehyde. Heretofore the most frequently used catalyst for this purpose has been calcium hydroxide. Unfortunately this system produces a wide array of products in addition to ribose which constitutes 1% or less of the final product. Attempts were made to find more mild conditions under which the formaldehyde could be reacted. Magnesium hydroxide suspensions were used for this purpose. Formaldehyde does not yield any sugars when incubated in magnesium hydroxide suspensions alone. However, if the magnesium hydroxide suspension was supplemented with doubly charged lead salts and catalytic amounts of any intermediate in the prebiotic pentose pathway, aldopentoses accounted for 30 per cent or more of the final product. The presence of lead in the incubation mixture also accelerated a number of other reactions including the interconversion of the four common aldopentoses, ribose, arabinose, lyxose and xylose.     

Prebiotic synthesis of histidyl-histidine

Summary Histidyl-histidine (His-His) has been synthesized in a yield of up to 14.4% under plausible prebiotic conditions using histidine (His), cyanamide, and 4-amino-5-imidazole carboxamide. A trace amount of His trimer was also detected. Because the imidazole group of His is involved in a number of important enzymatic reactions, and His-His has been shown to catalyze the prebiotic synthesis of glycyl-glycine, we expect this work will stimulate further studies on the catalytic activities of simple His-containing peptides in prebiotic reactions.     

Non-oxidative synthesis of pentose 5-phosphate from hexose 6-phosphate and triose phosphate by the L-type pentose pathway

1. Ribose 5-phosphate was non-oxidatively synthesized from glucose 6-phosphate and triose phosphate by an enzyme extract prepared from rat liver (RLEP). Analysis of the intermediates by GLC, ion-exchange chromatography and specific enzymatic analysis, revealed the presence of the following intermediates of the L-type pentose pathway: altro-heptulose 1,7-bisphosphate, arabinose 5-phosphate and D-glycero D-ido octulose 8-phosphate. 2. With either [1-14C] or [2-14C]glucose 6-phosphate as diagnostic substrates, the distribution of 14C in ribose 5-phosphate was determined. At early time intervals (0.5-8 hr), [1-14C]glucose 6-phosphate introduced 14C into C-1, C-3 and C-5 of ribose 5-phosphate, at 17 hr 14C was confined to C-1. With [2-14C]glucose 6-phosphate as substrate, 14C was confined to C-2, C-3 and C-5 of ribose 5-phosphate during early times (0.5-8 hr), while at 17 hr 14C was located in C-2. 3. The transketolase exchange reaction, [14C]ribose 5-phosphate + altro-heptulose 7-phosphate in equilibrium ribose 5-phosphate + [14C]altro-heptulose 7-phosphate, was demonstrated for the first time using purified transketolase, its activity was measured and it is proposed to play a major role in the relocation of 14C into C-3 and C-5 or ribose 5-phosphate during the prediction labelling experiments. 4. The coupled transketolase-transaldolase reactions, 2 fructose 6-phosphate in equilibrium altro-heptulose 7-phosphate + xylulose 5-phosphate and 2 altro-heptulose 7-phosphate in equilibrium fructose 6-phosphate + D-glycero D-altro octulose 8-phosphate were demonstrated with purified enzymes, but are concluded to play a minor role in the non-oxidative synthesis of pentose 5-phosphate and octulose phosphate by (RLEP). 5. The formation of gem diol and dimers of erythrose 4-phosphate is proposed to account in part for the failure to detect monomeric erythrose 4-phosphate in the carbon balance studies. 6. The equilibrium value for the pentose pathway acting by the reverse mode in vitro was measured and contrasted with the value for the pathway acting in the forward direction. The initial specific rates of the pentose pathway reactions in vitro for the reverse and forward directions are measured. 7. The study which includes carbon balance, time course changes and 14C prediction labelling experiments reports a comprehensive investigation of the mechanism of the pentose pathway acting reversibly.     

Prebiotic ribose synthesis: A critical analysis

The discovery of catalytic ability in RNA has given fresh impetus to speculations that RNA played a critical role in the origin of life. This question must rest on the plausibility of prebiotic oligonucleotide synthesis, rather than on the properties of the final product. Many cliams have been published to support the idea that the components of RNA were readily available on the prebiotic earth. In this article, the literature cited in support of the prebiotic availability of one subunit, D-ribose, is reviewed to determine whether it justifies the claim.Polymerization of formaldehyde (the formose reaction) has been the single reaction cited for prebiotic ribose synthesis. It has been conducted with different catalysts: numerous basic substances, neutral clays and heat, and various types of radiation. Ribose has been identified (yields are uncertain, but unlikely to be greater than 1%) in reactions run with concentrated (0.15 M or greater) formaldehyde. It has been claimed in reactions run at lower concentration, but characterization has been inadequate, and experimental details have not been provided.The complex sugar mixture produced in the formose reaction is rapidly destroyed under the reaction conditions. Nitrogenous substances (needed for prebiotic base synthesis) would interfere with the formose reaction by reacting with formaldehyde, the intermediates, and sugar products in undesirable ways.The evidence that is currently available does not support the availability of ribose on the prebiotic earth, except perhaps for brief periods of time, in low concentration as part of a complex mixture, and under conditions unsuitable for nucleoside synthesis.     

A possible prebiotic synthesis of thymine: uracil-formaldehyde-formic acid reaction.

When uracil is reacted with formaldehyde and formic acid in dilute aqueous solutions at 100-140 degrees C, 5-hydroxymethyluracil (5-HMU), methylenebiuracil (MBU) and thymine are formed. It has been shown that 5-HMU is an intermediate in the formation of MBU and thymine. In the presence of formic acid, 5-HMU gives MBU, thymine and in some cases uracil. The formation of thymine is generally favoured under acidic conditions, although small amounts of this base could also be obtained when the reactions were carried out under mildly basic conditions. A hydride ion transfer mechanism is suggested for some of these reactions. These results have relevance to the formation of thymine under prebiotic conditions.     

The prebiotic synthesis of carbohydrates: A reassessment

Summary Questions concerning the significance of previous work on the formose reaction have led us to reexamine the question of the prebiotic synthesis of sugars. The results of new experiments lead to the following conclusions: The formose reaction is a geochemically plausible reaction which depends on neither basic conditions nor on the presence of trace amounts of carbohydrate impurities. However, this process is not a plausible source of ribose nor of any other individual sugar. In contrast to the nonspecific formation of complex mixtures of sugars via the formose reaction, the reduced sugar pentaerythritol is formed with great selectivity by the ultraviolet irradiation of 0.1 M formaldehyde. This compound may have played an important role in chemical evolution.Offprint requests to: A.W. Schwartz     

The fate of 14C in glucose 6-phosphate synthesized from [1-14C]Ribose 5-phosphate by enzymes of rat liver.

1. Glucose 5-phosphate was synthesized from ribose 5-phosphate by an enzyme extract prepared from an acetone-dried powder of rat liver. Three rates of ribose 5-phosphate utilization were observed during incubation for 17 h. An analysis of intermediates and products formed throughout the incubation revealed that as much as 20% of the substrate carbon could not be accounted for. 2. With [1-14C]ribose 5-phosphate as substrate, the specific radioactivity of [14C]glucose 6-phosphate formed was determined at 1, 2, 5 and 30 min and 3, 8 and 17 h. It increased rapidly to 1.9-fold the initial specific radioactivity of [1-14C]ribose 5-phosphate at 3 h and then decreased to a value approximately equal to that of the substrate at 6 h, and finally at 17 h reached a value 0.8-fold that of the initial substrate [1-14C]ribose 5-phosphate. 3. The specific radioactivity of [14C]ribose 5-phosphate decreased to approx. 50% of its inital value during the first 3 h of the incubation and thereafter remained unchanged. 4. The distribution of 14C in the six carbon atoms of [14C]glucose 6-phosphate formed from [1-14C]ribose 5-phosphate at 1, 2, 5 and 30 min and 3, 8 and 17 h was determined. The early time intervals (1--30 min) were characterized by large amounts of 14C in C-2 and in C-6 and with C-1 and C-3 being unlabelled. In contrast, the later time intervals (3--17 h) were characterized by the appearance of 14C in C-1 and C-3 and decreasing amounts of 14C in C-2 and C-6. 5. It is concluded that neither the currently accepted reaction sequence for the non-oxidative pentose phosphate pathway nor the 'defined' pentose phosphate-cycle mechanism can be reconciled with the labelling patterns observed in glucose 6-phosphate formed during the inital 3 h of the incubation.     

Uracil salvage pathway in PC12 cells

The salvage anabolism of uracil to pyrimidine ribonucleosides and ribonucleotides was investigated in PC12 cells. Pyrimidine base phosphoribosyl transferase is absent in PC12 cells. As a consequence any uracil or cytosine salvage must be a 5-phosphoribosyl 1-pyrophosphate-independent process. When PC12 cell extracts were incubated with ribose 1-phosphate, ATP and uracil they can readily catalyze the synthesis of uracil nucleotides, through a salvage pathway in which the ribose moiety of ribose 1-phosphate is transferred to uracil via uridine phosphorylase (acting anabolically), with subsequent uridine phosphorylation. This pathway is similar to that previously described by us in rat liver and brain extracts (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273; Mascia et al., Biochim. Biophys. Acta 1472 (1999) 93). We show using intact PC12 cells that they can readily take up uracil from the external medium. The analysis of intracellular metabolites reveals that uracil taken up is salvaged into uracil nucleotides, with uridine as an intermediate. We propose that the ribose 1-phosphate-dependent uracil salvage shown by our in vitro studies, using tissues or cellular extracts, might also be operative in intact cells. Our results must be taken into consideration for the comprehension of novel chemotherapeutics' influence on pyrimidine neuronal metabolism.     

A proton magnetic resonance investigation of the glycosyl torsion angle of uracil nucleosides and nucleotides.

The use of line-shape decomposition techniques permitted the small 5-bond (5-J51') and 4-bond (4-J61') proton-proton coupling constants of a series of uracil nucleosides and nucleotides to be determined accurately. From an analysis of these coupling constants we have determined that the uracil base is in a predominantly anti conformation in aqueous solution and the mean position is not substantially altered by phosphate substitution at the 2', 3', or 5' positions, by changing the furanose stereochemistry from a ribose to a deoxyribose or an arabinose, or by an increase in temperature of 43 degree C.     

A new hypothesis for the origin of life

This hypothesis suggests that calcium chelating sugars, and especially ribose, have determined the nature of the first molecular systems. The self-organization capacities of these molecules enabled them to form regular arrays with certain salts. These arrays then evolved to form polysaccharides. In this first step, ribose and particularly -D-ribofuranose predominated over other prebiotic components. In a second step, the purines invaded these polysaccharides (3–5-polyribophosphodiester). The purines best suited for this were adenine and deoxyguanine, arising from the polymerization of HCN. Just as the polysaccharides reacted with purines, so the purines reacted with other small molecules and in particular, certain alkylating agents and water. After several methylation and oxidation reactions, adenine and deoxyguanine evolved to adenine, methylguanine, cytosine, uracil and thymine. Slow evolution of the prebiotic components gradually brought about a transition from a ribose world to an RNA world. The environment of this prebiotic RNA was different from that of modern RNA. For example, interaction of prebiotic RNA with water, calcium salts and certain zwitterionic molecules like the amino acids glycine and alanine was unavoidable. The interaction of these two small amino acids with calcium evolved to form transient anhydride bonds that quickly reverted to the initial state, or transformed to a peptide bond or to a more stable activated state, the oxazolone ring. The formation of this ring in double-stranded prebiotic RNA is the critical event that allowed the synthesis of new -L-amino acids. The positioning of the lateral sides of the amino acids inside the RNA suggests a stereochemical relationship that could explain the origin of the genetic code.     

Pentose phosphate pathway in rat colonic epithelium

The colonic cells of the large intestine are one of the most proliferative tissues of the animal body. The pentose pathway has an essential role in cell division and growth being the only pathway forming ribose 5-P necessary for all nucleotide and nucleic acid sunthesis. The pentose pathway may also provide reducing potential as NADPH for biosynthesis and C-3- C-8 glycolyl compounds. The maximum catalytic capacities of the reactions of the non-oxidative pentose pathway for the conversion of ribose 5-P to hexose and triose phosphates by the proximal and distal colon under feeding and starvation regimes are among the highest in the animal body. The qualitative presence of the oxidative pentose pathway was assessed by measurement of the C-1/C-6 ratio value of 1.67-1.82. Enzymes of the F-type and L-type pentose pathways are present in colonocytes and their maximum catalytic activities in colonocyte cytosol are reported. The contribution of the F-type pentose cycle to the total glucose metabolism of colonocytes, measured by the specific yield method, is negligibly low (approximately 1.5%). Colonic epithelial cells use glucose at a high rate (7.1 +/- 0.33 mumol min-1g-1 dry wt) and 79% of the glucose is converted to lactate. Arabinose 5-P has an intermediary role in the formation of keto pentose, sedoheptulose and hexose phosphates from ribose 5-P by colonocyte cytosol. The intermediary and reaction products of [1-13C] ribose 5-P dissimilation by colonocytes is investigated by 13C NMR spectroscopy. The 13C positional isotope distributions show labelling of C-1 and C-3 of hexose 6-phosphates consistent with either the theoretical predictions of the F-type pentose pathway or of the activities of exchange reactions catalysed by transketolase and/or transaldolase. Measurements of exchange reactions showed that the C-1/C-3 labelling of these compounds is mostly, if not wholly, attributable to exchange catalysis by these group transferring enzymes. The results suggest that the F-type PC has little role in the glucose metabolism of colonocytes and pentose phosphate formation may thus occur by a contribution (approx 20% of the total glucose metabolism) by the alternate L-type pathway.     

Syntheses of pyrimidine acyclic nucleoside phosphonates as potent inhibitors of thymidine phosphorylase (PD-ECGF) from SD-lymphoma

In the present study, we synthesized a series of pyrimidine acyclic nucleoside phosphonates bearing a number of substituents in C-5 position of uracil moiety and in the N-1-side chain. In addition, we have investigated in particular the novel syntheses of fluorinated derivatives substituted in the N-1-side chain and uracil C-5 position because fluorine-containing substituents are often powerful modifiers of chemical and biological properties. The obtained compounds exhibit a considerable inhibitory potency of thymidine phosphorylase from SD-lymphoma. In contrast, the synthesized phosphonates are not efficient inhibitors of E. coli and human thymidine phosphorylase.     

Prebiotic condensation reactions in an aqueous medium: A review of condensing agents

Biopolymers are formed by dehydration-type condensation reactions. In aqueous solutions dehydration reactions are very unlikely to happen spontaneously. However, coupling of dehydration-condensation to the hydrolysis of condensing agents could facilitate the synthesis of biopolymers in an aqueous solution. The literature shows that the peptides, nucleosides, nucleotides and oligonucleotides can be formed in this way. A careful study of the literature pertaining to prebiotic condensing agents was conducted in order to determine the most plausible prebiotic synthesis of biopolymers. The condensing agents taken into consideration are cyanamide, carbodiimide, dicyanamide, dicyandiamide, hydrogec-cyanide-tetramer, cyanogen and the linear- and cyclic polyphosphates. From both a chemical as well as biological point of view the polyphosphates appear to be the most plausible general prebiotic condensing agent.     

Nucleotide analogs based on pentaerythritol — An hypothesis

The synthesis of ribose and ribose-based nucleotides under reasonable prebiotic conditions has not been achieved. Glycerol has been suggested as a structural unit that might have preceded ribose in the evolutionary emergence of RNA. Template-directed oligomerizations of nucleotide analogs based on glycerol, however, have been only partially successful. Recent studies on the effect of ultraviolet irradiation of formaldehyde solutions have shown that the reduced sugar pentaerythritol is formed with great specificity. I argue that pentaerythritol is potentially capable of being converted by simple chemistry into a series of nucleoside analogs related to barbituric acid. These analogs may be able to take part in nucleic acid-like interactions and could therefore be of potential interest as a new class of candidates as RNA precursors.     

Incorporation of label from ribose into 5-(4'',5''-dihydroxypentyl) uracil of bacteriophage SP15 DNA.

Radioactively labeled ribose was incorporated into the glucosylated deoxynucleoside monophosphate of 5-(4',5'-dihydroxypentyl)uracil of bacteriophage SP15 DNA to a greater extent than into the other pyrimidine deoxynucleoside monophosphates. Results from formic acid hydrolysis of the deoxynucleoside monophosphates to their bases suggest that label from ribose is incorporated into the dihydroxypentyl side chain of 5-(4',5'-dihydroxypentyl)uracil.