Prebiotic synthesis: phosphorylation in aqueous solution.

Formation of uridine-5'-phosphate from uridine and inorganic phosphate in aqueous solution is effected by the following condensing agents: cyanogen, cyanoformamide, cyanate, cyanamide, thioformate, ethylisocyanide, and a watersoluble carbodiimide. The yields are always small, even when a large excess of condensing agent is used. The cyclization of uridine-3'-(2')-phosphate occurs under the same conditions but in much greater yield.

A nonenzymatic RNA polymerase model.

Polynucleotide templates containing C (cytidine) as the major component facilitate the synthesis of oligonucleotides from mixtures of the activated mononucleotide derivatives (as indicated by structure 1 in the text). A nucleotide is incorporated into oligomeric products if and only if its complement is present in the template. The reaction has a high fidelity and produces products with mean chain lengths of six to ten nucleotides. Bases other than guanosine are incorporated within oligomers or at their 3' termini, but rarely at their 5' termini.

Urea-inorganic phosphate mixtures as prebiotic phosphorylating agents.

Previous attempts to phosphorylate nucleosides by heating with inorganic phosphate succeeeded only when acid phosphates such as Ca(HPO(4))(2) were used. The addition of urea and ammonium chloride to the reaction mixture permits phosphorylation in high yield with neutral or basic phosphates at temperatures in the range of 65 degrees to 100 degrees C. Since the abundant mineral, hydroxylapatite, is a satisfactory substrate for this reaction, we believe that this procedure plausible model for prebiotic phosphorylation.

Efficient metal-ion catalyzed template-directed oligonucleotide synthesis.

The Pb2+ and Zn2+ ions are efficient catalysts for the polycytidylic acid-directed polymerization of an activated guanylic acid derivative, guanosine 5'-phosphorimidazolide. The products include oligomers of 30 to 40 units in length. The nucleotide residues are predominantly 2'-5' linked when Pb2+ is the catalyst, and predominantly 3'-5' linked in the presence of Zn2+. The significance of these results in the context of the prebiotic evolution of RNA polymerase is discussed.

Conditions for purine synthesis: did prebiotic synthesis occur at low temperatures?

The rate of polymerization of hydrogen cyanide to aminomalononitrile and the tetramer, diaminomalonodinitrile, is quadratic in the total cyanide concentration. Since the reactions form part of a plausible prebiotic purine synthesis and since they compete with hydrolysis, concentration of cyanide may have been important. This may be achieved usefully by cooling to separate out ice.

Phosphorylation with inorganic phosphates at moderate temperatures.

Uridine phosphates may be obtained by heating uridine with inorganic phosphates for 9 months at temperatures as low as 65 degrees C. Under similar conditions, uridine-5'-phosphate, in addition to forming uridine diphosphates, undergoes some dephosphorylation to give uridine.

Struvite and prebiotic phosphorylation.

Struvite, MgNH(4)PO(4).6H(2)O, rather than apatite or amorphous calcium phosphate is precipitated when phosphate is added to seawater containing more than 0.01M NH(4)(+) ions. Struvite may have precipitated from evaporating seawater on the primitive earth, and may have been important for prebiotic phosphorylation.

Template-directed synthesis of novel, nucleic acid-like structures.

In studying the origins of life, it is important to examine reactions of substrate mixtures that could plausibly have accumulated on the primitive earth. Nucleoside diphosphates would probably have been synthesized along with the standard nucleotides under prebiotic conditions. For these reasons, the template-directed reactions of activated derivatives of these diphosphates, alone or mixed with activated nucleotides, were investigated. An activated derivative of deoxyguanosine 3',5'-diphosphate condensed efficiently on a polycytidylate template to give oligonucleotide analogues in which each 3',5'-phosphodiester bond was replaced by a pyrophosphate linkage. Oligomers were formed even in the absence of a template, but much more slowly. Template-directed condensation occurred also with an analogous deoxyadenosine derivative on polyuridylic acid and with an analogous acycloguanosine derivative on polycytidylic acid.

Cyanoacetylene in prebiotic synthesis.

Cyanoacetylene is a major nitrogen-containing product of the action of an electric discharge on a mixture of methane and nitrogen. It reacts with simple inorganic substances in aqueous solution to give products including asparticacid, asparagine, and cytosine.

Partial resolution of DL-adenosine by template synthesis.

D-Adenosine-5'-phosphorimidazolide reacts very much more rapidly with D-adenosine than with L-adenosine on a poly-D-uridylic acid template. This permits the partial resolution of DL-adenosine. These experiments suggest that segregation of D- and L-nucleotides may have occurred at an early stage in biochemical evolution.

Template-directed synthesis with adenosine-5'-phosphorimidazolide.

Adenosine-5'-monophosphorimidazolide reacts efficiently with adenosine derivatives on a polyuridylic acid template, with the formation of internucleotide bonds.

Prebiotic synthesis of propiolaldehyde and nicotinamide.

We have identified propiololdehyde as a product of the action of an electric discharge on mixtures of methane and water or methane, nitrogen, and water. The aldehyde reacts with cyanoacetaldehyde and ammonia (other "prebiological molecules") to yield nicotinonitrile. This substance can be hydrolyzed to nicotinamide and nicotinic acid.

Search for organic and volatile inorganic compounds in two surface samples from the chryse planitia region of Mars.

Two surface samples collected from the Chryse Planitia region of Mars were heated to temperatures up to 500 degrees C, and the volatiles that they evolved were analyzed with a gas chromatograph-mass spectrometer. Only water and carbon dioxide were detected. This implies that organic compounds have not accumulated to the extent that individual components could be detected at levels of a few parts in 10(9) by weight in our samples. Proposed mechanisms for the accumulation and destruction of organic compounds are discussed in the light of this limit.

The origin of life--a review of facts and speculations.

Three popular hypotheses attempt to explain the origin of prebiotic molecules: synthesis in a reducing atmosphere, input in meteorites and synthesis on metal sulfides in deep-sea vents. It is not possible to decide which is correct. It is also unclear whether the RNA world was the first biological world or whether some simpler world preceded it.

Preparation of ligation intermediates and related polynucleotide pyrophosphates.

Unprotected oligonucleotides and oligodeoxynucleotides terminated with an unhindered 5'-phosphate group react with nucleoside 5'- phosphorimidazolides in aqueous solution to give 'capped' pyrophosphates in at least 70% yield. If adenosine 5'- phosphorimidazolide is used as a substrate in the reaction, ligase intermediates are obtained as products.

Reactions of polyfunctional amino acids with N,N'-carbonyldiimidazole in aqueous solution--oligopeptide formation.

1. Serine reacts with N,N'-carbonyldiimidazole in aqueous solution at 0 degrees C to yield Nalpha-[imidazolyl-(1)-carbonyl]-L-serine. This compound slowly transforms to L-2-oxo-oxazolidine-5-carboxylic acid. We found that L-2-oxo-oxazolidine-5-carboxylic acid was stable to hydrolysis under a variety of conditions and did not oligomerize to yield peptides. Threonine was found to react in an analogous manner with N,N'-carbonyldiimidazole, yielding Nalpha-[imidazolyl-(1)-carbonyl]-L-threonine and L-(+)-trans-5-methyl-2-oxo-oxazolidine-5-carboxylic acid. 2. Histidine reacts with N,N'-carbonyldimidazole in aqueous solution at 0 degrees C to yield a variety of histidine-containing intermidiates. These slowly transform to give 7-carboxy-imidazole-[1,5cotetrahydropyrimidin-5-one in up to 90% yield. 3. The polymerization of 7-carboxy-imidazo-[1,5c]-tetrahydropyrimidin-5-one in imidazole buffer at 30 degrees gives excellent yields of oligohistidines.

Non-enzymatic transcription of an oligodeoxynucleotide 14 residues long.

The 14-mer oligodeoxynucleotide d(C3GC3GC3GC2) acts as a template to facilitate the cooligomerization of guanosine 5'-phospho-2-methylimidazolide and cytidine 5'-phospho-2-methylimidazolide. The predominant products are a series of 3'-5'-linked oligonucleotides, complementary to the template, ranging in length from GGC to GGCGGGCGGGCGGG. Thus simple, non-enzymatic template-directed reactions can result in the accurate transfer of substantial amounts of information from template to products. The 15-mer oligodeoxynucleotide d(C3GC3GC3GC3) is also an efficient template, but directs the synthesis of the same family of products that are formed on the 14-mer template. This unexpected finding is explained by the preferential conversion of the dimer GG to GGC rather than to GGG. These results are interesting in the context of molecular evolution. They suggest that the detailed kinetics of template-directed synthesis could form the basis for the selection of one replicating oligonucleotide from a family of closely related oligonucleotides.

Template-directed synthesis on the oligonucleotide d(C7-G-C7).

When the deoxynucleotide template d(C7-G-C7) is incubated with the activated nucleotides 2-MeImpG and 2-MeImpC, a series of oligomers of G up to the sevenmer and a series of copolymers of composition GnC with n = 3 to 13 are formed. Oligomers GnC with n greater than 7 are completely degraded by pancreatic ribonuclease, establishing that they contain a 3' to 5' internucleotide bond between 5'-C and 3'-G within a sequence of the form (pG)ipC(pG)j. As expected, (pG)7-Cp and (pG)6-Cp are major hydrolysis products. Detailed analysis of the product distribution shows that a substantial fraction of the oligomeric products are of the type (pG)ipC(pG)j with i less than 7. This shows that product synthesis does not necessarily begin at the 3' terminus of the template. The significance of this finding in terms of the origin of molecular replication is discussed.

Non-enzymatic template-directed synthesis on RNA random copolymers. Poly(C,A) templates.

Poly(C,A) random copolymer templates direct the oligomerization of 2-MeImpG (2-MeImpX is the 5'-phospho-2-methylimidazolide of the nucleoside X) and 2-MeImpU, resulting in the production of a variety of oligo (G,U)s. This reaction is less efficient than comparable reactions involving poly(C,U) or poly(C,G) templates. The efficiency of monomer incorporation into newly synthesized oligomers is lower for 2-MeImpU than 2-MeImpG, and cannot be improved by increasing the concentration of 2-MeImpU relative to 2-MeImpG. This suggests that RNA templates containing runs of consecutive adenine residues would not be suitable for use in a chemical self-replicating system. The distribution of oligomeric products can be characterized in detail using high-pressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition, and phosphodiester-linkage isomerism. Oligomers up to about the 13-mer, with base composition Gn, Gn-1, U, and Gn-2, U2, have been identified.

Non-enzymic template-directed synthesis on RNA random copolymers. Poly(C, G) templates.

Poly(C, G) random copolymer templates direct the oligomerization of 2-Me-ImpG and 2-MeImpC, resulting in the production of a variety of oligo(G, C)s. The efficiency of monomer incorporation into newly synthesized oligomers is greater for 2-MeImpG than for 2-MeImpC, and decreases for both monomers as the guanine content of the template increases. The relatively low efficiency of oligomerization on guanine-rich templates is largely a consequence of intra- and intermolecular template self-structure. The problem of template self-structure is clearly a major obstacle to the development of a system of self-replicating polynucleotides. The distribution of oligomeric products can be characterized in detail using high-pressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition and phosphodiester-linkage isomerism. Oligomers up to about the 12-mer, with base composition Gn, Gn-1C and Gn-2C2, have been identified. The 3' to 5' regiospecificity of the products is high, particularly for oligomers with base composition Gn.