The origin of life on Earth comprised a long series of steps: from the synthesis of small molecules within the primordial atmosphere or near hydrothermal vents, through the formation of biomonomers and biopolymers, culminating in the emergence of a self-replicating, autonomous organism. This philosophical outlook, if not the intimate details, began with the Russian biochemist Alexander Oparin and the British biologist J. B. S. Haldane, who, in the 1920s independently proposed a sequential model for the origin of life (1). Although the process of Darwinian selection may have modulated the populations of genetic macromolecules once the stage of an RNA (or "pre-RNA") world developed, the term "prebiotic evolution" is used here to describe the presumed earlier era of synthesis and degradation that preceded self-replication. A common theme is that the ingredients for life were generated by the flow of energy (sunlight, lightning, or thermal radiation) through the primordial hydrosphere so that the putative mechanisms for the origin of life should be compatible with the conditions that would have prevailed in the early atmosphere and oceans.
The time frame for the emergence of life must be constrained by the physical and biological history of the Earth, but firm dates are difficult to establish. The age of our planet is approximately 4.5 billion years, and life cannot be more ancient unless it came from an extraterrestrial environment. The latter possibility should not be dismissed and may gain credence if the existence of past (or current) life on other planets is confirmed (2). Regardless of the source, organisms would not have survived until the Earth cooled and meteoritic bombardment subsided sufficiently such that the oceans remained in a liquid state. The conditions necessary for sustainable life may not have persisted until about four billion years ago. However, evidence of extant organisms appears in fossilized stromatolites in Western Australia from 3.5 billion years ago, and possibly in apatite inclusions from rocks in Greenland dated at almost 3.9 billion years, suggesting that the appearance of life occurred quite rapidly on a geologic time scale once the conditions were favorable (3).
A historic demonstration of the feasibility of prebiotic simulations was performed by Stanley Miller (4) during the fall of 1952 in the laboratory of Harold Urey at the University of Chicago. Based on Urey’s cold-accretion theory for the origin of the planets, Miller subjected a gaseous mixture of methane, ammonia, hydrogen, and water to an electrical discharge, analogous to the effect of lightning in the atmosphere of the young Earth. Chromatographic analysis revealed the presence of three biological amino acids (glycine, alanine, and aspartic acid) along with other products. Further work by Miller and by many other research groups extended the suite of presumed prebiotic amino acids to include glutamic acid, leucine, isoleucine, serine, and threonine. All of the chiral amino acids were obtained as a racemic mixture of left- and right-handed forms, as expected from the achiral starting materials.
The apparent success of these early experiments, which depended on the accessibility and sensitivity of assays specific for amino acids, heightened interest in the nascent discipline of origins-of-life studies. However, prescient objections to the reducing atmosphere of the Miller-Urey simulations were raised first by Philip Abelson, a geochemist at the Carnegie Institution, who argued that the hydrogen-rich gases would have been rapidly replaced by a secondary atmosphere in which carbon was present as carbon dioxide or carbon monoxide, while nitrogen was probably present as molecular dinitrogen (5). By the early 1980s, a growing body of computational and experimental evidence slowly led to a revised view of the dominant atmosphere during the era before life began. Unfortunately, as shown by Miller and others, these non-reducing gas mixtures (CO2/N2/H2O or CO/N2/H2O) give dramatically lower yields and less variety in amino acids produced by electric discharge (6). If gas-phase syntheses of the Miller-Urey design were important in the origin of life, they must either have proceeded during a brief period when the Earth was very rich in hydrogen, or there may have been another unidentified source of reducing equivalents that maintained a source of hydrogen over a longer stretch of geologic time. Alternatively, the formation of amino acids and other organic molecules may have been favored near submarine hydrothermal vents, where reducing equivalents would have been present in the extruded gases; the technical challenges inherent in high pressures and temperatures have necessarily restricted the number of such simulations, but compounds as complex as pyruvic acid have been detected using formic acid as the carbon source (7). Nevertheless, the existence of both thermodynamic and kinetic barriers to the reduction of CO2 (the preferred starting point for any prebiotic synthesis) raises many questions about the availability of the organic precursors in hydrothermal models (8).
A very different source of the vital ingredients may exist beyond the Earth, in the form of comets, interplanetary dust, and asteroidal debris (9). These materials are believed to contain carbon compounds, some of which can survive passage through the atmosphere when such objects approach our planet. An intriguing possibility is that their cargo could already be enriched in the left-handed form of amino acids that are required to make modern proteins (10). While much remains to be understood about the chemical processing of these extraterrestrial bodies, there is great interest in their possible contribution to the primordial soup.
1. The Cyanide Paradigm
John Oro at the University of Houston in 1960 made a startling observation: adenine, a constituent of RNA as well as the nicotinamide and flavin cofactors, was formed after acid hydrolysis of ammonium cyanide solutions, which were deemed prebiotic starting materials based on the prior detection of HCN in the Miller-Urey electric discharge reactions (11). Although the conditions involved high concentrations of both cyanide and ammonia, James Ferris and his coworkers (12) at Rensselaer Polytechnic Institute obtained a yield of 0.03% after acid hydrolysis of a six-month reaction at room temperature of 0.1 M HCN, adjusted to pH 9.2 with ammonia and kept in the dark; adenine was also detected after hydrolysis at pH 8.5 (12). Ferris, working with Leslie Orgel (13) at the Salk Institute, demonstrated that adenine could be synthesized photochemically using near-UV light, via an intermediate known as 4-amino-5-cyanoimidazole (AICN, Fig. 1). This imidazole derivative can be prepared through a dark reaction by combining diaminomaleonitrile (DAMN) with formamidine (14). Urea, widely regarded as a prebiotic compound formed (among other ways) by the hydrolysis of HCN oligomers, reacts with AICN to give another important purine, guanine (15).
Figure 1. Synthesis of purines from HCN, showing two possible routes to the intermediate 4-amino-5-cyanoimidazole (AICN) from the HCN tetramer, DAMN.
The adenine that is released upon alkaline hydrolysis of the HCN oligomers is probably formed by a separate pathway (not shown in Fig. 1) that does not require light. The initial products in the oligomerization are now well-characterized, proceeding through a stepwise self-addition to give the tetramer, DAMN, which is a precursor to the imidazole derivatives (Fig. 1). The higher oligomers constitute a heterogenous mixture that is poorly defined, but their composition probably does not correspond to any known biopolymers (12).
HCN oligomerization also provides a source of the RNA component, uracil, which was first identified in such a mixture by Alan Schwartz and Andries Voet (16) at the University of Nijmegen. Ferris and Joshi (17) showed the presence of orotic acid after alkaline hydrolysis of HCN oligomers and further demonstrated that this pyrimidine undergoes a photochemical decarboxylation (Fig. 2) to yield uracil. Cytosine is more difficult to synthesize, but Miller and Robertson (18) have published an efficient pathway based on urea and cyanoacetaldehyde as starting materials; cyanoacetaldehyde is formed by hydrolysis of cyanoacetylene (a product of methane and dinitrogen atmospheres subjected to an electrical discharge, which thus requires a source of reduced carbon). Hydrolysis of cytosine yields uracil so that these syntheses provide an indirect route to this pyrimidine.
Figure 2. Possible steps in the prebiotic synthesis of pyrimidines.
Finally, HCN also provides a route to amino acids, notably glycine, alanine (alpha and beta isomers) and aspartic acid (12). Thus, an especially attractive feature of cyanide as a precursor to biomolecules is that it yields constituents of proteins and nucleic acids by a common reaction system. Moreover, Leslie Orgel and his colleagues (14) proposed an innovative mechanism for concentrating HCN by eutective freezing: at -23.4°C, a solution of 74.5 mole percent HCN (25 M) is obtained. Despite the low temperature, oligomerization is quite rapid under such conditions, which might have occurred in glacial climates.
An unresolved dilemma is how HCN itself might have been made on the early Earth (19). As Miller and others have shown, cyanide is readily formed when reducing gas mixtures of methane and ammonia are subjected to an electrical discharge, and this species was proposed as an important intermediate in the Miller-Urey syntheses of amino acids. By the same token, HCN is not formed from more oxidized atmospheres of CO2/N2/H2O or CO/^/H^O. One possibility proposed by Fujio
Egami (20) at the Mitsubishi-Kasei Institute of Life Sciences is that HCN resulted from the reaction of formaldehyde with hydroxylamine. Formaldehyde is a plausible photoproduct of oxidized atmospheres (CO2 and H2O), but the source of NH2OH remains to be elucidated (21). If the synthesis of HCN can ultimately be reconciled with a non-reducing environment on the primordial Earth, this achievement would greatly strengthen the paradigm that cyanide played an important role in the synthesis of amino acids, purines, and pyrimidines.
2. Activation Processes
Modern biochemistry exploits the unique properties of phosphate as an activating group to drive unfavorable reactions, and the earliest biosynthetic processes may have used phosphate in an analogous fashion. A striking illustration of the activating power of this group is found in glycolaldehyde phosphate, which Albert Eschenmoser (22) of the Federal Technical Institute (FTH) in Zurich has proposed as a precursor of the RNA sugar, ribose. Under strongly alkaline conditions, glycolaldehyde phosphate reacts with formaldehyde to give ribose 2,4-diphosphate as the major product, with glyceraldehyde 2-phosphate serving the role of intermediate; by contrast, formaldehyde alone gives a complex mixture with only a trace of ribose. More recently, Gustaf Arrhenius and his colleagues (23) at the Scripps Institution of Oceanography have shown that certain mineral catalysts (known as layered double hydroxides) can effect the formation of ribose 2,4-diphosphate from glyeraldehyde 2-phosphate and glycolaldehyde phosphate at near-neutral pH. While the subsequent steps from this derivative to nucleotides have yet to be elucidated, the work of Eschenmoser represents a significant advance in stereoselective synthesis.
A formidable challenge in prebiotic research has been the demonstration of possible condensation mechanisms by which the elements of water (H2O) could be removed in order to couple two molecules. One approach has involved the heating of the ingredients in the dry state to drive off the water. This method yields nucleosides in 2% to 10% yield when purines (adenine or guanine) are heated in the presence of ribose, but the reaction fails when the pyrimidines cytosine or uracil are substituted (24). Repeated wet-dry cycles, which simulate the periodic flooding and evaporation of a tidal lagoon, effect the self-condensation of amino acids to form modest amounts of oligopeptides up to the pentamer in a process that is catalyzed by a common clay mineral called kaolinite (25). While such a reaction system is relatively inefficient, with total yields near 1% after 2 months, it represents a more natural model than studies carried out with transient condensing agents as the activating species (26).
Phosphorylation of nucleosides and other substrates represents another step that has met with limited success. Organic condensing agents such as cyanamide and cyanoacetylene are effective but not under the low micromolar concentrations of phosphate that likely prevailed in the primordial ocean (26). Inorganic polyphosphates including the trimer, which has been detected in volcanic fumaroles, can convert adenosine into a mixture of mono-, di-, and triphosphates, but the high temperatures associated with volcanic environments render them unsuitable for complex organic synthesis (27). A structurally related compound, cyclotriphosphate or "trimetaphosphate," has been employed in the preparation of glycolaldehyde phosphate and other phosphate esters (28); however, trimetaphosphate concentrations would be limited by the geothermal formation of longer polymers from which it is derived. Mixtures of urea and inorganic phosphate give high yields of phosphorylated products under mild heating, but this approach requires high concentrations of urea that may not have been available on the early Earth (29). Further work must be done to elucidate plausible prebiotic pathways to phosphate esters.
In summary, a significant body of literature (see Suggestions for Further Reading) has emerged that demonstrates the feasibility of prebiotic syntheses of specific compounds under particular conditions. The efficacy of cyanide as a precursor to purines, amino acids, and, to a lesser extent, pyrimidines suggests that HCN likely had a role in the formation of the first biomolecules. However, much work remains to reconcile such pathways with geochemical conditions that might have prevailed on the early Earth and to elucidate how a genetic macromolecule might have formed from a dilute primordial soup. Despite the many problems in nucleotide assembly, the postulate of a so-called RNA world has provided remarkable insights into the interrelated roles of replication and catalysis in the origins of life.
