Downloaded from http://cshperspectives.cshlp.org/ on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Ribonucleotides John D. Sutherland School of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL, United Kingdom Corespondence: [email protected] It has normally been assumed that ribonucleotides arose on the early Earth through a process in which ribose, the nucleobases, and phosphate became conjoined. However, under plaus- ible prebiotic conditions, condensation of nucleobases with ribose to give b-ribonucleosides is fraught with difficulties. The reaction with purine nucleobases is low-yielding and the reaction with the canonical pyrimidine nucleobases does not work at all. The reasons for these difficulties are considered and an alternative high-yielding synthesis of pyrimidine nucleotides is discussed. Fitting the new synthesis to a plausible geochemical scenario is a remaining challenge but the prospects appear good. Discovery of an improved method of purine synthesis, and an efficient means of stringing activated nucleotides together, will provide underpinning support to those theories that posit a central role for RNA in the origins of life. hether RNA first functioned in isolation, derivation of RNA. Notwithstanding these Wor in the presence of other macromole- caveats, however, the self-assembly of polymeric cules and small molecules is still an open ques- RNA on the early Earth most likely involved tion, and a question that is addressable through activated monomers (Verlander et al. 1973; chemistry (Borsenberger et al. 2004). If syner- Ferris et al. 1996). These activated monomers gies are found between RNA assembly chemis- could either have come together sequentially try and that associated with the assembly of to make RNA one monomer at a time, or short lipids and/or peptides, the purist RNA world oligoribonucleotides formed by such a process concept (Woese 1967; Crick 1968; Orgel 1968) could have joined together by ligation in what might have to be loosened to allow other such would thus amount to a two-stage assembly molecules a role in the origin of life. Metabo- of RNA polymers. Replication of RNA would lism, or the roots of metabolism, could also then have involved template-directed versions potentially have coevolved with RNA if organic of these or related chemistries (Orgel 2004). chemistry happened to work in a particular The details of the polymerization processes way on a set of plausible prebiotic feedstock that might plausibly have given rise to the first molecules in a dynamic geochemical setting. RNA molecules can only be investigated when Such considerations point to the need for an there is some evidence as to the specific chemi- open mind when considering the chemical cal nature of the activated monomers. Broadly Editors: David Deamer and Jack W. Szostak Additional Perspectives on The Origins of Life available at www.cshperspectives.org Copyright # 2010 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a005439 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a005439 1 Downloaded from http://cshperspectives.cshlp.org/ on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press J.D. Sutherland speaking, however, it is possible to differentiate cyanoacetaldehyde 9), and the purines to hydro- different polymerization chemistries on the gen cyanide and a C(IV) oxidation level mole- basis of the bonds formed in the polymerization cule such as 6 or 8. This retrosynthetic analysis step. P–O bond forming polymerization chem- ultimately breaks ribonucleotides down into istry is reasonable to consider first because of the molecules that are sufficiently simple so that simplicity of P–O retrosynthetic disconnections they can be deemed prebiotically plausible feed- of RNA (Corey 1988). In the case of the simplest stock molecules (Fig. 2) (Sanchez et al. 1966; P–O bond forming polymerization chemistry, Pasek and Lauretta 2005; Bryant and Kee 2006; the monomeric products of preceding prebiotic Thaddeus 2006). chemistry would either be activated ribonucleo- It is not just because the simplest retrosyn- side-50-phosphates 1—activated through having thetic disconnections of ribonucleotides proceed a leaving group attached to the phosphate— by way of ribose, nucleobases, and phosphate or ribonucleoside-20,30-cyclic phosphates 2, that people have tried to synthesize them via wherein the activation is intrinsic to the cyclic these three building blocks under prebiotically phosphate (Fig. 1). If all potential routes from plausible conditions for the last 40–50 years— prebiotic feedstock molecules to such mono- in terms of their appearance to the human mers were to be investigated experimentally eye, ribonucleotides undoubtedly consist of without success, then the potential for prebiotic these three building blocks. Experimentally, self-assembly of monomers associated with there have been several notably successful reac- more complicated polymerization chemistries, tions that ostensibly support this nucleobase would additionally have to be investigated (this ribosylation approach: Orgel’s and Miller’s syn- would include alternative P–O bond forming theses of cytosine 10 (Ferris et al. 1968; Robert- polymerization chemistry, as well as C–O and son and Miller 1995); Benner’s and Darbre’s C–C bond forming chemistries). However, con- syntheses of ribose 3 by aldolization of glycolal- tinuing with the simplest P–O bond forming dehyde 4 and glyceraldehyde 5 (Ricardo et al. polymerization chemistry, and the assumption 2004; Kofoed et al. 2005); Pasek’s and Kee’s that it seems reasonable to follow the simplest demonstration of phosphate synthesis by dis- retrosynthetic disconnections first, 1 and 2 can proportionation of meteoritic metal phos- then be conceptually reduced to ribose 3,a phides (Pasek and Lauretta 2005; Bryant and nucleobase and phosphate (Joyce 2002; Joyce Kee 2006); and Orgel’s urea-catalyzed phos- and Orgel 2006). Ribose 3 can then be discon- phorylation of nucleosides (eg., 11 (BvC)!2 nected to glycolaldehyde 4 and glyceraldehyde (BvC)) (Lohrmann and Orgel 1971). Indeed, 5 through aldol chemistry, and the nucleobases for many years, a prebiotically plausible synthe- disconnected to simpler carbon and nitrogen sis of ribonucleotides from ribose 3, the nucle- containing molecules—the pyrimidinesto cyan- obases, and phosphate has been tantalizingly amide 6 and cyanoacetylene 7 (conventionally close but for one step of the assumed synthe- through the hydration products urea 8 and sis—the joining of ribose to the nucleobases. O P O– H X –O O B O P O O B O B O O 1 OOH 2 O O O P H OOH P –O O B – O O O RNA (X = a leaving group) OOH Figure 1. Activated ribonucleotides in the potentially prebiotic assembly of RNA. Potential P–O bond forming polymerization chemistry is indicated by the curved arrows. 2 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a005439 Downloaded from http://cshperspectives.cshlp.org/ on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Ribonucleotides This reaction works extremely poorly for the (Fig. 3). Ribose 3 exists as an equilibrating mix- purines and not at all in the case of the pyrimi- ture of different forms in aqueous solution dines (Fuller et al. 1972a, 1972b; Orgel 2004). (Fig. 3A) (Drew et al. 1998). The mixture is So, why does the ribosylation chemistry not dominated by b- and a-pyranose isomers work with free nucleobases and ribose 3 when, (3 [b–p] and 3 [a–p]) with lesser amounts using the protecting and controlling groups of of b- and a-furanose isomers (3 [b–f ] and 3 conventional synthetic chemistry, nucleobase [a–f ]). The various hemiacetal ring forms ribosylation is possible? The reasons are pre- equilibrate via the open chain aldehyde form 3 dominantly kinetic and can be appreciated (a), which is a very minor component along by consideration of the structure and reactiv- with an open chain hydrate. The purine nucle- ity of ribose 3 and representative nucleobases obase adenine 12 also exists in various equili- brating forms in aqueous solution (Fig. 3B) (Fonseca Guerra et al. 2006). In this case, the HO O O isomers differ in the position of protonation, the major tautomer, 12 has N9 protonated, 4 OH OH HO OH HO O but other tautomers such as 13—in which N1 3 (α-f) H N CN is protonated—exist at extremely low concen- 2 5 OH 6 tration. To connect adenine 12 to ribose 3 to v + H2O give a natural RNA ribonucleoside 11 (B A) it is necessary for N9 of adenine to function as H2N NH2 a nucleophile and C1 of 3 (a–f ) to function O NH2 8 as an electrophile. The latter is possible under N –H O –H O 2 acidic conditions when a small amount of 2 N O H 14—a selectively protonated form of 3 (a– O X CN 10 f )—is present at equilibrium. The protonation 9 NH2 converts the anomeric hydroxyl group into a +H2O N better leaving group and enhances the electro- CN HO N philicity of C1. The major tautomer of adenine, O O 7 12, is not nucleophilic at N9 because the lone HO OH pair on that atom is delocalized throughout 11 (B=C) the bicyclic ring structure. N9 of several minor HO OH tautomers such as 13 is nucleophilic because P –2H2O O O– NH the nitrogen lone pair is localized, and so reac- 2 tion with 14 is possible, though slow because N of the low concentrations of the productively HO N O O reactive species. To compound this sluggish- ness, the reaction is plagued with additional O O P problems. First, the acid needed to activate 3 O O– (a–f ) also substantially protonates adenine, 2 (B=C) giving the cation 15, which is not nucleophilic Figure 2. One of the synthetic routes to b-ribocyti- on N9 (Christensen et al.