Ribonucleotides (1)

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March 10, 20102010; doi: 10.1101/cshperspect.a005439 originally published onlineCold Spring Harb Perspect Biol

John D. SutherlandRibonucleotides

Subject Collection The Origins of Life

Constructing Partial Models of Cells

et al.Norikazu Ichihashi, Tomoaki Matsuura, Hiroshi Kita,

The Hadean-Archaean EnvironmentNorman H. Sleep

Ribonucleotides

John D. Sutherland

An Origin of Life on Mars

Christopher P. McKay

Characterize Early Life on EarthHow a Tree Can HelpDeep Phylogeny

RandallEric A. Gaucher, James T. Kratzer and Ryan N.

Primitive Genetic PolymersAaron E. Engelhart and Nicholas V. Hud

Medium to the Early EarthCosmic Carbon Chemistry: From the Interstellar

Pascale Ehrenfreund and Jan Cami

Membrane Transport in Primitive CellsSheref S. Mansy

Origin and Evolution of the RibosomeGeorge E. Fox

The Origins of Cellular LifeJason P. Schrum, Ting F. Zhu and Jack W. Szostak

BiomoleculesPlanetary Organic Chemistry and the Origins of

Kim, et al.Steven A. Benner, Hyo-Joong Kim, Myung-Jung

From Self-Assembled Vesicles to ProtocellsIrene A. Chen and Peter Walde

the Origins of LifeMineral Surfaces, Geochemical Complexities, and

Robert M. Hazen and Dimitri A. Sverjensky

The Origin of Biological HomochiralityDonna G. Blackmond

Historical Development of Origins ResearchAntonio Lazcano

Earth's Earliest AtmospheresKevin Zahnle, Laura Schaefer and Bruce Fegley

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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 theearly Earth through a processin which ribose, the nucleobases, and phosphate became conjoined.However, under plaus-ible prebioticconditions, condensation of nucleobases with ribose to giveb-ribonucleosidesis fraught with difficulties. The reaction with purine nucleobases is low-yielding and thereaction with the canonical pyrimidine nucleobases does not work at all. The reasons forthese difficulties are considered and an alternative high-yielding synthesis of pyrimidinenucleotides is discussed. Fitting the new synthesis to a plausible geochemical scenario isa remaining challenge but the prospects appear good. Discovery of an improved methodof purine synthesis, and an efficient means of stringing activated nucleotides together, willprovide underpinning support to those theories that posit a central role for RNA in theorigins of life.

Whether RNA first functioned in isolation,

or in the presence of other macromole-cules and small molecules is still an open ques-tion, and a question that is addressable through

chemistry (Borsenberger et al. 2004). If syner-

gies are found between RNA assembly chemis-try and that associated with the assembly oflipids and/or peptides, the purist RNA worldconcept (Woese 1967; Crick 1968; Orgel 1968)

might have to be loosened to allow other such

molecules a role in the origin of life. Metabo-lism, or the roots of metabolism, could alsopotentially have coevolved with RNA if organic

chemistry happened to work in a particularway on a set of plausible prebiotic feedstock

molecules in a dynamic geochemical setting.Such considerations point to the need for anopen mind when considering the chemical

derivation of RNA. Notwithstanding these

caveats, however, the self-assembly of polymericRNA on the early Earth most likely involvedactivated monomers (Verlander et al. 1973;

Ferris et al. 1996). These activated monomers

could either have come together sequentiallyto make RNA one monomer at a time, or shortoligoribonucleotides formed by such a processcould have joined together by ligation in what

would thus amount to a two-stage assembly

of RNA polymers. Replication of RNA wouldthen have involved template-directed versionsof these or related chemistries (Orgel 2004).

The details of the polymerization processesthat might plausibly have given rise to the first

RNA molecules can only be investigated whenthere is some evidence as to the specific chemi-cal nature of the activated monomers. Broadly

Editors: David Deamer and Jack W. SzostakAdditional Perspectives on The Origins of Life available at www.cshperspectives.org

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speaking, however, it is possible to differentiatedifferent polymerization chemistries on the

basis of the bonds formed in the polymerizationstep. PO bond forming polymerization chem-

istry is reasonable to consider first because of thesimplicity of P O retrosynthetic disconnectionsof RNA (Corey 1988). In the case of the simplest

PO bond forming polymerization chemistry,

the monomeric products of preceding prebioticchemistry would either be activated ribonucleo-side-50-phosphates1activated through having

a leaving group attached to the phosphateor ribonucleoside-20,30-cyclic phosphates 2,

wherein the activation is intrinsic to the cyclicphosphate (Fig. 1). If all potential routes fromprebiotic feedstock molecules to such mono-

mers were to be investigated experimentally

without success, then the potential for prebioticself-assembly of monomers associated withmore complicated polymerization chemistries,

would additionally have to be investigated (thiswould include alternative PO bond forming

polymerization chemistry, as well as CO andC C bond forming chemistries). However, con-tinuing with the simplest PO bond forming

polymerization chemistry, and the assumptionthat it seems reasonable to follow the simplest

retrosynthetic disconnections first,1and 2 canthen be conceptually reduced to ribose 3, a

nucleobase and phosphate (Joyce 2002; Joyce

and Orgel 2006). Ribose 3can then be discon-nected to glycolaldehyde 4 and glyceraldehyde

5through aldol chemistry, and the nucleobasesdisconnected to simpler carbon and nitrogen

containing moleculesthepyrimidinesto cyan-amide 6 and cyanoacetylene 7 (conventionallythrough the hydration products urea 8 and

cyanoacetaldehyde 9), andthe purines to hydro-gen cyanide and a C(IV) oxidation level mole-

cule such as 6 or 8. This retrosynthetic analysisultimately breaks ribonucleotides down into

molecules that are sufficiently simple so thatthey can be deemed prebiotically plausible feed-stock molecules (Fig. 2) (Sanchez et al. 1966;

Pasek and Lauretta 2005; Bryant and Kee 2006;

Thaddeus 2006).It is not just because the simplest retrosyn-

thetic disconnections of ribonucleotides proceed

by way of ribose, nucleobases, and phosphatethat people have tried to synthesize them via

these three building blocks under prebioticallyplausible conditions for the last 40 50 yearsin terms of their appearance to the human

eye, ribonucleotides undoubtedly consist of

these three building blocks. Experimentally,there have been several notably successful reac-tions that ostensibly support this nucleobase

ribosylation approach: Orgels and Millers syn-theses of cytosine 10(Ferris et al. 1968; Robert-

son and Miller 1995); Benners and Darbressyntheses of ribose 3by aldolization of glycolal-dehyde 4 and glyceraldehyde 5 (Ricardo et al.

2004; Kofoed et al. 2005); Paseks and Keesdemonstration of phosphate synthesis by dis-

proportionation of meteoritic metal phos-phides (Pasek and Lauretta 2005; Bryant and

Kee 2006); and Orgels urea-catalyzed phos-

phorylation of nucleosides (eg., 11 (BvC)!2(BvC)) (Lohrmann and Orgel 1971). Indeed,

for many years, a prebiotically plausible synthe-sis of ribonucleotides from ribose 3, the nucle-

obases, and phosphate has been tantalizinglyclose but for one step of the assumed synthe-sisthe joining of ribose to the nucleobases.

O

O P O

OO

BO

O

O OH

BO

PO

OX

RNA(X = a leaving group)

2

O

O OH

BO

O

O OH

BO

POO

POO

H

H

1

Figure 1.Activated ribonucleotides in the potentially prebiotic assembly of RNA. Potential P O bond formingpolymerization chemistry is indicated by the curved arrows.

J.D. Sutherland

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This reaction works extremely poorly for thepurines and not at all in the case of the pyrimi-

dines (Fuller et al. 1972a, 1972b; Orgel 2004).So, why does the ribosylation chemistry not

work with free nucleobases and ribose3

when,using the protecting and controlling groups ofconventional synthetic chemistry, nucleobase

ribosylation is possible? The reasons are pre-

dominantly kinetic and can be appreciatedby consideration of the structure and reactiv-ity of ribose 3 and representative nucleobases

(Fig. 3). Ribose 3exists as an equilibrating mix-ture of different forms in aqueous solution

(Fig. 3A) (Drew et al. 1998). The mixture isdominated by b- and a-pyranose isomers

(3

[bp] and 3

[ap]) with lesser amountsofb- and a-furanose isomers (3 [bf ] and 3[af ]). The various hemiacetal ring forms

equilibratevia the open chain aldehyde form 3(a), which is a very minor component alongwith an open chain hydrate. The purine nucle-obase adenine 12 also exists in various equili-

brating forms in aqueous solution (Fig. 3B)(Fonseca Guerra et al. 2006). In this case, the

isomers differ in the position of protonation,the major tautomer, 12 has N9 protonated,but other tautomers such as 13in which N1

is protonatedexist at extremely low concen-

tration. To connect adenine 12 to ribose 3 togive a natural RNA ribonucleoside 11 (BvA)it is necessary for N9 of adenine to function as

a nucleophile and C1 of 3 (af ) to functionas an electrophile. The latter is possible under

acidic conditions when a small amount of

14a selectively protonated form of 3 (af )is present at equilibrium. The protonation

converts the anomeric hydroxyl group into abetter leaving group and enhances the electro-

philicity of C1. The major tautomer of adenine,

12, is not nucleophilic at N9 because the lone

pair on that atom is delocalized throughout

the bicyclic ring structure. N9 of several minortautomers such as 13 is nucleophilic because

the nitrogen lone pair is localized, and so reac-tion with 14 is possible, though slow because

of the low concentrations of the productivelyreactive species. To compound this sluggish-ness, the reaction is plagued with additional

problems. First, the acid needed to activate 3(af ) also substantially protonates adenine,giving the cation 15, which is not nucleophilicon N9 (Christensen et al. 1970;Zimmer and Bil-

tonen 1972; Majoret al.2002). Second,the mostnucleophilic nitrogenthe 6-amino groupof

the major tautomer of adenine 12 reacts with 3(a)the most reactive form of 3 despite itsscarcityresulting in N6-ribosyl adducts as by

far the major products (Fuller et al. 1972a,1972b). Third, the other isomeric forms of

ribose, 3 (bp), 3 (ap), and 3 (bf ), can

2 (B=C)

11 (B=C)

3 (-f) HO

OH

O

5

OH

O

4

HOP

OH

O O

NH

N

O

NH2

10

NH2

NH2

NH2

O

8

OCN

9

H2N

H2N

CN

6

CN

7

+ H2O

+H2O

H2OH2O

2H2O

O

HO

HO

OH

N

N

O

OHO N

N

O

OP

O

O O

O

HO OH

HO

OH

X

Figure 2. One of the synthetic routes to b-ribocyti-dine-20,30-cyclic phosphate 2 (BvC) implied bythe assumption that nucleosides can self-assemble

by nucleobase ribosylation. The general syntheticapproach has been supported by the experimentaldemonstration of most of its steps. However, pre-biotically plausible conditions under which the keynucleobase ribosylation step works have not beenfound despite numerous attempts over severaldecades.

Ribonucleotides

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by the resonance canonical structure 17). Ex-perimentally, cytosine 10 cannot be ribosylated

on N1 even using the conditions established forunselective ribosylation of adenine 12 (Fuller

et al. 1972a). If there is any tautomeric formsuch as 18with a localized N1 lone pair presentat equilibrium, it must be in such a low concen-

tration as to be effectively unreactive. The same

considerations hold true for uracil.In conventional synthetic chemistry, the

aforementioned difficulties in nucleobase ribo-

sylation can be overcome with directing, block-ing, and activating groups on the nucleobase

and ribose (Ueda and Nishino 1968). Thus, thecytosine derivative 19 is directed to function asa nucleophile at N1 by alkylation of O2. The

ribose-derived intermediate 20 is constrained

to the furanose form by benzoylation of theC5-hydroxyl group, and neighboring groupparticipation from an O2-benzoyl group directs

b-glycosylation. These molecular interventionsare synthetically ingenious, but serve to empha-

size the enormous difficulties that must be over-come if ribonucleosides are to be efficientlyproduced by nucleobase ribosylation underpre-

biotically plausible conditions. This impassehas led most people to abandon the idea that

RNA might have assembled abiotically, andhas prompted a search for potential pre-RNA

informational molecules (Joyce et al. 1987;

Eschenmoser 1999; Schoning et al. 2000; Zhanget al. 2005; Sutherland 2007). However, we real-

ized that there were other possible syntheticapproaches that although less obvious, still

had the potential to make the ribonucleotides

1 and 2 (Anastasi et al. 2007). Furthermore,and as pointed out earlier, there are also alterna-

tive bond forming polymerization chemistries

imaginable. Our plan was to work throughthese possibilities by systematic experimenta-tion before deciding whether the abiogenesis

of RNA is possible or not.

RECENT RESULTS

As building blocks for pyrimidine ribonucleo-

tides, we have investigated the chemistry of gly-colaldehyde 4, glyceraldehyde 5, cyanamide 6,

cyanoacetylene 7, and phosphatethe same

feedstock molecules ultimately invoked in thenucleobase ribosylation approach (Fig. 2) (in

ongoing work on the prebiotic synthesis ofpurine nucleotides, the systems chemistry of

mixtures containing hydrogen cyanide, is addi-tionally being investigated). However, we havenot presumed a particular order of assembly,

and have systematically investigated many op-

tions over the last decade or so (Sutherlandand Whitfield 1997; Ingar et al. 2003; Smithet al. 2004;Smith and Sutherland 2005; Anastasi

et al. 2007, 2008). Furthermore, aware of thepotential forcertain moleculesto act as catalysts

of reactions in which other molecules arejoined, an important aspect of our approachhas been the study of the chemistry of multi-

component chemical systems, including mixed

oxygenous and nitrogenous chemistry (Szostak2009). It has long been held in prebiotic chemi-stry that the aldol chemistryof aldehydespoten-

tially required to make sugars should not bemixed with, for example, the oligomerization

of hydrogen cyanide and cyanamide potentiallyrequired to make purine nucleobases (Shapiro1988). This is because the two chemistries

have been assumed to interfere with each other,suggesting the potential for a combinatorial

explosion of minor by-products. However,given the impasse of preformed nucleobase

ribosylation, and the conceptual difficulties

with which a transition from a pre-RNA worldto the RNA world is fraught, taking a few syn-

thetic risks seemed justified.Cutting straight to the chase, we found that

mixed oxygenous-nitrogenous chemistryof gly-colaldehyde 4-cyanamide 6 mixtures can betamed by the inclusion of phosphate such that

the heterocycle 2-aminooxazole 21is obtained

in .80% yield (Fig. 4) (Powner et al. 2009).The phosphate functions both as a pH bufferand as a general acid-base catalyst. The hetero-

cycle 21 readily sublimes, potentially offering aprebiotically plausible purification through

sublimation and precipitation or rain-in. In asmooth CC bond forming reaction in waterat neutral pH, 21 then adds to glyceraldehyde

5, giving the four pentose aminooxazolines, butwith high stereoselectivity for the ribo- and

arabino-configured materials (Anastasi et al.

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2006). Remarkably, theribo-isomer then selec-tively crystallizes out of solution, leaving the

arabino-isomer 22the most abundant in solu-tion. Reaction of22 with cyanoacetylene 7 fur-nishesthe anhydronucleoside23 ifthepHofthe

reaction is maintained at pH6.5 through

the use of phosphate as a buffer (Powner et al.2009). Heating 23, inorganic phosphate, andurea 8 in the dry-state or in formamide

solution then gives the activated ribonucleotide

2(BvC). Finally, irradiation of2 (BvC) with

UV light in solution, followed by heating(Powner and Sutherland 2008), converts it to

a mixture of 2

(Bv

C) and2

(Bv

U), the twoactivated pyrimidine nucleotides needed forRNA synthesis.

The presumed mechanisms of the various

reactions that make up the self-assembly se-quence (Fig. 5) reveal some remarkable aspectsof selectivity, and show the synthetic power of

systems chemistry (Anastasi et al. 2006; Powneret al. 2009). In the first step, we found that the

reaction of glycolaldehyde 4 and cyanamide 6generated a series of dead end adducts and asmall amount of 2-aminooxazole 21 in the

absence of phosphate, but in its presence 21

was produced cleanly in extremely high yield.This improvement in the yield of 21 is attrib-uted to general acid base catalysis of two

stepsthat in which the nitrile carbon under-goes attack by a hydroxyl group nucleophile,

and the CH deprotonation step that generatesthe aromatic ring of 21 (Fig. 5A). Phosphateis an ideal general acid-base catalyst in this

context as the reaction is ideally conducted atnear neutral pH values close to the second pKaof phosphoric acid. Phosphate is also knownto catalyze both the dimerization and the

hydrolysis of cyanamide 6 (Lohrmann and

Orgel 1968), but these reactions are slowerthan the formation of21. However, if an excess

of6 is allowed to react with glycolaldehyde 4 inthe presence of phosphate, the formation of21is followed by the formation of cyanoguanidineand urea 8.

In the second step of the self-assembly proc-

ess, 2-aminooxazole 21adds to glyceraldehyde

5, giving all four pentose aminooxazolinesbut, as mentioned previously, with high stereo-selectivity for the ribo-isomer, and the key

arabino-isomer 22(Fig. 5B). The reaction ini-tially follows the course of an electrophilic aro-

matic substitution reaction with the formationof a cationic intermediate. However, ratherthan rearomatizing by C H deprotonation,

this intermediate instead undergoes intramo-lecular addition of a hydroxyl group (Anastasi

et al. 2006). Although the C50-hydroxyl group

2 (B=C)

HO

OH

O

5OH

O

4

HOP

OH

O O

CN

6

CN

7

OHO N

N

O

OP

O

O O

OHO

HO

O

N N

OHO

HO

O

N

O

N

H2O

H2O

21

22

23

2 (B=U)

OHO N

NH

O

O

OP

O

O O

h, H2O

partial

conversion

NH2

NH2

NH2

NH2

H2N

Figure 4. The recently uncovered route to activatedpyrimidine nucleotides 2. The nucleobase ribosyla-tion problem is circumvented by the assembly pro-ceeding through 2-aminooxazole 21, which can bethought of as the chimera of half a pentose sugarand half a nucleobase. The second half of the pen-toseglyceraldehyde 5and the second half of thenucleobasecyanoacetylene 7are then addedsequentially to give the anhydronucleoside23. Phos-phorylation and rearrangement of23 then furnishes2(BvC), and UV irradiation effects the partial con-version of2 (BvC) to 2 (BvU).

J.D. Sutherland




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and glyceraldehyde 5more selective for the for-mation of the desiredarabino-aminooxazoline

22(Powner et al. 2009).In addition to its role as a pH buffer in the

addition of22

to cyanoacetylene7

(at pH valueshigher than 6.5, or in the absence of phosphate,the anhydronucleoside 23undergoes hydrolysis

of the C2O20 bond givingb-arabinocytidine

[Sanchez and Orgel 1970]), phosphate almostcertainly functions as a general acid catalystfor the first addition step, thereby allowing the

reaction to proceed by a path that does notrequire the intermediacy of a high-energy cya-

novinyl anion (Fig. 5C). Furthermore, reactionof excess cyanoacetylene with the hydroxylgroups of the anhydronucleoside product 23 is

prevented by phosphate since HPO422 is more

nucleophilic than these hydroxyl groups. Inthe course of acting as a chemical buffer inthis way, the phosphate is partly transformed

into cyanovinyl phosphate, and this latter com-pound, over time, reacts with more phosphate

to give pyrophosphate (Ferris et al. 1970).The urea 8 produced as a by-product in the

reaction of glycolaldehyde 4 with cyanamide 6,

if the latter is in excess, then acts as a catalyst forthe incorporation of phosphate in the fourth

step of the self-assembly sequence (Fig. 5D).At high temperatures in the dry-state (Lohr-

mann and Orgel 1971), or in formamide solu-

tion (Schoffstall 1976), the weakly nucleophilicurea oxygen atom attacks a minor tautomer

of H2PO42, displacing water via a dissociative,

monomeric metaphosphate-like transition state

to give an imidoyl phosphate. Although thisreaction is reversible in principle, water is lostat high temperature, driving the formation of

the high-energy intermediate, which can then

phosphorylate the hydroxyl groups of nonvola-tile compounds such as the anhydronucleoside

23. Again, this reaction is reversible in principle,

though it is unlikely to be fully so in urea meltsbecause the viscosity of the reaction medium

likely prevents free diffusion. It transpires that

23 adopts a conformation that potentiallymakes the C50-hydroxyl group more hindered

than the C30-hydroxyl group; thus, phosphory-lationof the latter is apparently preferred kineti-

cally (Powneret al.2009). Thedianionicform of

the phosphate group of the O30-phosphorylatedanhydronucleoside24can then attack C20 in an

intramolecular SN2 reaction, thereby invertingthe configuration of that carbon, and giving

the ribocytidine-20

,30

-cyclic phosphate 2

(BvC) (Tapiero and Nagyvary 1971). Alterna-tively, if kinetic selectivity is not fully operative,

the O50- and O30-phosphorylation products of

23could both be sampled at equilibrium, andthe equilibrium could then be displaced to thedetriment of the former through irreversible

conversion of the latter to 2 (BvC). Pyrophos-phatethe product of reaction of the cyano-

vinyl phosphate by-product in the precedingstep with additional phosphate (Ferris et al.1970)can also function as a phosphorylating

agent in urea melts. In this case, the imidoyl

phosphate is generated by nucleophilic dis-placementofphosphate(cf.,water)viaadissocia-tive, monomeric metaphosphate-like transition

state. Pyrophosphate is not soluble in forma-mide, but there is another twist to phosphoryla-

tion of23with inorganic phosphate with ureain formamide solution, and that is that forma-mide is also weakly nucleophilic on its oxygen

atom, and so can also react to give imidoylphosphate intermediatesindeed, phosphory-

lation can be accomplished in formamide inthe absence of urea (Schoffstall 1976). Forma-

mide can be considered to be prebiotically

available because it is the hydration productof hydrogen cyanide. The latter is inevitably

also produced when cyanoacetylene 7 is madeby high-energy atom recombination chemistry

(Sanchez et al. 1970). Given that the purinenucleobases are constitutionally derivable fromhydrogen cyanide, the case for the involvement

of this compound and its hydration product

in the prebiotic synthesis of pyrimidine andpurine nucleotides seems extremely strong(Sanchez et al. 1967).

In the last step of the self-assembly seque-nce, 2 (BvC) is partly converted to 2(BvU)

by UV irradiation. Remarkably, the irradiationconditions destroy other cytidine nucleotides,and since some such compounds willlikely arise

from similar assembly chemistry proceedingfrom aminooxazolines other than22, the irradi-

ation might also serve a sanitizing function such

Ribonucleotides




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that 2 could be produced in the absence ofstereoisomeric contaminants. The proposed

mechanism of the photochemistry (Fig. 5E)is speculative, but supported by a significant

body of fact. It has long been known thatirradiation of other cytidine nucleosides andnucleotides leads to C5-photohydrates that

eliminate water to regenerate the original

cytosine derivative (Miller and Cerutti 1968).During prolonged irradiation, such cytosinederivatives go through many cycles of photoex-

citation and thermal relaxation. Deleteriousphotochemistrynucleobase loss, C10- and

C20-epimerization, hydrolysis to uracil deriva-tives, etc.increases with increased irradiation,presumably because high energy intermediates

are thus accessed for longer (Powner et al.

2007; Powner and Sutherland2008). The molec-ular details of these processes have been elusive,but recent work has suggested plausible mecha-

nisms that can be further tested. It is thoughtthat irradiation of the cytosine ring leads to

the formation of a Dewar pyrimidine 25through a disrotatory 4p electrocyclic process(Shaw and Shetlar 1990). Steric inhibition of

resonance is then expected to make C5-proto-nation facile, giving an amidinium ion that

can undergo an electrocyclic ring-openingmost easily visualized, as shown, for the reso-

nance canonical form 26 (Fig. 5E). The conse-

quence of this second pericyclic process is theformation of an N1,C6-iminium ion 27. In

most molecular contexts, an N1,C6-iminiumion would be expected to undergo attack by

hydroxide anion, giving a cytidine photohy-drate, but in the irradiation of 2 (BvC), the20,30-cyclic phosphate allows ready access to

otherwise inaccessible western sugar ring con-

formers (Saenger 1984) in which the C50-hydroxyl group is proximal to C6 and canthus add to it instead. Thus, the specific nature

of the cyclic phosphate of2(BvC) controls thecytosine photochemistry through enforced

intramolecular nucleophilic addition. Theresultant C5,O50-anhydronucleotide 28 isthought to undergo thermal elimination back

to 2 (BvC) more slowly than other cytidinephotohydrates eliminate water because of con-

formational restriction about the C5C6

bond. This has the effect that 2 (BvC) under-goes fewer photochemical excitation and ther-

mal relaxation cycles than other cytidinenucleosides and nucleotides. The consequences

of this are twofold. Firstly, deleteriouschemistryfrom high-energy (charged) intermediates isreduced because these intermediates are

accessed less often. Secondly, the persistence of

the C5,O50-anhydronucleotide allows forgreater hydrolysis to the corresponding uracilderivative29. Relaxation of this uracil derivative

is then responsible for the high yield of 2(BvU) relative to the yield of the correspond-

ing uridine nucleosides and nucleotides in theirradiation of other cytidine nucleosides andnucleotides. Indeed, most other pyrimidine

nucleosides and nucleotides are destroyed by

the prolonged irradiation that partly converts2 (BvC) to 2 (BvU). This has the beneficialeffect that 2 (BvC) and2 (BvU) can be effec-

tivelysanitized by destruction of nucleoside andnucleotide by-products of the assembly process

described herein.

TOWARD A GEOCHEMICALLYPLAUSIBLE SYNTHESIS

The contextually specific chemoselectivity, andthe systems chemistry aspects of this newly

uncovered self-assembly route to activated py-

rimidine nucleotides suggest that the chemistryshould be viewed as predisposed. However, the

route as operated thus far in the laboratory isassociated with several steps, and the conditions

for these steps are different. Furthermore, puri-fication in between certain steps was carried outto make analysis of the chemistry easier. Clearly,

these issues need to be addressed beforethe syn-

thesis can be seen as geochemically plausible.First, the sequence of different conditions

needs considering. Millers iconic experiment

(Miller 1953) has apparently conditioned manyin the field to think that a prebiotic synthesis

needs to occur under one set of conditions.But, think of chemistry occurring now on theEarth. There is no doubt that in many locations,

conditions vary with timethere are lightperiods and dark periods, hot periods and

cold periods, dry periods and wet periods.

J.D. Sutherland




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If the same were true on the primordial Earth,then why should we expect prebiotic synthesis

just to occur in one particular period? Further-more, the frequent impacts of asteroids, mete-

orites, and comets on the early Earth wouldsurely have exacerbated the changeability ofgeochemical conditions. Given that there are

so many geochemically plausible conditions,

and sequences of conditions, it is difficult totry and predict solely from geochemistry theactual conditions that pertained for the pre-

biotic synthesis of any particular compoundor class of compounds. Surely, it makes more

sense to first find predisposed chemical routesto molecules of interest and then ask whetherthe sequence of conditions is geochemically

plausible. If it then turns out that this sequence

of conditions additionally supports the synthe-sis of other important molecules, then evidencemight accumulate that the sequence of condi-

tions actually did take place during the originof life on Earth. Furthermore, the search for fur-

ther relevant prebiotic synthesis would then beexpedited. From what is now known aboutpyrimidine nucleotide self-assembly, one geo-

chemically plausible sequence of conditionscoincidentally redolent of Darwins suggestion

(Darwin 1871)involves a warm pond thatevaporates and dries out, stays dry and is heated

for a time, is then filled again by rain, and sub-

sequently bathed in sunshine.Secondly, one has to wonder if any purifi-

cation steps could reasonably be invoked insuch a scenario, and ask if the other chemistry

would work in the absence of purification. 2-Aminooxazole 21 sublimes readily on beingwarmed at atmospheric pressure, and resolidi-

fies on cooling surfaces. This property suggests

that 21 could plausibly be purified prebioti-callyif necessaryand be relocated by pre-cipitation. If delivered to glyceraldehyde 5and

phosphate, then reaction of 21 and 5 givingthe pentose aminooxazolines, including the

keyarabino-isomer 22, certainly seems plausi-ble. Subsequent delivery of cyanoacetylene 7produced by atmospheric chemistryby rain-

in also seems plausible, though there is nodoubt that it would be accompanied by hydro-

gen cyanide. Will hydrogen cyanide interfere

with the reaction of22 and 7, giving the anhy-dronucleoside 23? The answer awaits experi-

mental evaluation. If the hydrogen cyanidedoes not interfere, but slowly hydrolyses to for-

mamide,thenwhen the water in the pond evap-orates, a formamide solution would remain. If

this solution gets heated, the phosphorylationand rearrangement of23to 2 (BvC) will take

place whether the pond additionally happenedto contain urea or not. The issue seems notto be whether the chemistry could take place

or not, but the yield and purity of products.Because of the other aminooxazolines, there

would doubtless be other stereoisomeric prod-ucts at this point, but this would not matter assubsequent dilution with water and irradiation

would not just partly convert 2 (BvC) to 2

(Bv

U), but also destroy these other products.So, there is more work to be done, and althoughit is premature to conclude that the newly

discovered self-assembly route to activatedpyri-midine nucleotides (Powner et al. 2009) is

geochemically plausible, the signs are lookinggood! What is now needed on top of furtherexperimental work to assess the geochemical

plausibilityof the synthesis, is a thorough inves-tigation of the synthesis of activated purine

nucleotides.

FUTURE CHALLENGESIf further experimentation supports nucleo-

side-20,30-cyclic phosphates 2 as the activatedmonomers for prebiotic RNA synthesis, then

an obviousnextquestion is howsuchmonomersoligomerise. Pioneering work from the 1970spartly addresses this and suggests that general

acid-base catalysis is the key (Verlander et al.

1973). In the dry-state, mixtures of aliphaticamines and the corresponding ammonium saltsare effective oligomerization catalysts. It is

thought that the amines deprotonate theC50-hydroxyl group of one nucleoside-

20,30-cyclic phosphate 2, enabling it to attackthe phosphate group of another. The ammo-nium salts serve to protonate the oxygen

leaving group generating a C20-orC 30-hydroxylgroup. Short oligomers are easily produced,

but the chemistry generates both 30,50- and

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20,50-internucleotide links, and further work isneeded to address this selectivity issue.

The oligomerization of racemic mixturesof nucleoside-20,30-cyclic phosphates 2 clearly

has the potential to generate extremely complexmixtures of diastereoisomers, so a major goalhas to be the generation of these monomers

in enantiopure form. This issue has not yet

been solved, but it is apparent from the self-assembly chemistry that if glyceraldehyde 5can be obtained in enantiopure form then the

resultant pyrimidine nucleotides will be simi-larly enantiopure. How glyceraldehyde 5might

be obtained in enantiopure is a question thatcan only be answered when a prebiotically plausi-blesynthesis of5 is shown. The conditions poten-

tially required to generate glyceraldehyde5 by the

aldolization of glycolaldehyde 4 and formalde-hyde are the same conditions that favour the con-version of5to the more stable dihydroxyacetone.

A synthesis of5and indeed 4from formalde-hyde involving umpolung would therefore be

preferable, but prebiotically plausible carbonylumpolung has not yet been shown.

Finally, the potential for simultaneous self-

assembly of RNA and other molecules such aslipids and peptides must be investigated. Com-

partmentalization of RNA leading to protocellsis seen as a crucial step towards systems that can

undergo Darwinian evolution (Szostak et al.

2001), so can lipids be produced alongsidenucleotides? Peptides, even dipeptides, offer ad-

ditional scope for catalysis, and their prebioticsynthesis and use could help drive the evolution

of the ribosome and the genetic code, so canpeptides also be produced under the same con-ditions? Experimental investigation of such

questions will rely heavily on analytical chemis-

try because of the complexity of the multicom-ponent systems that will have to be investigated.There is cause for optimism, however, as pre-

biotic systems chemistry, though still in itsinfancy, is already suggesting new solutions to

old problems.

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