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org/ on June 20, 2017

Phil. Trans. R. Soc. B (2011) 366, 2870–2877

doi:10.1098/rstb.2011.0134

Research

Prebiotic chemistry: a new modus operandi

Matthew W. Powner† and John D. Sutherland*,‡
School of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL, UK
A variety of macromolecules and small molecules—(oligo)nucleotides, proteins, lipids and meta-
bolites—are collectively considered essential to early life. However, previous schemes for the
origin of life—e.g. the ‘RNA world’ hypothesis—have tended to assume the initial emergence of
life based on one such molecular class followed by the sequential addition of the others, rather
than the emergence of life based on a mixture of all the classes of molecules. This view is in part
due to the perceived implausibility of multi-component reaction chemistry producing such a
mixture. The concept of systems chemistry challenges such preconceptions by suggesting the possi-
bility of molecular synergism in complex mixtures. If a systems chemistry method to make mixtures
of all the classes of molecules considered essential for early life were to be discovered, the significant
conceptual difficulties associated with pure RNA, protein, lipid or metabolism ‘worlds’ would be
alleviated. Knowledge of the geochemical conditions conducive to the chemical origins of life is
crucial, but cannot be inferred from a planetary sciences approach alone. Instead, insights from
the organic reactivity of analytically accessible chemical subsystems can inform the search for the
relevant geochemical conditions. If the common set of conditions under which these subsystems
work productively, and compatibly, matches plausible geochemistry, an origins of life scenario
can be inferred. Using chemical clues from multiple subsystems in this way is akin to triangulation,
and constitutes a novel approach to discover the circumstances surrounding the transition from
chemistry to biology. Here, we exemplify this strategy by finding common conditions under
which chemical subsystems generate nucleotides and lipids in a compatible and potentially
synergistic way. The conditions hint at a post-meteoritic impact origin of life scenario.
Keywords: prebiotic chemistry; systems chemistry; chemoselectivity; phosphorylation

1. INTRODUCTION has been an anathema to most organic chemists until

Although life is ineluctably associated with the recently.
chemistry of complex mixtures, the majority of organic When it comes to origins of life chemistry, studies
chemistry thus far practised in the laboratory has been tend to be of two sorts: conventional A þ B ! C
of the two component A þ B ! C variety. This is for a reactions directed towards prebiotically important
number of reasons: early organic chemists had limited molecules; or multi-component reactions such as
purification and analytical tools, and so found such Miller–Urey type syntheses that lead to hugely complex
reactions—especially if they proceeded in high product mixtures [2]. In reality, neither sort of reaction
yields—easiest to manage; single products of such scheme is likely to be conducive to the emergence of
reactions can themselves be mixed with other single life. The former involves implausibly simple mixtures
components in a subsequent step of a linear multi- of starting materials and gives only one of many
step synthesis; and, commercially, single products required product molecules. The latter, though often
can conveniently be sold as such. For these and involving more plausible reactant mixtures, lacks
other reasons, much of modern chemistry has focused intrinsic chemical control, and results in mixtures so
‘above the arrow’ i.e. not on facilitating multi-reactant complex that useful products are obtained in minimal
experiments, but on the reagents of transformation. yield interspersed with numerous by-products.
There are a few multi-component reactions known— What is needed is Goldilocks chemistry: chemistry
e.g. the Ugi reaction [1]—but these again tend to be that works on plausible reactant mixtures to give mul-
directed towards single products, and the concept of tiple required products in good yield without creating
a multi-component reaction giving many products too many deleterious by-products. This is because
diminutive yields of myriad products leads to system
degeneration, as subsequent productive multi-molecu-
* Author for correspondence ([email protected]). lar reactions are kinetically disfavoured through
†
Present address: Department of Molecular Biology, Massachusetts
General Hospital, Boston, MA 02114, USA.
dilution raised to the power of the molecularity. The
‡
Present address: MRC Laboratory of Molecular Biology, Hills early work perhaps closest to being Goldilocks chem-
Road, Cambridge CB2 0QH, UK. istry is a study of the Strecker multi-component
One contribution of 17 to a Discussion Meeting Issue ‘The chemical reaction and associated chemistry in a prebiotic con-
origins of life and its early evolution’. text by authors who can also be credited with the
2870 This journal is q 2011 The Royal Society
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Prebiotic chemistry M. W. Powner & J. D. Sutherland 2871

first use of the systems chemistry concept in this area to find conditions for an overall systems chemistry
[3]. Finding similar chemistry leading to a broader synthesis of multiple prebiotic target molecules.
spectrum of biologically relevant structures—includ- Accordingly, we are investigating other subsystems to
ing nucleotides and lipids—has been the goal of our synthesize lipids, peptides and metabolites. Should
laboratory in recent years [4,5]. we find that the conditions under which these other
Our approach has been to study chemical subsys- subsystems are synthetically productive match any of
tems, and ultimately to assemble these subsystems the conditions of ribonucleotide synthesis, it will
into a prebiotically plausible overall system with the strengthen the case for the occurrence of the
goal of observing emergent behaviour. In prebiotic common conditions on the early Earth, and further
chemistry, as in traditional biochemistry, catalysis has support the prebiotic plausibility of the syntheses.
proved to be key to directing reaction kinetics; even Thus far we have investigated the synthesis of alkyl
low-level catalysis by components of mixtures can phosphates as lipids, and report our results herein.
have a profound effect on reaction outcomes [4]. If To appreciate the results of these latter experiments
products of subsystems turn out to be catalysts of reac- in the context of our prebiotic ribonucleotide syn-
tions in other subsystems, a complex temporally thesis, it is first necessary to briefly review that
controlled series of reactions can ensue in the com- synthesis.
plete system. Reactions need not occur in cycles for Cyanamide 1 reacts with glycolaldehyde 2 in the
such control, though cycles can provide a mechanism presence of phosphate to give 2-aminooxazole 3. If
for molecular (auto-)amplification and control [6]. the stoichiometry of 1 : 2 is greater than 1 : 1, excess
The chemical behaviour of the components of such a 1 is then hydrated to urea 4 in a slower phosphate-
system may appear to be choreographed, but it is cru- catalysed reaction [7]. Addition of 2-aminooxazole 3
cial to recognize that the behaviour is implicit in the to glyceraldehyde 5 then gives the pentose aminooxa-
structures of the input reagents under the defined con- zolines 6 with ribo-6 and arabino-6 predominant
ditions through the laws of chemistry. [4,8]. Arabino-6 turns out to be the desired intermedi-
The behaviour of the system can also be influenced ate for pyrimidine ribonucleotide synthesis, but there
by variation in reaction conditions over time. This is is a stereochemical twist to the story. It is not the
an important point in the prebiotic chemistry field arabino-6 initially formed by this addition reaction
because too often it has been assumed that only that ends up as the pyrimidine ribonucleotides; if it
fixed conditions prevailing over a long time should were, then there would be no way to produce enantio-
be considered for prebiotic synthesis. Temperature, merically pure ribonucleotides unless a prebiotic route
humidity and light flux must have varied on the early to enantiopure glyceraldehyde 5 existed. Routes to
Earth, and so it is perfectly reasonable to invoke vari- non-racemic glyceraldehyde can be envisaged [9],
ations in conditions over the course of a prebiotic but obtaining this three-carbon sugar enantiopure
systems chemistry experiment. One is not seeking to looks very difficult. A means of enantioenrichment
recreate exactly what happened on early Earth, but is, therefore, needed at some point in the synthetic
to show what is plausible. Given the huge range of route. Ribo-6 is highly crystalline [10– 12] and the
possible conditions, and sequences of conditions, we least soluble of the pentose aminooxazolines 6 [12].
caution against the tendency to rigidly invoke one set Furthermore, when ribo-6 crystallizes from non-race-
or sequence of conditions, and then to limit the chem- mic solutions it becomes enantioenriched [8]. At a
istry one explores to only that which is possible under threshold enantiomeric excess (ee) of about 60 per
the chosen conditions. cent in solution, the crystalline ribo-6 that forms is
What seems a much better approach is to explore all enantiopure. Thus, if 2-aminooxazole 3 adds to glycer-
sorts of different geochemically plausible (sequences aldehyde 5 with an ee of 60 per cent, enantiopure
of ) conditions in an attempt to elucidate scenarios in ribo-6 crystallizes out of the reaction mixture.
which chemistry giving all the products needed for Now the twist—ribo-6 can be equilibrated with
life takes place. If one set, range, or sequence of con- arabino-6 by incubation in phosphate buffer solution
ditions is thereby uncovered that gives all the [13]; moreover, if enantiopure ribo-6 as purified by
products, then it is akin to triangulation in navigation crystallization is incubated in such a solution, it is con-
wherein multiple pointers provide better evidence verted into a mixture of enantiopure arabino-6 and
than a single pointer to define the relevant chemical enantiopure ribo-6. The enantiopure arabino-6 in this
space. mixture can then react with cyanoacetylene 7 to give the
anhydronucleoside 8 in a reaction in which phosphate
serves to keep the pH from rising above neutrality,
2. PREBIOTIC RIBONUCLEOTIDE SYNTHESIS IN and thereby prevents base-catalysed hydrolysis and
A SUBSYSTEM other reactions of 8 [4]. The enantiopure ribo-6 in the
We recently demonstrated a prebiotically plausible sys- mixture also reacts with cyanoacetylene 7 to give anhydro-
tems chemistry route to pyrimidine ribonucleotides in nucleoside 9. Conditions whereby 9 might be converted
which phosphate, a dormant reagent in the early steps, into purine ribonucleotides are currently being sought.
acts as a general acid – general base catalyst, pH buffer However, if they are not found then it transpires that
and chemical buffer (scheme 1) [4]. The sequence of subsequent phosphorylation and irradiation steps,
reaction conditions—mild heating in water, followed which convert the isomer 8 into pyrimidine ribonucleo-
by evaporation, dry state heating, rehydration and tides, will otherwise destroy the material deriving from
UV irradiation—under which this synthesis proceeds 9 because of a remarkable series of chemoselectivity
serves as a starting point in our triangulation strategy effects [4,14].
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2872 M. W. Powner & J. D. Sutherland Prebiotic chemistry

N
H2 N N + HO NH2 3
O
1 2 O
HO OH
Pi ee > 60%

O 5
H2 N NH2 HO
O
O N
ee > 60%
4
O NH2
HO
6
H2 N NH2 crystallization
O HO
O HO
4 P OH 2 O O
OO N N
12 O NH2 O NH2
HO HO
enantiopure arabino-6 enantiopure ribo-6
H2 N NH2
CN
7
13 O P O
O O Pi

+ H 2O
HO HO
O O
N N
NH2 NH2
N N
HO O HO O
8 9

partially via:
HO O HO
P O Cyt
HO O O
O base O Cyt
+ HO OH
14
O O O O
P 10 P 11 products
O O O O
(base = Cyt)
h

10 (base = Cyt, Ura) predominant

photodestruction

Scheme 1. ‘Systems chemistry’ prebiotic synthesis of the canonical pyrimidine ribonucleotides. An indication of the dissocia-
tive mechanism for the phosphorylation of anhydronucleosides 8 and 9 is shown in the box. Pi, inorganic phosphate.

The phosphorylation of 8 by inorganic phosphate, Mechanistically (scheme 1, box), a carbonyl group lone-
which is key to the synthesis, takes place either in pair of 4 attacks a rare tautomer 12 of the phosphate
urea 4 melts, or in formamide – urea 4 mixtures at elev- mono-anion to displace water and generate the imidoyl
ated temperatures, due to nucleophilic catalysis by phosphate 13. Although 12 is a minor species, it is
urea 4, as mentioned previously, a co-product of the highly reactive towards nucleophilic substitution because
first step of the reaction sequence. Chemoselective the phosphorus atom bears a good leaving group—
phosphorylation of the 30 -hydroxyl group of anhydro- water—and two negatively charged oxygen substituents,
nucleoside 8 followed by rearrangement of the so that reaction can occur dissociatively via a meta-
resultant 30 -phosphate to ribo-cytidine-20 ,30 -cyclic phosphate-like transition state. Imidoyl phosphate 13
phosphate 10 (base ¼ Cyt) is observed. 50 -Phosphoryl- can then undergo attack at phosphorus either by
ation also takes place, though to a lesser degree if the water—in which case, the formation reaction is
ratio of 8 : phosphate is 1 : 1, resulting in the con- reversed—or by various alcohols. In the latter case, rare
comitant production of the diphosphate 11 [4]. tautomers of alkyl monophosphate mono-anions are

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Prebiotic chemistry M. W. Powner & J. D. Sutherland 2873

produced that then reversibly tautomerize to singly 4. A LINKED SOURCE OF PHOSPHATE AND
charged alkylphosphate mono-anions. The phosphoryla- LIPIDS: TOWARDS A HIGHER ORDER SYSTEM
tion chemistry is slowly reversible, though the evaporation The most plausible prebiotic source of phosphate is
of water results in predominant alcohol phosphorylation. from anoxic corrosion of schreibersite [22,23], a min-
This slow reversibility means that chemoselectivity eral found in iron– nickel meteorites. By a series of
can result from thermodynamic and kinetic effects. disproportionation reactions, this iron– nickel phos-
Kinetically, 30 -phosphorylation is favoured because phide ((Fe,Ni)3P) produces ferrous and nickel
0
an nO ! p * CN stereoelectronic effect between the O5 phosphates as well as more reduced phosphorus-con-
atom and C2 of the nucleobase in 8 reduces electron taining species. The phosphate can be mobilized in
density on the O50 atom and increases its steric bulk solution if ferrous and nickel ions are precipitated or
relative to the O30 atom [14]. Thermodynamically, sequestered in some way. Experimentally, sulphide
30 -phosphorylation is favoured because the 30 -phosphate addition causes precipitation of FeS and NiS, and
can rearrange to the more stable cyclic nucleotide 10 this is probably prebiotically relevant as it is thought
(base ¼ Cyt) [15], whereas the 50 -phosphate cannot. It that significant H2S was available from volcanic emis-
transpires that phosphate can also bring about the sions. Alternatively—or additionally—it is possible
hydrolysis of the anhydronucleoside C2–O20 bond via that hydrogen cyanide could sequester the ferrous
nucleophilic addition at C2 followed by dissociative iron as hexacyanoferrate(II) [24]. This is an attractive
nucleophilic attack at phosphorus by urea 4; another possibility as it would both liberate phosphate and
consequence of the nO ! p CN * stereoelectronic effect concentrate hydrogen cyanide; subsequent irradiation
in anhydronucleoside 8 is that this hydrolysis is slowed could then release the hydrogen cyanide [24].
relative to that of isomer 9 because the electrophilicity In keeping with the approach outlined in the intro-
of C2 of 8 is lower than that of C2 of 9 due to the partial duction, this meteoritic source of phosphate suggests
donation of electron density from O50 [14]. The resul- that geochemistry associated with iron/nickel meteorite
tant in situ hydrolysis of 9 to a-ribonucleoside 14 then impacts should be considered. Our modus operandi has
allows phosphorylation of this free nucleoside to give a it that the resultant sequence of geochemical conditions
range of products all destined to be destroyed by UV should be assessed on its own merits, but also with the
irradiation in the next step, whereas 10 (base ¼ Cyt) requirements of our systems chemistry synthesis of
simply converts to 10 (base ¼ Cyt, Ura) [4]. ribonucleotides in mind, and triangulation our goal.
Large impactors would deliver considerable energy
with meteorite and ejecta vapour persisting in the
atmosphere after the impact. This would in turn
3. THE NEED FOR MEMBRANES cause significant ocean vapourization through down-
The importance of membranes in the origin of life ward thermal radiation, and production of atom and
was noted by Haldane in the late 1920s [16]. The molecular fragment recombination products such as
self-assembly of bilayer vesicles (including those hydrogen cyanide and cyanoacetylene. Later, after
encapsulating macromolecules) from lipids has been partial cooling, hot iron/nickel particles in the water
amply demonstrated [16] and various reactions have vapour- and CO2-rich atmosphere would catalyse
been shown to take place in, or be catalysed by, such first reduction of atmospheric CO2 to CO [25], and
structures [17]. Lipids are amphiphilic molecules nor- then Fischer – Tropsch (FT) chemistry (2CO þ H2
mally composed of long chain aliphatic hydrocarbons ! – CH2 – þ CO2) on a huge scale [26].
with polar head groups. In a general sense, bilayer A prolonged hydrogen-rich atmosphere on the early
formation requires a lipid composition ratio of one Earth seems unlikely because this lightest of elements
polar head to two non-polar tails. Each polar head is unfettered by Earth’s gravity. Accordingly, the par-
can have either one or two non-polar tails attached. ticular type of FT chemistry that is most likely
For those polar heads attached to only one non-polar following iron/nickel meteorite impact is the Kölbel –
tail, a second non-polar tail molecule lacking a Engelhardt (KE) variant (3CO þ H2O ! – CH2 – þ
highly polar head is needed in roughly equimolar 2CO2) as this does not require an atmosphere rich in
quantity. Single chain amphiphiles can form bilayers hydrogen per se. Instead, the hydrogen is synthesized
under conditions where an ionizable head group is in situ by a water gas shift reaction (CO þ H2O !
only partly ionized—the unionized head group not CO2 þ H2) [27]. Under optimal laboratory con-
being highly polar—otherwise such amphiphiles ditions, the reaction proceeds with more than 80 per
usually form micelles rather than bilayer structures cent CO conversion. Hydrocarbon yields in the KE
such as vesicles [18,19]. reaction are optimal when the CO : H2O ratio is ca
In considering the possible prebiogenesis of lipids, 2.6—an excess of CO leads to carbon deposition,
we have realized that one likely synthetic sequence is whereas an excess of H2O causes catalyst oxidation
linked to the production of phosphate on the early with concomitant production of H2 [27]. In the pres-
Earth (vide infra). Because of the central role of phos- ence of a large amount of iron, these stoichiometric
phate in our prebiotic synthesis of ribonucleotides, processes would be expected to shift the local CO :
and the growing appreciation of the evolutionary H2O ratio towards the optimal for hydrocarbon
advantages that accrue if (self-)replicating informa- synthesis.
tional molecules are compartmentalized in (dividing) Significantly, the KE reaction produces a higher
vesicles [20,21], we have sought prebiotic chemistry fraction of oxygenates than the FT reaction. Straight
that leads to both ribonucleotides and lipids in a chain alkanols, ketones, aldehydes (including H2CO)
single system. and carboxylic acids are formed in addition to straight
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2874 M. W. Powner & J. D. Sutherland Prebiotic chemistry

NH4 H2 PO 4 (1 eq.)

(H2 N)2 CO (10 eq.),

100o C, 24 h

+ + O + O
OH OH O O
OH P P
HO HO
1 eq. 1 eq. >50 eq. O NH 4 + O NH 4 +

7:3
Scheme 2. Selective formation of long chain alkyl phosphates in the phosphorylation of mixed chain length alkanols.

chain hydrocarbons [27]. This array of products is geochemical events. The reason for attempting this is
produced in the temperature range 180 – 2808C; at that such considerations point to the likely co-location
higher temperatures, CO conversions drop slowly but of anhydronucleosides and mixed chain alkanols when
methane production becomes predominant (methana- phosphorylation conditions occurred.
tion). The percentage CO conversion is essentially
constant over the pressure range 1 – 100 bar, but the
oxygenate content rises with pressure (3% alcohols 5. ALKANOL PHOSPHORYLATION
by weight at 3 bar, 30% by weight at 100 bar). In light of the foregoing, we investigated the
Given these attributes of the KE reaction, the presence phosphorylation of mixed length alkanols under
of mixed length straight chain alkanols on the early the conditions previously established for anhydro-
Earth seems highly likely. nucleoside phosphorylation and rearrangement [4]
The presence of NiS on the Earth’s surface in a (scheme 2). Short chain alkanols tend to predominate
CO-containing atmosphere could have led to the syn- over medium and long chain alkanols in the oxygenate
thesis of other FT-type products—including thioacids fraction of products synthesized by the KE variant of
and the corresponding carboxylic acids—by the same the FT reaction [27], and so, as a representative KE
sorts of chemistry that have been invoked by other oxygenate fraction for phosphorylation, we employed
authors for deep-sea hydrothermal vent scenarios a mixture of ethanol, hexanol and decanol, with the
[28,29]. However, the surface scenario we describe C2 alkanol in a large molar excess over the longer
here has the added advantage that the other chem- chain species. Because ethyl phosphate is not amphi-
istries required to make nucleotides, inter alia, are philic, significant chemoselectivity would have to
possible in the same location, as conditions naturally operate for membrane-forming amphiphiles to be
change over time. In contrast, deep-sea vents do not produced by phosphorylation of such a mixture
allow drying down, dry state heating or UV irradiation. using sub-stoichiometric phosphate. In the event, we
Thus, when conditions calmed down after an impact, were delighted to find that when such an oxygenate
phosphate and organic nucleotide precursors could fraction was mixed with ammonium phosphate and
have been present along with mixed alkanols. Gases urea, dried down, and heated on a glass fibre sup-
such as cyanoacetylene and hydrogen cyanide would port—to mimic spreading out on an (inert) mineral
partition between the gaseous atmospheric phase and surface—no ethyl phosphate was apparent in the 1H-
aqueous solution surface phase. As mentioned, hydro- NMR spectrum of the products after washing of the
gen cyanide could be concentrated during phosphate solid support and lyophilization of the washings.
mobilization, whereas cyanoacetylene would be Downfield, phosphorus-coupled methylene signals
expected to gradually dissolve in surface water and resonated as a quartet indicative of longer chain alkyl
react with nucleophilic species dissolved therein. phosphate(s) (the methylene group of ethyl phosphate
During such a period, the chemistry from 1, 2, 5 and has a higher multiplicity). Furthermore, the next most
7 to anhydronucleosides 8 and 9 could have taken downfield signal for the b-methylene protons was a
place. Then a temperature increase—perhaps associ- quintet with no trace of the triplet expected for ethyl
ated with another impact—could cause aqueous phosphate. Integration of all other methylene signals
surface phases to dry out and the residues to be indicated that decyl phosphate and hexyl phosphate
heated; this is when the conversion of 8 and 9 to 10, were both present in a ratio of 7 : 3 (no residual long
11 and other nucleotides could have taken place. chain alcohols were detectable—either the excess alka-
To reiterate: this sequence of conditions and the nol vapourized, or it is possible that these alcohols
scenario causing it were not arbitrarily selected ahead were not eluted from the solid phase during the wash-
of our experimental investigations into prebiotic ing protocol). Precise yield determination based on
nucleotide chemistry. The sequence of conditions is weight was not possible because of uncertainties con-
empirically realized by the chemistry we, and others, cerning how much of the various reagents remained
have discovered. The scenario is an attempt to match after the washing and lyophilization steps. However,
this sequence of conditions to a plausible sequence of related phosphorylations have been found to approach
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Prebiotic chemistry M. W. Powner & J. D. Sutherland 2875

quantitative yields based on phosphate incorporation hopefully prove possible to thereby adduce a sequence,
with only traces of inorganic phosphate, pyrophos- set or range of geochemically plausible conditions that
phate and higher polyphosphates, if the alcohol to be could have simultaneously and chemoselectively deliv-
phosphorylated is in (even slight) excess over ered a broad spectrum of biologically relevant
phosphate. molecules on the early Earth. Delivery of these mol-
The chemoselective formation of decyl phosphate ecules within the full system under such conditions
over hexyl phosphate, and the lack of ethyl phosphate can then be experimentally reproduced with high
can be attributed to the reversibility of the phosphoryl- expectations of emergent properties.
ation, and the progressively lower volatility of ethanol,
hexanol and decanol. It is highly significant that
the temperature that was found experimentally to 7. EXPERIMENTAL SECTION
be ideal for the phosphorylation of the anhydro- (a) Synthesis of decyl and hexyl phosphate
nucleosides 8 and 9 also results in a chain length monoesters via the diallyl phosphotriesters
chemoselectivity for alkanol phosphorylation favour- (i) Diallyl decyl phosphate
ing the formation of amphiphilic medium and long Decanol (36 ml, 0.19 mmol) was dissolved in anhy-
chain alkyl phosphates. If such alkyl phosphates were drous acetonitrile (1 ml) over 3 Å molecular sieves
to be mixed with similarly thermally fractionated (30 mg, dried over P2O5 at 3008C) under a nitrogen
alkanols, or if the phosphorylation reaction used atmosphere and the suspension was stirred for
less phosphate than mixed single chain amphiphile, 30 min. N,N-Bis-iso-propyl-diallyl phosphoramidite
long chain non-amphiphile combinations with the (0.1 ml, 0.38 mmol) was added and the reaction was
potential to form bilayer vesicles would result. stirred for 30 min, before 1H-tetrazole solution
This is currently being investigated experimentally. (0.45 M in acetonitrile, 3.4 ml) was added and the
Furthermore, mixed alkanol/anhydronucleoside phos- mixture was stirred for 24 h. t-Butyl hydrogen per-
phorylations are being studied because of the potential oxide solution (6.0 M in decane, 0.76 ml) was added
for forming compartmentalized nucleotides as well as and the mixture was stirred for 2 h. The reaction mix-
an additional aspect of chemoselectivity. As mentioned ture was filtered through a pad of Celite (10 mm),
above, when 8 undergoes phosphorylation, significant concentrated in vacuo and the residue purified by
quantities of the diphosphate 11 are produced in silica gel flash column chromatography (eluting with
addition to the desired product 10. In fact, when pyr- cyclohexane : EtOAc 3 : 2) to give 45 mg (75%) of
ophosphate– urea mixtures are employed as the the title compound as a clear oil. Rf: 0.5 (cyclo-
phosphorylating agent with a pyrophosphate:anhydro- hexane : EtOAc 4 : 1). 1H-NMR (500 MHz, CDCl3)
nucleoside ratio of 5 : 2, 11 can be produced in yields dH 5.86 ppm (ddtt, J ¼ 17.2, 10.3, 5.5, 0.8 Hz, 2H,
of 70 per cent ( J.D.S and M.W.P unpublished data). CH ¼ CH2); 5.30 (dq, J ¼ 17.2, 1.5 Hz, 2H, CH ¼
It thus seems likely that phosphorylation of a CH2); 5.19 (dq, J ¼ 10.3, 1.3 Hz, 2H, CH ¼ CH2);
mixture comprising anhydronucleoside 8, excess pyro- 4.47 (ddt, J ¼ 8.1, 5.5, 1.3 Hz, 4H, CH2CH ¼
phosphate, and an even greater excess of mixed chain CH2); 3.98 (q, J ¼ 6.8 Hz, 2H, POCH2CH2CH2);
length alkanols will give 10—rather than 11—in 1.64 (q, J ¼ 2.5 Hz, 1H, POCH2CH2CH2); 1.62 (q,
increased yield along with long chain alkyl phosphates J ¼ 6.6 Hz, 1H, POCH2CH2CH2); 1.12– 1.37 (m,
and long chain alkanols. This expectation is based on 14H, (CH2)7); 0.81 (t, J ¼ 7.1 Hz, 3H, Me). 13C-
the fact that an excess of pyrophosphate over 8 will NMR (101 MHz, CDCl3) dC 132.5 ppm (2C, CH ¼
drive phosphorylation of both the 50 - and 30 -hydroxyl CH2); 118.1 (2C, CH ¼ CH2); 68.1 (2C, CH ¼
groups, but phosphorylation of the former is reversible CH2CH2); 68.0 (POCH2CH2CH2); 31.9 (POCH2-
whereas phosphorylation of the latter is rendered CH2CH2); 30.3 (POCH2CH2CH2); 30.2 (CH2);
irreversible by the rearrangement that generates the 29.5 (CH2); 29.3 (CH2); 29.1 (CH2); 25.4 (CH2);
20 ,30 -cyclic phosphate. The reversible phosphorylation 22.7 (CH2); 14.1 (Me). 31P-NMR (162 MHz,
of the 50 -hydroxyl groups should then compete with CDCl3) dP – 0.70 ppm (h, J ¼ 7.8 Hz). 1H-decoupled
31
reversible long chain alkanol phosphorylation, and if P-NMR (162 MHz, CDCl3) dP – 0.70 ppm. ES –
the alkanols are in an excess over the 50 -hydroxyl MS (pos. m/z): 341 (100%, [M þ Naþ]þ). HRMS
groups, the latter should not end up being extensively (m/z): [M þ Naþ]þ calcd for C16H31O4NaP,
phosphorylated at equilibrium. When the analytical 341.1852; found, 341.1855 (figure 1).
methods for fully characterizing such mixtures with
respect to individual product yield and assessment of (ii) Sodium decyl phosphate
nucleotide-containing vesicle formation are estab- Diallyl decyl phosphate (30 mg, 0.10 mmol) was dis-
lished, we will carry out such experiments and report solved in CH2Cl2 (5 ml), trimethylsilyl bromide
the results. (64 ml, 0.5 mmol) was added and the solution was stir-
red under a nitrogen atmosphere for 15 h. The
reaction mixture was then concentrated in vacuo. The
6. THE WAY FORWARD residue was dissolved in H2O and the resultant sol-
The modus operandi for prebiotic chemistry adumbrated ution was adjusted to pH ¼ 9.0 with 1 M NaOH and
in this paper and our results thus far suggest a clear way lyophilized to give 20 mg (84%) of the title compound
forward. Through investigation of multi-component as a white semi-solid. 1H-NMR (400 MHz, D2O) dH
reactions and sequential one-pot multi-step synthetic 3.57 ppm (q, J ¼ 7.0 Hz, 2H, POCH2CH2CH2);
sequences, productive subsystems will be further 1.43 (quin., J ¼ 6.9 Hz, 2H, POCH2CH2CH2);
defined. After interrelation of these subsystems, it will 1.04 –1.22 (m, 14H, (CH2)7); 0.71 (t, J ¼ 7.1 Hz,
Phil. Trans. R. Soc. B (2011)
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2876 M. W. Powner & J. D. Sutherland Prebiotic chemistry

O O
P P
O O O O
O O

Figure 1. Diallyl decyl phosphate. Figure 3. Diallyl hexyl phosphate.

O O
P P
O OH
ONa O OH
ONa
Figure 2. Sodium decyl phosphate.
Figure 4. Sodium hexyl phosphate.

3H, Me). 31P-NMR (162 MHz, D2O) dP 3.86 ppm (t, (14.2 mg, 0.12 mmol) and urea (74 mg, 1.20 mmol)
J ¼ 5.9 Hz). 1H-decoupled 31P-NMR (162 MHz, were dissolved in H2O (0.5 ml). The solution was
D2O) dP 3.86 ppm. ES – MS (neg. m/z): 237 (100%, applied evenly to two glass fibre discs (25 mm 
[M –Naþ]2). HRMS (m/z): [M –Naþ]2 calcd for 0.68 mm), the discs were dried at 408C for 2 days
C10H22O4P, 237.1261; found, 237.1263 (figure 2). and then heated at 1008C for 24 h. The discs were
then washed with H2O (10 ml) and the washings
(iii) Diallyl hexyl phosphate were lyophilized. The lyophilisate was then dissolved
Following the procedure described above for the prep- in D2O (3 ml) and adjusted to pD ¼ 9.0 to give com-
aration of diallyl decyl phosphate, hexanol (23 ml, plete solubility. The sample was analysed by 1H- and
31
0.19 mmol) was converted to diallyl hexyl phosphate P-NMR and spiked with authentic standards of
41 mg (83%) as a clear oil. Rf: 0.5 (cyclohexane : hexyl phosphate and decyl phosphate as prepared
EtOAc 4 : 1). 1H-NMR (500 MHz, CDCl3) dH above. Relative integration of methylene, methyl and
5.89 ppm (ddtt, J ¼ 17.2, 11.1, 5.5, 1.0 Hz, 2H, O-methylene protons showed no presence of ethyl
CH ¼ CH2); 5.30 (dq, J ¼ 17.2, 1.5 Hz, 2H, CH ¼ phosphate, and a product ratio of decyl phosphate :
CH2); 5.19 (dq, J ¼ 10.3, 1.3 Hz, 2H, CH ¼ CH2); hexyl phosphate 7 : 3 was observed. 1H-NMR
4.44 – 4.50 (m, J ¼ 8.1, 5.5, 1.3 Hz, 4H, (500 MHz, D2O) dH 3.71 ppm (q, J ¼ 6.6 Hz, 2H,
POCH2CH ¼ CH2); 3.99 (q, J ¼ 6.8 Hz, 2H, POCH2); 1.50 (quin., J ¼ 6.6 Hz, 2H, POCH2CH2),
POCH2CH2CH2); 1.61 (quin., J ¼ 6.9 Hz, 2H, 1.12– 1.27 (m, 12H, (CH2)n); 0.75 (t, J ¼ 6.8 Hz,
POCH2CH2CH2); 1.18 –1.36 (m, 8H, (CH2)); 0.82 3H, Me).31P-NMR (162 MHz, D2O) dP 0.90 ppm
(t, J ¼ 7.1 Hz, 3H, Me). 13C-NMR (101 MHz, (m). 1H-decoupled 31P-NMR (162 MHz, D2O) dP
CDCl3) dC 131.6 ppm (2C, CH ¼ CH2); 117.1 (2C, 0.90 ppm.
CH ¼ CH2); 67.5 (2C, CH ¼ CH2CH2); 67.0 This work was supported by the Engineering and Physical
(POCH2CH2CH2); 30.3 (POCH2CH2CH2); 29.2 Sciences Research Council through the provision of a
(POCH2CH2CH2); 24.1 (CH2CH2CH3); 21.5 studentship to M.W.P. We thank Samuel Bjork,
(CH2CH3); 12.9 (Me). 31P-NMR (162 MHz, Christopher Chan, Dr Colm Duffy and Dr Dougal Ritson
CDCl3) dP –0.70 ppm (h, J ¼ 7.8 Hz). 1H-decoupled for helpful suggestions.
31
P-NMR (162 MHz, CDCl3) dP – 0.70 ppm. ES –
MS (pos. m/z): 285 (100%, [M þ Naþ]þ). HRMS
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