A Light-Releasable Potentially Prebiotic Nucleotide Activating Agent

Article abstract of DOI:10.1021/jacs.8b05189

Investigations into the chemical origin of life have recently benefitted from a holistic approach in which possible atmospheric, organic, and inorganic systems chemistries are taken into consideration. In this way, we now report that a selective phosphate activating agent, namely methyl isocyanide, could plausibly have been produced from simple prebiotic feedstocks. We show that methyl isocyanide drives the conversion of nucleoside monophosphates to phosphorimidazolides under potentially prebiotic conditions and in excellent yields for the first time. Importantly, this chemistry allows for repeated reactivation cycles, a property long sought in nonenzymatic oligomerization studies. Further, as the isocyanide is released upon irradiation, the possibility of spatially and temporally controlled activation chemistry is thus raised.

Full text of DOI:10.1021/jacs.8b05189

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Communication
A light-releasable potentially prebiotic nucleotide activating agent
Angelica Mariani, David A. Russell, Thomas Javelle, and John D. Sutherland
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b05189 • Publication Date (Web): 02 Jul 2018
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A light-releasable potentially prebiotic nucleotide activating agent
Angelica Mariani‡, David A. Russell‡, Thomas Javelle, John D. Sutherland*
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MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH,
UK.
Supporting Information Placeholder
phosphorimidazolides via interrupted Passerini
chemistry in a four component system.
ABSTRACT: Investigations into the chemical origin of
life have recently benefitted from a holistic approach
in which possible atmospheric, organic, and inor-
ganic systems chemistries are taken into considera-
tion. In this way, we now report that a selective phos-
phate activating agent, namely methyl isocyanide,
could plausibly have been produced from simple
prebiotic feedstocks. We show that methyl isocyanide
drives the conversion of nucleoside monophosphates
to phosphorimidazolides under potentially prebiotic
conditions and in excellent yields for the first time.
Importantly, this chemistry allows for repeated re-ac-
tivation cycles, a property long sought in non-enzy-
matic oligomerization studies. Further, as the isocya-
nide is released upon irradiation, the possibility of
spatially and temporally controlled activation chem-
istry is thus raised.
Incubation of adenosine 5′ -monophosphate
(AMP) with imidazole 2 (Im), acetaldehyde 3 and me-
thyl isocyanide (pH 6) resulted in the formation of
adenosine 5′ -phosphorimidazolide (ImpA) in 54%
yield after only 30 min (Figure 1, Table S1 and Figure
S1a). The reaction could also be performed with other
prebiotically relevant aldehydes, such as formalde-
hyde or glycolaldehyde (41 and 48% yields of ImpA
were obtained after 30 min, respectively, data not
shown). Screening the reaction with acetaldehyde
over a pH range revealed a pH optimum of 6.5 (yield:
71%, Figure 1d and Table S1), presumably reflective of
the simultaneous requirements of having imidazole
as its free base (pKa of imidazolium ≈ 7.0), dianionic
AMP (pKa of AMP monoanion ≈ 6.5), and sufficient
acid to protonate acetaldehyde.12 Mechanistically, we
speculate that the transient imidoyl phosphate 4 (un-
detectable by NMR spectroscopy), generated from the
reaction of AMP with the nitrilium ion 5, is attacked
by imidazole at phosphorus, with the consequent for-
mation of the phosphorimidazolide (Figure 1a).
From the pioneering works of Orgel1,2 and Ferris,2,3
through to the most recent breakthroughts of Szostak
and co-workers,4,5 nucleoside 5′ -phosphorimidaz-
olides have been used as long-lived activated nucleo-
tides for the non-enzymatic oligomerization and tem-
plated copying of RNA. Yet, a prebiotically plausible
synthesis of such species remains elusive.6,7 Isocya-
nides are known phosphate activating agents8 and we
have previously described how they could have been
involved in a fleeting prebiotic activation of nucleo-
side monophosphates by Passerini-type chemistry.9
This chemistry requires both isocyanides and alde-
hydes, and while the latter species arise from the pho-
toredox pathways that lead from HCN to amino acids,
lipids, and ribonucleotides10,11 a prebiotic pathway to
the former remains to be found. Herein, following
along systems chemistry lines, we describe a poten-
tially prebiotic synthesis of methyl isocyanide 1 and
demonstrate its use in the in situ formation of 5′ -
Following its initial formation, and in the absence
of a primer-template complex (when polymerization
can be expected),4,5 ImpA is progressively hydrolyzed
to AMP and thence formation of AMP pyrophosphate
(AppA, Figure 2a,b). It is interesting to note that alt-
hough these outcomes are reflective of the known re-
activity of ImpA under these conditions, a connection
between 5′ ,5′ -pyrophosphate ribodinucleotides and
modern cofactors has been previously suggested.13,14
The other canonical mononucleotides, GMP, CMP,
and UMP, displayed analogous behaviors, with ImpN
yields ranging from 69 to 75% (Table S1 and Figure
S2). Importantly, no modifications occurred to any of
the nucleobases, revealing isocyanides to be selective
phosphate activating agents.9,15 The requirement for
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Mg2+ in ribonucleotide polymerization prompted us
to investigate the effect of this additive on the synthe-
sis of ImpA. Mg2+ catalysis, as expected, enhanced the
rate of both ImpA hydrolysis and AppA production
(Figure 2b), but did not affect the initial yield of im-
idazolide (Table S1).
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Figure 1. Synthesis of ImpA and spiking experiments. a, Suggested mechanism for the methyl isocyanide-
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mediated synthesis of ImpA. H (left, magnification showing the H-C(1′ ) region) and P (right) NMR spectra
confirming the formation of b, ImpA (100 mM AMP, 400 mM 1, 100 mM 2, 400 mM 3, pH 6.5, 3.5 h) and c, AppA
(100 mM AMP, 400 mM 1, 100 mM 2, 400 mM 3, 20 mM Mg2+, pH 6.5, 72 h; 5-fold dilution before spiking), by
spiking with authentic samples (top). d, Plot of the maximum % yield of ImpA vs. pH of the reaction. (Green:
AMP, blue: ImpA, orange: AppA).
A desirable feature in prebiotic nucleotide activa-
tion chemistry is the possibility of repeatedly acti-
vating the spent monomers that derive from imid-
azolide hydrolysis to allow for further rounds of
polymerization.15 Although it was beyond the scope
of this study to investigate polymerization, we
sought to establish repeated activation chemistry.
Importantly, adding methyl isocyanide in aliquots,
followed by intervals in which hydrolysis and pyro-
phosphate formation served as a proxy for polymer-
ization in a more complex system, resulted in cycles
of AMP activation, in which every fresh portion of
the activating agent triggered the regeneration of
ImpA with comparable efficiency (Figure 2c).
the presence of 2-aminoimidazole 6 resulted in its
conversion to the corresponding 2NH2ImpA at an
optimum pH of 7 (Table S1 and Figures S1b and S3).
Further optimization resulted in improvements to
the production of both ImpA and 2NH2ImpA, with
89 and 76% yields obtained, respectively (Table S1).
For this selective phosphate activation chemistry
to have occurred on the primordial Earth, a prebiotic
syn-
Recently, Szostak and co-workers4,16 have reported
the superiority of nucleoside 5′ -phosphoro-2-ami-
noimidazolides (2NH2ImpN) in the non-enzymatic
copying of oligoribonucleotides, as a result of more
efficient formation of a transient imidazolium
bridged dinucleotide. In our system, activation of
AMP with methyl isocyanide and acetaldehyde in
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CO2 + N2 + H2O
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lightning+
meteorites
impact
CO + CN + NO
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HCN
x6
dissolution
FeII
+
6H
7
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-
-
-
N2
[Fe(CN)6]4
7
NO2
NO3
hn
, 2H+
-
[Fe(CN)6]4
7
-
-
CN
CN
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+ H2O
low pH
-
[Fe(CN)5N2]3
11
-
[Fe(CN)5NO]2
8
-
[Fe(CN)5CNCH3]3
-
H2PO2
12
CH3NH2
9
HCN
sponge Ni
-
CN
-
[Fe(CN)6]4
hn
-
[Fe(CN)5N2CH3]2
H2O
7
CH3NC
1
10
Figure 2. Synthesis and recycling of ImpA. a,
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31P NMR spectra showing ImpA formation and prod-
ucts thereof (100 mM AMP, pH 6.5; reaction times as
labeled on each spectrum). b, Plot of % ImpA and
AppA (from a) vs. time and effect of Mg2+. c, Plot of
% ImpA and AppA vs. time for the (re)cycling of
(spent) AMP, by iterative cycles of activation.
(Green: AMP, blue: ImpA, orange: AppA).
Figure 3. Schematic representation of the sys-
tems chemistry network producing methyl iso-
cyanide (1).21 High-energy atmospheric chemistry
generates HCN (via CN.), CO and NO.. NO. under-
goes various disproportionation reactions upon dis-
solution,23 providing NO2 and NO3−. HCN is stored
−
−
in solution as 7, which reacts with NO2 (or NO3−) to
provide 8.37 HCN is also a source of CH3NH2 9, which
is diazotized by 8. Alkylation of 7 by 10 gives 12 and
11. Irradiation of 12 in the presence of CN− releases 1
and returns 7; reaction of 11 with CN− returns 7, re-
leasing N2.
thesis of methyl isocyanide would have been re-
quired. We thus considered its possible formation as
a result of metallo-organic chemistry.10,17,18 Iron, as
the most abundant transition metal on Earth, prob-
ably played a fundamental role in the emergence of
life’s building blocks.19 Upon exposure to a primor-
dial atmosphere containing HCN, CO, and NO., iron
would have formed stable homoleptic complexes
such as [Fe(CN)6]4− (ferrocyanide 7, Figure 3),20 but
also mixed ligand complexes with ligands isoelec-
tronic with cyanide of the form [Fe(CN)5L]n−, where
L = CO (n = 3) or NO (n = 2).19 Continuing a study
initiated by Beck,19,21 we investigated the chemistry
of the nitrosyl complex [Fe(CN)5NO]2− (nitroprus-
side, 8), known, inter alia, to mediate the diazotiza-
tion of amines.21,22 To determine whether nitroprus-
side could plausibly have formed under prebiotic
conditions on Earth, we considered its possible for-
mation from ferrocyanide and the nitrogen oxides
Combining literature reports that nitroprusside24
can be formed from NO2 and [Fe(CN)5H2O]3−, and
−
that the latter can be produced by photoaquation of
ferrocyanide,25-27 we found that irradiating a mixture
−
of ferrocyanide and NO2 in the pH range 7-9.8 with
365 nm light afforded nitroprusside as the only new
FeII complex detectable by C NMR spectroscopy
13
(Figure S4a). Importantly, irradiation of the same
mixture with 254 nm light also afforded nitroprus-
side, albeit less efficiently, suggesting that broad
band irradiation from the young sun could have sup-
ported the formation of this complex.
We next investigated the reactions of nitroprus-
side with methylamine 9, which would have been
delivered to the primordial Earth in comets.28 Alter-
natively, hydrogenation of HCN to methylamine
would have been expected to occur in a geochemical
scenario in which iron-nickel meteorites containing
schreibersite (Fe,Ni)3P underwent corrosion in
HCN-containing pools. In an anoxic environment,
corrosion of schreibersite has been shown to give
−
NO., NO2 (nitrite), and NO3− (nitrate). Lightning
and meteorite impacts in the N2-rich primordial at-
mosphere would have produced NO. (nitric oxide,
Figure 3), which could have been directly absorbed
into ferrocyanide containing pools, producing nitro-
−
prusside in situ. Alternatively, NO2 and NO3− could
have accumulated in solution following dispropor-
tionation of NO..23
soluble FeII, H2PO2 (hypophosphite), HPO3
−
2−
(phosphite) and HPO42− (phosphate), while the bulk
iron-nickel alloy matrix gives insoluble porous
nickel (sponge nickel) or nickel-enriched alloy.29,30
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As hypophosphite is known to decompose to H2 and
[Fe(CN)4(CNCH3)2]2− (signals unassigned, 9%). Fur-
ther irradiation did not improve the yield of methyl
isocyanide, which we attribute to a photostationary
equilibrium having been reached.
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4
5
6
7
8
phosphite in the presence of Raney Nickel,31 we have
recently investigated the hypophosphite-sponge
Nickel combination as a plausible prebiotic hydro-
genation system in keeping with the general geo-
chemical scenario described above. Accordingly, we
found that reduction of HCN with hypophosphite
and sponge nickel afforded methylamine as the sole
organic product, identified by 1H and 13C NMR spec-
troscopy (Figure S4b,c).
Next, we sought to link the diazotization chemis-
try and the photolysis of complex 12, in order to
demonstrate that methyl isocyanide could be syn-
thesized in a plausible geochemical setting. Accord-
ingly, a mixture of methylamine, ferrocyanide and
9
2−
nitroprusside, in the presence of CN− and HPO4
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The reaction of methylamine with nitroprusside
(pH 9.8), was allowed to react for 20 h. The mixture
of products obtained was then irradiated for 2 h in
2−
in the presence of CN− and HPO4 (pH 9.8) pro-
1
ceeded with slow evolution of a gas, presumably N2,
the presence of excess CN−. Analysis by H NMR
indicative of diazotisation chemistry. Analysis of the
spectroscopy showed the expected mixture of prod-
ucts, including methyl isocyanide (Figure 4d).
1
31
reaction mixture by H and P NMR spectroscopy
confirmed the presence of products expected from
the trapping of [Fe(CN)5N2CH3]2− 10 by the nucleo-
philes H2O, CN−, and HPO42−, namely CH3OH
(methanol), CH3CN (acetonitrile), and CH3OPO32−
(methyl phosphate, Figure 4a,b and Figure S5a), re-
spectively, together with an initially unidentified
species. Reasoning that ferrocyanide is also pro-
duced under the above reaction conditions (by reac-
tion of cyanide with [Fe(CN)5N2]3− 11, Fig. 3, or
[Fe(CN)5H2O]3−), and realizing that it could act as a
nucleophile in its own right, we tentatively assigned
the species as the isocyanide complex
[Fe(CN)5CNCH3]3− 12. This assignment was
strengthened by repeating the above reaction with
ferrocyanide (1 equivalent) present from the outset,
which led to an increase in the intensity of the new
signal, and unambiguously confirmed by compari-
Interestingly, the by-product of the imidazolide
synthesis described above is 2-hydroxy-N-
methylpropanamide 13, hydrolysis of which would
regenerate methylamine and thus feed back into a
new cycle for the production of fresh methyl isocya-
nide. The other product of the hydrolysis would be
lactate, a major player in extant and maybe early me-
tabolism.
Overall these findings depict a common plausible
scenario in which HCN is central not only to the syn-
thesis of protein, lipid and RNA building blocks, but
also drives chemical pathways that ultimately lead
to nucleotide activation. In this scenario, methyl iso-
cyanide could have been produced in a ferrocyanide
and nitroprusside containing environment upon de-
livery of methylamine. Pools containing different
accumulated materials (possibly at different pHs),
could have occasionally been linked by streams,34 al-
lowing the methyl isocyanide and nucleotide pro-
ducing subsystems to mix, thereby enabling nucleo-
tide activation and polymerization chemistry. The
lack of high-yielding and prebiotically plausible
phosphate activating agents has been a central prob-
lem in origin of life research for nearly 60 years,
prompting the use of pre-activated nucleotide sub-
strates,4,7 synthetic surrogates of ineffective prebi-
otic reactants,6,35 or prebiotically questionable syn-
theses of desirable activating agents.36 Here, for the
first time, we describe a prebiotically plausible syn-
thesis of methyl isocyanide, a storable and light-re-
leasable activating agent, and demonstrate its use in
the efficient in situ activation of nucleotide mono-
phosphates.
1
son with the H NMR spectrum of an authentic
standard prepared by addition of methyl isocyanide
to a solution of [Fe(CN)5H2O]3− (Figure 4c,e and Fig-
ure S6b-d). Although initially concerned by the pH
discontinuity between the activation chemistry and
methyl isocyanide synthesis, we found that complex
12 could be generated in a pH range between 7-9.8,
as the diazotization chemistry still proceeds, albeit
more slowly, at neutral pH.
Mixed ligand isocyanide complexes are known to
undergo isocyanide ligand exchange in coordinating
organic solvents upon irradiation at 365 nm.32 Irra-
diation of an aqueous solution of complex 12 at this
wavelength, in the presence of excess CN− provided
free methyl isocyanide (yield: 50% after 2 h, Figure
S6), clearly identified by its characteristic 1:1:1 triplet
1
at δ 3.16 in the H NMR spectrum, and ferrocya-
1
nide.33 The remaining materials detectable by H
NMR spectroscopy were residual complex 12 (41%),
and two new complexes, tentatively assigned as cis-
[Fe(CN)4(CNCH3)2]2−
and
trans-
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Angelica Mariani: 0000-0002-2547-4224
1
2
3
4
David A. Russell: 0000-0002-2182-4178
John D. Sutherland: 0000-0001-7099-4731
Author Contributions
5
‡These authors contributed equally.
6
7
Funding Sources
This work was supported by the Medical Research
Council (grant no. MC_UP_A024_1009) and a grant
from the Simons Foundation (grant no. 290362 to
J.D.S.). The authors declare no competing financial in-
terests.
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ACKNOWLEDGMENT
We thank L. Wu and Z. Liu for authentic standards of
ImpA and AppA, and all J.D.S. group members for
fruitful discussions.
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website.
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role of transition-metal complexes in the chemical evolu-
tion. NASA TM-75381, 1979, originally published in Kemiai
Közlemenyek 1978, 50, 223-240.
to aldehydes. J. Chem. Soc. 1962, 3961-3963.
10
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32. Costanzo, L. L.; Giuffrida, S.; De Guidi, G.; Condorelli, G.
Photochemical and thermal behaviour of isocyanide com-
plexes. V*. Photolysis of Fe(CNCH3)4(CN)2 in acetonitrile.
J. Organomet. Chem. 1986, 315, 73-77.
33. The full wavelength dependence of this ligand exchange re-
action has not been studied, but, as expected, the release of
free methyl isocyanide did not occur upon irradiation at
254 nm.
34. Ritson, D. J.; Battilocchio, C.; Ley, S. V.; Sutherland, J. D.
Mimicking the surface and prebiotic chemistry of early
Earth using flow chemistry. Nat. Commun. 2018, 9, 1821.
35. Jauker, M.; Griesser, H; Richert, C. Copying of RNA se-
quences without pre-activation. Angew. Chem. Int. Ed.
2015, 54, 14559-14563.
36. During the preparation of this manuscript, a report on the pur-
portedly prebiotic synthesis of nucleoside 5'- phosphorimidaz-
olides from 1 M sodium cyanide and 1 M sodium hypochlorite
(bleach) was published: Yi, R.; Hongo, Y.; Fahrenbach, A. C.
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gen-15 and carbon-13 NMR study of Roussin salts and esters
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of pentacyanonitrosylferrate(II) with aliphatic amines. In-
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ous solution: hydrated electron formation and aquation. Is-
rael J. Chem. 1970, 8, 691-697.
26. Shirom, M.; Stein, G. Excited state chemistry of the ferro-
cyanide ion in aqueous solution. II. Photoaquation. J.
Chem. Phys. 1971, 55, 3379-3382.
27. Gáspár, V.; Beck, M. T. Kinetics of the photoaquation of
hexacyanoferrate(II) ion. Polyhedron 1983, 2, 387-391.
6
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Page 7 of 11
Journal of the American Chemical Society
SYNOPSIS TOC
1
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17
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7
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N
a
b
N
Journal of the American Chemical Society
Page 8 of 11
N
N
2
H
O
O
HO
AMP
O
P
OH
N
O
O
P
O
P
O
O
H+
H2O
N
N
O
O
N
O
N
+
O
O
NH2
O
NH2
O
N
NH2
N
N
N
3
1
5
1
2
3
O
N
N
N
N
N
N
HO
OH
HO
OH
HO
OH
AMP
N
H
ImpA
4
13
OH
c
+ 4ImpA
5
6
7
8
1H
31P
1H
31P
+ ImpA
+ AppA
+ AppA
NH2
H2N
N
N
N
N
N
N
N
N
O
O
O O
OH
HO
O
O
P
P
O
O
OH
O
HO
9
AppA
10
11
12
13
14
15
16
d 100
ImpA
AppA
50
ACS Paragon Plus Environment
0
pH
17
6.1
6.0
5.9
5
0
-5
-10
6.1
6.0
5
0
-5
-10 -15
δ
/
ppm
δ/ppm
δ/ppm
δ/ppm
18

100
a
b
ImpA
Page 9Jooufr1n1al of the American Chemical SocietAyppA
72h
ImpA (+Mg2+
AppA (+Mg2+
)
)
15h
50
1
2
5h
3
4
0
2.55h
6
Time (h) 20
40
60
80
100
c
1.75h
8
0.5h
9
50
10
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11
12
10
5
0
-5
-10 -15
δ/ppm
0
Time (h) 20
40
60
80
13

Journal of the American Chemical Society
Page 10 of 11
CO2 + N2 + H2O
lightning+
meteorites
impact
1
2
3
4
CO + CN + NO
5
6
7
8
HCN
x6
dissolution
FeII
+
9
6H
10
11
12
13
14
15
16
17
18
-
-
-
N2
[Fe(CN)6]4
7
NO2
h , 2H+
NO3
-
[Fe(CN)6]4
7
-
-
CN
CN
+ H2O
low pH
-
[Fe(CN)5N2]3
11
-
[Fe(CN)5NO]2
8
19
-
[Fe(CN)5CNCH3]3
-
20
21
H2PO2
12
CH3NH2
HCN
sponge Ni
22
-
9
CN
23
24
-
[Fe(CN)6]4
h
ACS Paragon Plus Environment
-
[Fe(CN)5N2CH3]2
H2O
7
25 CH3NC
26
10
1

[Fe(CN)6]4- (7)
a
CN
2-
NC
CN
[Fe(CN) NO]2- (8)
CN-
Page 1J1ooufr1n1al of the American Chemical Society
5
CH3NH2
CH3NC + [Fe(CN)6]4-
Fe
NC
12
CN
CNCH3
2-
CN-, HPO4
h
9
1
7
2-
+ CH3OH, CH3CN, CH3OPO3
1
b
2
3
4
CH3OH
CH3CN
2-
CH3OPO3
5
6
7
8
c
CH3OH
9
10
11
CH3OPO3
CH3CN
2-
12
13
14
d
CH3OH
15
16
CH3CN
2-
CH3OPO3
17
18
19
20
e
21
22
23
24
25
26
27
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28
3.25
3.00
2.75
2.50
2.25
δ2/9ppm