Common and Potentially Prebiotic Origin for Precursors of Nucleotide Synthesis and Activation

Article abstract of DOI:10.1021/jacs.7b01562

We have recently shown that 2-aminoimidazole is a superior nucleotide activating group for nonenzymatic RNA copying. Here we describe a prebiotic synthesis of 2-aminoimidazole that shares a common mechanistic pathway with that of 2-aminooxazole, a previously described key intermediate in prebiotic nucleotide synthesis. In the presence of glycolaldehyde, cyanamide, phosphate and ammonium ion, both 2-aminoimidazole and 2-aminooxazole are produced, with higher concentrations of ammonium ion and acidic pH favoring the former. Given a 1:1 mixture of 2-aminoimidazole and 2-aminooxazole, glyceraldehyde preferentially reacts and cyclizes with the latter, forming a mixture of pentose aminooxazolines, and leaving free 2-aminoimidazole available for nucleotide activation. The common synthetic origin of 2-aminoimidazole and 2-aminooxazole and their distinct reactivities are suggestive of a reaction network that could lead to both the synthesis of RNA monomers and to their subsequent chemical activation.

Full text of DOI:10.1021/jacs.7b01562

Communication
pubs.acs.org/JACS
Common and Potentially Prebiotic Origin for Precursors of
Nucleotide Synthesis and Activation
Albert C. Fahrenbach,†,§,∇ Constantin Giurgiu,†,‡,∇ Chun Pong Tam,†,‡ Li Li,† Yayoi Hongo,§
Masashi Aono,§,⊥ and Jack W. Szostak*,†,‡,§
†Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology,
Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States
‡Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United
States
§Earth-Life Science Institute, Tokyo Institute of Technology, 2-12-1-IE-1 Ookayama, Meguro-ku, Tokyo, 152-8550, Japan
⊥Faculty of Environment and Information Studies, Keio University, 5322 Endo, Fujisawa, Kanagawa 252-0882, Japan
S
* Supporting Information
Scheme 1. Common Prebiotic Synthetic Pathway for 2-
ABSTRACT: We have recently shown that 2-amino-
imidazole is a superior nucleotide activating group for
nonenzymatic RNA copying. Here we describe a prebiotic
synthesis of 2-aminoimidazole that shares a common
mechanistic pathway with that of 2-aminooxazole, a
previously described key intermediate in prebiotic
nucleotide synthesis. In the presence of glycolaldehyde,
cyanamide, phosphate and ammonium ion, both 2-
aminoimidazole and 2-aminooxazole are produced, with
higher concentrations of ammonium ion and acidic pH
favoring the former. Given a 1:1 mixture of 2-amino-
imidazole and 2-aminooxazole, glyceraldehyde preferen-
tially reacts and cyclizes with the latter, forming a mixture
of pentose aminooxazolines, and leaving free 2-amino-
imidazole available for nucleotide activation. The common
synthetic origin of 2-aminoimidazole and 2-aminooxazole
and their distinct reactivities are suggestive of a reaction
network that could lead to both the synthesis of RNA
monomers and to their subsequent chemical activation.
Aminooxazole (2NH2Ox) and 2-Aminoimidazole
a
(2NH2Im)
a
Pi: inorganic phosphate.
Other than prebiotic chemistry, compounds containing
imidazole motifs, including 2-aminoimidazole and 2-thioimida-
zole, are known for their medicinal properties.15
We became interested in finding a prebiotic synthesis of 2-
aminoimidazole (2NH2Im), because we recently demonstra-
ted16 that ribonucleoside 5′-monophosphates activated with
2NH2Im offer a 10−100-fold enhancement in the rate of
nonenzymatic template-directed RNA primer extension
compared to those activated with 2-methylimidazole, i.e., in
this context 2NH2Im is the most effective leaving group known
to date. Furthermore, the conjugate base of 2NH2Im is isosteric
and isoelectronic with 2-aminooxazole17 (2NH2Ox) − a key
intermediate in the prebiotic synthesis of cytidine and uridine
2′,3′-cyclic phosphates, as reported by Sutherland and co-
workers.18 The prebiotic synthesis18 of 2NH2Ox is known, and
begins with the addition of glycolaldehyde and cyanamide,
followed by a series of steps that are facilitated by inorganic
phosphate acting as both a pH buffer and general-base catalyst.
In this pathway, cyanamide first undergoes addition to the
carbonyl of glycolaldehyde. Next, an intramolecular general-
base-catalyzed attack of the glycolaldehyde-derived hydroxyl on
the cyanamide-derived nitrile carbon leads to a five-membered
ring. General-base catalysis by phosphate also likely accelerates
C−H deprotonation in the following dehydration step leading
to the aromatic 2NH2Ox.
midazoles are thought to have played1 important roles in
Iprebiotic chemistry prior to their current biological
significance as components of the histidine residues in proteins,
where they function as, for example, charge-relay agents in
catalytic triads.2 One of the most important potential roles of
imidazoles in prebiotic chemistry came to light through the
efforts of Orgel and co-workers,3,4 who demonstrated that
nucleoside 5′-phosphoro-imidazolides, and especially nucleo-
tides activated with 2-methylimidazole, allow for nonenzymatic
template-directed5,6 RNA synthesis yielding predominantly the
canonical 3′-5′ phosphodiester linkage. These phosphoro-
imidazolides are more reactive than nucleoside triphosphates
as a result of their labile P−N bonds7 and their propensity to
form a reactive imidazolium-bridged8 dinucleotide. In addition
to prebiotic activation chemistry, imidazoles have also been
suggested to play catalytic roles,9 assisting in the oligomeriza-
tion of amino acids,10 phosphates11,12 and nucleotides,13,14 and
a number of plausible mechanisms for the prebiotic synthesis of
imidazoles have been reported1,9 (further discussion in SI).
Received: February 14, 2017
© XXXX American Chemical Society
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1
other major products could be observed in the H NMR
spectrum.
Understanding how the ratio of 2NH2Im to 2NH2Ox varies
under different pH regimes and ammonium ion concentrations
is important for evaluating the reaction in the context of
potential geochemical scenarios. We systematically examined
the reaction at pH 4, 5.5, 7 and 8.5 with NH4Cl concentrations
of 0, 0.5, 1, 2, 3, 4 and 5 M. All reactions were monitored by 1H
NMR after 1, 2 and 3 h, and the ratios of 2NH2Im to 2NH2Ox
were measured by resonance integration (Table S1). The ratios
after 3 h are plotted in Figure 2. For all pH values, the ratio of
2NH2Im to 2NH2Ox increases with increasing NH4Cl, whereas
mildly acidic pH also tends to increase this ratio. At least part of
this effect of pH can be explained by the fact that increasing
acidity does not tend to favor dehydration of the 4-hydroxy
intermediate of 2NH2Ox, a minor species that can be detected
under the acidic conditions tested. A maximum ratio of
[2NH2Im]/[2NH2Ox] = 56 was obtained at a pH value of 5.5
with 5 M NH4Cl after 3 h. Analysis by Q-TOF LC-MS
confirmed these results (Table S2, Figures S3 and S4 for further
details). Similar yields for both 2NH2Ox and 2NH2Im were
obtained when either 100 or 50 mM concentrations of the
starting materials were used. We carried out a preparative scale
reaction for the synthesis of 2NH2Im at a pH of ∼5.3
employing 1 M NH4H2PO4 and 5 M NH4HCO2 and obtained
a 41% isolated yield (Scheme S2). Given the general
mechanistic features of the above synthetic reactions, we
anticipated the prebiotic formation of imidazoles may be rather
general. We set out to make 2-thioimidazole (Figure S5).
Heating a 1 M solution of NH4SCN and glycolaldehyde at pH
4 in 4 M NH4Cl at 60 °C for 24 h led to the formation of 2-
Figure 1. (a) Possible mechanism for the synthesis of 2NH2Ox and
2NH2Im. The initial formation of α-aminoacetaldehyde is thought to
be a potential intermediate on the path to 2NH2Im, although this
exchange reaction may also occur after addition of cyanamide. (b)
1
Partial H NMR spectrum (400 MHz, D2O) showing the H4/H5
aromatic resonances for 2NH2Ox (red) and 2NH2Im (blue) after
reaction of glycolaldehyde, cyanamide, sodium phosphate and
ammonium chloride, all at 1 M, for 3 h at pH 7 and 60 °C.
Given their structural similarities, we wondered whether
2NH2Im and 2NH2Ox could share a common prebiotic
synthetic pathway (Scheme 1). Previously reported (nonpre-
biotic) syntheses of 2NH2Im make use of the dimethyl- or
diethyl-acetal of α-aminoacetaldehyde,19−21 which presumably
stabilizes the amino group by preventing enolization and
subsequent exchange with water. We reasoned that in order to
synthesize 2NH2Im, we would need to find a plausible route for
the synthesis of α-aminoacetaldehyde. We hypothesized simply
adding a sufficiently high concentration of an ammonium salt to
the mixture of glycolaldehyde, cyanamide and inorganic
phosphate, would lead to the formation of at least some of
this intermediate at equilibrium through an Amadori-type enol-
mediated exchange mechanism (Figure 1a, Scheme S1).
Alternatively, exchange of the glycolaldehyde-derived hydroxyl
to an amine could take place after the addition of cyanamide to
glycolaldehyde.
1
thioimidazole with no other major products observable by H
NMR, although the yield was relatively low at 6.2%.
Knowing that 2NH2Ox and 2NH2Im can be made in the
same reaction flask in approximately equimolar ratios at pH 7
and ∼1 M NH4Cl, we asked whether these two compounds
would display sufficiently different reactivities such that
2NH2Ox could be channeled toward nucleotide synthesis,
whereas 2NH2Im would be preserved for later nucleoside 5′-
phosphate activation. The next step on the pathway from
To test this hypothesis, we carried out reactions with
aqueous solutions of cyanamide and glycolaldehyde in the
presence of 1 M phosphate at varying concentrations of NH4Cl
at pH 7, 60 °C for 3 h. In the absence of NH4Cl, analysis of the
reaction mixture by 1H NMR spectroscopy revealed that
2NH2Ox is produced nearly exclusively (Figures S1 and S2), as
expected based on previous reports by Sutherland et al.17,18
The addition of 1 M NH4Cl (Figure 1b) resulted in the
appearance of another resonance in the aromatic region of the
proton NMR spectrum. Addition of an authentic standard of
2NH2Im confirmed that this resonance arises from the H4 and
H5 protons of 2NH2Im. Increasing the concentration of
NH4Cl to 5 M resulted in the almost exclusive formation of
2NH2Im. Quantification of the yield was carried out using a
calibrated solution of 5′-cytidine monophosphate (CMP),
which was added directly to the NMR tube; we used its H5
and H6 resonances as standards for integration. After 1 h, the
reaction was complete, and the yield was determined to be
15%. Although this yield is not as high as that previously
reported18 for 2NH2Ox (>80%), it is important to note that no
Figure 2. Bar graph displaying the ratios of 2NH2Im to 2NH2Ox at
varying pH and NH4Cl concentrations. All ratios were determined by
1H NMR spectroscopy from reactions that were carried out at 60 °C
with 1 M sodium phosphate, and monitored over a period of 3 h.
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Figure 3. Selective cyclization of rac-glyceraldehyde with 2NH2Ox in the presence of 2NH2Im. The reaction was carried out at 1 M of each
component and was monitored by high-resolution (Q-TOF) LC-MS using a C18 column. All traces are extracted ion chromatograms for m/z values
that correspond to the [M + H]+ ions for 2NH2Ox (red), 2NH2Im (blue) and the mixture of aminooxazoline stereoisomers (green). All
chromatograms were extracted with a tolerance 0.1 Da. The 24 h chromatograms have been displayed offset by ∼10 s for clarity.
2NH2Ox to the cytidine and uridine cyclic phosphates involves
a cycloaddition reaction with glyceraldehyde, which generates a
mixture of ribo- and arabino-furanosyl aminooxazolines18 as the
major products. For 2NH2Im to be preserved for subsequent
nucleotide activation, it would have to be significantly less
reactive with glyceraldehyde than 2NH2Ox. To address this
question, we reacted a 1:1 mixture of 2NH2Ox and 2NH2Im
with glyceraldehyde at 40 °C in the presence of inorganic
phosphate for 24 h while monitoring the reaction mixture by
high-resolution LC-MS (Figure 3). The mixture of pentose
aminooxazolines was detected in the reaction even after only
∼10 min of reaction time, and their concentration reached a
maximum after approximately 3 h (Figure S6). After 24 h,
nearly all the 2NH2Ox had been depleted from the reaction
mixture, whereas ∼80% of the 2NH2Im remained. Analysis by
1H NMR spectroscopy confirmed this result (Figure S7). We
also detected lesser amounts of a product with an m/z of
174.09, a value that is consistent with the [M + H]+ mass of the
analogous product but cyclized with 2NH2Im. Although it
would appear that the 2NH2Im can also react in some fashion
with glyceraldehyde, based on the initial rates measured from
control experiments reacting 2NH2Im and 2NH2Ox with
glyceraldehyde individually in separate solutions, we estimate
that the reaction with 2NH2Ox is about 1 order of magnitude
faster. Indeed, the reaction of 2NH2Im with glyceraldehyde is
sufficiently slow such that the reaction stops after the
consumption of only ∼20% of 2NH2Im. The likely reason
for this effect is that the isomerization of glyceraldehyde to
dihydroxyacetone is a competing process occurring at a similar
rate, preventing the reaction from going to a completion. We
suspect that the slower reaction kinetics of 2NH2Im compared
to 2NH2Ox are at least in part a result of the greater aromatic
stability of 2NH2Im, the greater nucleophilicity of 2NH2Ox, or
both. We also examined the case of a “one-pot” reaction, in
which a solution of 1 M cyanamide, glycolaldehyde,
glyceraldehyde, ammonium chloride and sodium phosphate at
pH 7 was heated to 40 °C and monitored by LC-MS and NMR
over time (Figures S8 and S9). These results provide additional
evidence that the greater stability of 2NH2Im allows it to
persist and potentially accumulate for later activation chemistry.
Finally, we demonstrate that N-cyano-2-aminoimidazole
(2NH2ImCN) is capable of activating CMP to furnish (Scheme
2) cytidine-5′-phosphoro-(2-aminoimidazole) (2NH2ImpC).
Several reports in the literature suggest that N-cyanoimidazole
can serve22,23 as an activating agent for the formation of
phosphodiester bonds. 2NH2ImCN appeared attractive as a
potentially prebiotic activating agent, because it represents one
of the simplest possible extensions of the chemistry reported
here, i.e., oxidative coupling with hydrogen cyanide. We formed
2NH2ImCN by reacting24 2NH2Im with cyanogen bromide in
acetone at room temperature for 15 min (Figure S10). The
reaction was concentrated to near dryness, at which point an
aqueous solution of CMP with 10% D2O was added and the
pH adjusted between 5.5 and 6. After about 10 min at room
temperature, 31P NMR spectroscopic analysis revealed ∼20%
conversion to 2NH2ImpC (Figure S11 and S12). A maximum
of about 40% conversion was obtained after 1.6 h. By addition
of another freshly prepared batch of 2NH2ImCN, a final
conversion of ∼75% was achieved. While the prebiotic
synthesis of 2NH2ImCN has not yet been achieved, this
synthesis of 2NH2ImpC serves as a proof of concept,
highlighting the potential usefulness of N-cyanoimidazoles as
prebiotic activating agents.
In summary, we have demonstrated a prebiotically plausible
synthetic pathway for 2-aminoimidazole that shares a common
origin with the synthesis of 2-aminooxazole. Recently, Powner
et al. showed 2-aminothiazole can be efficiently synthesized in a
Scheme 2. Synthesis of Cytidine-5′-phosphoro-(2-
aminoimidazole) Making Use of N-cyano-2-aminoimidazole
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similar fashion as 2NH2Ox and 2NH2Im, starting from
cyanamide and β-mercaptoacetaldehyde.25 2-Aminothiazole
forms stable crystalline aminals with aldehydes, but not with
ketones, allowing for the concomitant accumulation and
purification of reactive aldehydes, and the chemical selection
of proteinogenic amino acids. Remarkably, all three compounds
in the series 2-amino- oxazole/imidazole/thiazole seem to have
important potential prebiotic roles. In the shared pathway for
2NH2Ox and 2NH2Im production, the relative yield of each
species depends on the pH and ammonium chloride
concentration. At neutral pH, this proposed pathway for the
prebiotic synthesis of 2NH2Im requires high concentrations of
aqueous ammonia, on the order of 1 M to generate comparable
amounts of 2NH2Im and 2NH2Ox. There are several scenarios
in which ammonium ions could have been generated in a
concentrated form within an ancient aqueous reservoir. In one
scenario proposed by Sutherland,26 cyanide produced in the
atmosphere rains out and is captured by ferrous ions as
ferrocyanide; salts of ferrocyanide then precipitate and
accumulate over long periods of time. Subsequent thermal
processing of deposits of magnesium ferrocyanide by magma or
impacts would generate magnesium nitride, which upon
moistening would hydrolyze, releasing ammonia. Other path-
ways to ammonia sources have also been suggested (see SI).
Finally, the greater stability/slower reactivity of 2NH2Im in
comparison to 2NH2Ox suggests that the former could
accumulate over time even as the latter is continuously
processed into intermediates on the path to nucleotide
synthesis. Having a mechanism for the simultaneous production
of 2NH2Im and 2NH2Ox suggests the tantalizing prospect of
an ancient prebiotic reaction network that could have led to
both nucleotide synthesis and to the subsequent chemical
activation of those nucleotides in a manner suitable for efficient
nonenzymatic template-directed replication. Although we have
shown that N-cyano-2-aminoimidazole can serve as an
activating agent, one of the great challenges ahead is
understanding how such an activating agent, or another
mechanism altogether for the prebiotic activation of nucleoside
5′-monophosphates, could have arisen from such a network.
Simons Foundation to J.W.S. (290363) and from the NSF
(CHE-1607034) to J.W.S. A.C.F. is supported by a Research
Fellowship from the Earth-Life Science Institute at the Tokyo
Institute of Technology. L.L. is a Life Sciences Research
Foundation Fellow. Part of this work was supported by JSPS
KAKENHI Grant-in-Aid for Scientific Research on Innovative
Areas “Hadean Bioscience”, Grant Number JP26106003.
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ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b01562.
Experimental details (PDF)
̌
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AUTHOR INFORMATION
Corresponding Author
*szostak@molbio.mgh.harvard.edu
■
ORCID
Albert C. Fahrenbach: 0000-0002-8315-8836
Chun Pong Tam: 0000-0001-6381-9011
Jack W. Szostak: 0000-0003-4131-1203
Author Contributions
∇These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
J.W.S. is an Investigator of the Howard Hughes Medical
Institute. This work was supported in part by grants from the
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DOI: 10.1021/jacs.7b01562
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX