Angewandte
Research Articles
Chemie
available for utilization. These thermodynamic predictions
guided us to experimentally consider the behavior of iron
phosphates and hydroxyapatite as a source of phosphate
under prebiotic conditions and how the presence of cyanide
could impact phosphorylation under our reaction conditions.
As mentioned above, struvite, newberyite, and hydrox-
yapatite are likely phosphate sinks on a prebiotic Earth,[8,12]
and have been shown to be effective phosphorylation sources
when used with a urea-rich solvent. Along with Mg2+ and
Ca2+, Fe2+ was a major component of the prebiotic hydro-
sphere,[36] making it likely that similar minerals would have
been formed with Fe2+ as the major divalent cation. In order
to investigate the formation of ferrous-iron-based minerals
and their impact on prebiotic phosphorylation, we synthe-
sized iron phosphate minerals under anaerobic conditions.
This synthesis was conducted following protocols that had
previously been used to synthesize hydroxyapatite and
struvite, but with the divalent cation (Ca2+ or Mg2+) replaced
with Fe2+. These protocols produced vivianite (Figure S5),
and an amorphous iron phosphate mineral lacking any
incorporated ammonium (Figure S6a,b). This lack of nitro-
gen was confirmed through Nesslerꢀs test and further
supported through modeling, which showed the stability of
iron phosphate and could explain the lack of an iron-based
struvite analogue in nature (Figure S7).
The prebiotic hydrosphere was also significantly different
from that of the modern Earth, free of abundant oxygen and
enriched in reduced chemical species. Small lakes and ponds
of water were likely enriched in urea, formed through
multiple prebiotic reactions,[37–39] along with formamide and
ammonium formate, resulting from the hydrolysis of cyanide
and cyanamide.[40,41] These small bodies of water, through
repeated heating and evaporation, could become enriched in
these dissolved organics, which would oscillate in composi-
tions and concentrations depending on changing levels of
water activity. At some points this could create a viscous,
multicomponent solvent during times of particularly low
water activity.[8,22] Thus, the components of this pond would be
expected to exist in a dynamic equilibrium upon repeated
drying and rewetting with their ratios depending, at any point
in time, on the temperature, water activity, and the recent
history of the pond. The formation of this low-water-activity
solvent would inhibit further loss of some volatile compo-
nents, including ammonium, formate, and formamide, but
might protect some molecules that are labile in high-water-
activity mediums.[8,22,41] The interaction between this urea-
and organic-enriched prebiotic milieu and the minerals from
the local geosphere were likely a key step in the formation of
secondary minerals that have proven to be more favorable for
organic molecule phosphorylation.[8]
of crystallization.[42] Previously, a urea-rich solvent composed
of urea, ammonium formate, and water initially in a 1:2:4
molar ratio (UAFW) has been shown to promote reactions
between mineral phosphate sources and organic substrates,
leading to the generation of organophosphates under dehy-
dration conditions with moderate heating.[8,22] Here, phos-
phorylation of adenosine in the UAFW solvent was tested
under anaerobic, dehydrating conditions, with synthetic
vivianite and iron phosphate as phosphate sources along with
sodium phosphate monobasic, newberyite, and hydroxyapa-
tite.
LC-MS and 31P NMR analysis were used to distinguish
between phosphorylated and non-phosphorylated products
from these reactions, with total phosphorylation being
quantified by LC-MS (Figures S8–S11). The detailed analysis
by 31P NMR spectroscopy shows that all studied phosphate
sources produced a similar composition of phosphorylated
products dominated by 5’-AMP, 2’-AMP and 3’-AMP, 2’,3’-
cAMP, phosphorylated adenosine esterified with additional
phosphates, and carbamoylated adenosine phosphates ex-
pected from urea degradation and formation of isocyanate
under our reaction conditions. In spite of the significant
formation of pyrophosphate during the reaction, no evidence
of formation of pyrophosphate esters (ADP or ATP) was
found. These results also demonstrate that pyrophosphate
could reach significant concentrations at moderate temper-
ature in a prebiotic pond under dehydration and formation of
urea-rich eutectic fluids. All five of our phosphate sources
were found to be effective in producing adenosine phos-
phates, with the ferrous iron phosphate minerals showing
reduced yields when compared to their non-ferrous counter-
parts (Figure 2). In the cyanide-rich environment that could
have led to UAFW formation, it is also likely that small
amounts of cyanide ions would exist in solution. Cyanide
could reach high concentrations in these small pools through
evaporation or complexation with metals such as iron. Metal
complexation is particularly interesting as it would not only
allow for the accumulation of cyanide, but also allow for its
slow release from these organic “minerals” to be utilized for
key prebiotic reactions.[33,43,44] Thus, we decided to investigate
the possibility that the presence of cyanide ions would further
alter the availability of phosphate, perhaps by forming
a complex with the iron of the iron phosphate minerals.
We observed that the addition of NaCN (600 mm, in
excess to the phosphate sources) to the reactions containing
ferrous iron phosphate minerals resulted in a marked im-
provement in phosphorylation (Figure 2). After 9 days, phos-
phorylation yields from the synthetic iron phosphate in-
creased almost twofold when compared to the non-NaCN
reaction, showing 14% more phosphorylated adenosine.
Vivianite showed an even more remarkable change, exhibit-
ing a sixfold increase (15% more phosphorylated adenosine)
in phosphorylation yields when NaCN was added, making
vivianite a significantly better phosphate source than hydrox-
yapatite. Sodium phosphate and newberyite showed lower
phosphorylation yields when NaCN was added, likely because
of the excess sodium present in solution (Figure S12).
Intriguingly, phosphorylation using hydroxyapatite with
When allowed to evolve anaerobically at ambient temper-
ature and in the presence of indirect sunlight, this amorphous
iron phosphate mineral phase was observed to crystallize into
the mixed-iron-valence phosphate phase beraunite
[(Fe2+Fe3+)5(OH)5(PO4)4·4H2O; Figure S6c,d]. The forma-
tion of beraunite and vivianite demonstrates that it is possible
to create mixed-valence-iron minerals on a prebiotic Earth
(without oxygen) through sunlight-driven oxidation in
a Schikkorr-like reaction, producing H2 and OHÀ from waters
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Angew. Chem. Int. Ed. 2019, 58, 2 – 9
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