Effects of Pressure and pH on the Hydrolysis of Cytosine: Implications for Nucleotide Stability around Deep-Sea Black Smokers

Article abstract of DOI:10.1002/cbic.201700555

The relatively low chemical stability of cytosine compared with other nucleobases is a key concern in origin-of-life scenarios, but the effect of pressure on the rate of hydrolysis of cytosine to uracil remains unknown. Through in situ NMR spectroscopy measurements, it has been determined that the half-life of cytosine at 373.15 K decreases from (18.0±0.7) days at ambient pressure (0.1 MPa) to (8.64±0.18) days at high pressure (200 MPa). This yields an activation volume for hydrolysis of (?11.8±0.5) cm³ mol^?1; a decrease that is similar to the molar volume of water (18.0 cm³ mol^?1) and consistent with a tetrahedral 3,3-hydroxyamine transition-state/intermediate species. Similar behaviour was also observed for cytidine. At both ambient and high pressures, the half-life of cytosine decreases significantly as the pH decreases from 7.0 to 6.0. These results provide scant support for the notion that RNA-based life forms originated in high-temperature, high-pressure, acidic environments.

Full text of DOI:10.1002/cbic.201700555

Accepted Article
Title: The effects of pressure (0.1 - 200 MPa) and pH on the hydrolysis
of cytosine at 373 K: implications for nucleotide stability around
deep-sea black smokers
Authors: Christopher P Lepper, Martin AK Williams, David Penny,
Patrick JB Edwards, and Geoffrey Brind Jameson
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To be cited as: ChemBioChem 10.1002/cbic.201700555
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The effects of pressure (0.1 – 200 MPa) and pH on the hydrolysis
of cytosine at 373 K: implications for nucleotide stability around
deep-sea black smokers
Christopher P. Lepper,[b] Martin A.K. Williams,[c] David Penny,[b] Patrick J.B. Edwards,[b] and Geoffrey
B. Jameson*[a]
Abstract: The relatively low chemical stability of cytosine compared
to other nucleobases is a key concern in origin-of-life scenarios, but
the effect of pressure on the rate of hydrolysis of cytosine to uracil has
remained unknown. We have determined by in situ NMR
measurements that the half-life of cytosine at 373.15 K decreases
from 18.0 (±0.7) days at ambient pressure (0.1 MPa) to 8.64 (±0.18)
days at high pressure (200 MPa). This yields an activation volume for
hydrolysis of -11.8 (±0.5) cm3 mol-1, a decrease that is similar to the
molar volume of water (18.0 cm3 mol-1) and consistent with a
tetrahedral 3,3-hydroxyamine transition-state/intermediate species.
Very similar behavior was observed also for cytidine. At both ambient
and high pressures, the half-life of cytosine decreases significantly as
pH is dropped from 7.0 to 6.0. These results provide scant support for
the notion that RNA-based life forms originated in high-temperature,
high-pressure, acidic environments.
that, at least at ambient pressure of one atmosphere (0.1 MPa),
folded RNA structures denature at temperatures less than 70 oC
in vitro.[8] With respect to chemical stability, studies at mesophilic
conditions[9] have revealed the susceptibility of cytosine and
cytidine to hydrolytic deamination, both as individual molecules
and when incorporated into ~5000 base-pair bacteriophage
DNA.[10] Subsequently, Levy and Miller[11] found that at 100 oC and
ambient pressure the nucleobase cytosine hydrolysed to uracil
(Scheme 1) with a half-life of just 19 days, compared to 340 years
at 25 oC. This rate of hydrolysis is at least 15 times faster than the
rates of decomposition of the other nucleobases (the half-life of
guanine and adenine are, respectively, 0.8 and 1 yr at 100 °C).
These studies have been augmented very recently by Lewis et
al.,[12] who reported rate constants and an enthalpy of activation
of ~23 kcal mol-1 for deamination of cytosine and various
derivatives at pH 2.4 and 7.0 over the temperature range
~90−~200 oC.
There are many, varied and hotly debated scenarios for the origin
of life on earth. Many scenarios have RNA-based life forms
preceding the extant protein-based life forms[1], or co-evolution of
RNA alongside DNA.[2] However, these scenarios are vigorously
disputed in favour of protein-first FeS-templated origins.[3] There
are acid hot-start[4] versus alkaline warm-start[5] versus cold-
start[6] scenarios with hot-start scenarios popularistically featuring
the energy- and chemically-rich deep-ocean black smokers where
hyperthermophiles from the kingdom Archaea, often taken as the
closest living descendants of the first protein-based life forms,[7]
thrive at temperatures between 80 °C and 110 °C and often at
high pressure (> 50 MPa). A single point of agreement in current
(i.e., since ~2012) origin-of-life scenarios invokes a role for FeS
species in redox reactions. Separately, it has long been known
[a]
Professor Dr Geoffrey B Jameson
Institute of Fundamental Sciences, the MacDiarmid Institute and the
Riddet Institute
Massey University
Palmerston North
New Zealand
E-mail: g.b.jameson@massey.ac.nz
Scheme 1. The hydrolysis of cytosine and cytidine to uracil and uridine,
respectively. The atom labelling scheme is shown for NMR-monitored protons
H5 and H6.
[b]
[c]
Dr Christopher P Lepper, Professor Dr David Penny, Dr Patrick JB
Edwards
Institute of Fundamental Sciences
Massey University
Palmerston North
Assuming that earliest life forms were rather inefficient and
not adapted and stripped down for speedy turnover, in contrast to
many extant Prokarya and Archaea, these results on the
diminished chemical stability of cytosine at elevated temperatures
and the physical instability of folded RNA structures have
rendered unlikely a hot-start scenario for RNA-based life forms. In
this context, it must also be noted that although phosphodiester
bonds have extreme chemical stability, with a half life for
hydrolysis at 25 oC and pH 7 of ~130,000 to 30,000,000 years,[13]
in the presence of strong Lewis-acid species, featuring usually
New Zealand
Professor Dr Martin (Bill) AK Williams
Institute of Fundamental Sciences, the MacDiarmid Institute and the
Riddet Institute
Massey University
Palmerston North
New Zealand
Supporting information for this article is given via a link at the end of
the document.
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dizinc(II), dinickel(II) complexes or lanthanide species, hydrolysis
rates are orders of magnitude higher than those for the
deamination of cytosine.[14] Modern cytidine deaminases,
including the APOBEC3 family of single-stranded DNA mutator
proteins all feature a Zn(his)(cys)2 active site.[15]
MPa. The half- lives for cytidine at 0.10 MPa and 150 MPa and
373 K are (18.2 ± 0.7) days and (9.3 ± 0.4 days), respectively. A
similar small enhancement in decomposition rate at 373 K for
cytidine compared to cytosine was noted by Lewis et al.[12]
The rate constants for hydrolysis of cytosine at 373 K are
plotted in Figure 1b as ln k vs. pressure p. From equation 1
However, these studies on the stability of cytosine have not
explicitly addressed the effect of pressure, and whether or not
high pressure in the absence of strong Lewis-acid metal ions
might counter or exacerbate the deleterious effects of high
temperatures on RNA’s physical and chemical stability. It has
been argued that high pressure may have had an important role
in the development of life on earth,[6, 16], even though the
syntheses of the building blocks of life, nucleotides,
carbohydrates, and amino acids, have recently been reported to
be facilitated by ultraviolet radiation, which is conspicuously
absent at depth.[17] We report here the effects of pressure, and of
pH, on the chemical stability of cytosine and cytidine at 100 oC.
The hydrolysis of cytosine (Scheme 1) was monitored at 373
K and at pressures of 0.10, 50, 100, 150 and 200 MPa (Figure
1a). In Figure S1, the corresponding data for hydrolysis of cytidine
at 373 K and 0.10 and 150 MPa are shown. The process is
pseudo-first order in all cases, as expected from ambient pressure
measurements,[9, 11-12, 18]. All plots of -ln[cytosine] vs. time are
linear over the practically accessible observation times. The NMR
spectra, especially of cytidine, were closely inspected for
evidence of decomposition additional to the deamination to uracil.
No evidence was found by way of peaks that were not assignable
to either uracil (uridine) or cytosine (or cytidine). Moreover, the
summed integrals of the H5 and H6 proton signals for uracil and
cytosine (or cytidine), normalised to that of reference TMSP, were
constant within 4.0% over the time course of the experiment.
Representative NMR spectra at the start and end of the reaction
are shown in Figure S2 at both 0.10 MPa and 200 MPa (see
Supporting Information).
ꢀln ꢁ
∆ꢃ‡
ꢄꢅ
= −
Equation 1
ꢀꢂ
where R is the gas or universal constant and T is the absolute
temperature in K, volume of activation, ΔV‡, was calculated to be:
for cytosine (−11.8 ± 0.5) cm3 mol-1, and for cytidine, −14.6 cm3
mol-1. Given the changing isothermal compressibility of water with
pressure, linearity of this plot was not necessarily to be expected.
A brief examination of the pH dependence of cytosine
hydrolysis was carried out at pressures of 0.1 and 150 MPa
(Figure 2). It is seen in both curves that the rate of hydrolysis is
the slowest in the region of pH 7, and increases significantly,
especially at ambient pressure, on dropping the pH to 6.0. Initial
measurements made at pH 4.8 and 373 K (data not shown)
showed further enhancement of the rate of hydrolysis compared
to that at pH 7.0 and pH 6.0. Rates of hydrolysis at pH 8.0 were
only marginally greater than those at pH 7.0.
Figure 2. (a) Fraction of cytosine remaining, Nf = [cytosine]t/[cytosine]0, plotted
as –ln Nf vs time (t) at 373 K and pH 6.0 (red), 7.0 (green) and 8.0 (blue) at 0.1
MPa (empty shapes and dotted lines) and 150 MPa )filled shapes and solid
lines). The pH is corrected for the effects of pressure and temperature. The rate
constant for hydrolysis of cytosine is the slope of the line. (b) The pH
dependence of the rate constants for the hydrolysis of cytosine at 50 MPa (red)
and 200 M Pa.
The chemical instability of biomolecules has long been
recognised as a weakness in the argument for a hot start origin of
life theory[19]. The relatively quick rate of hydrolysis of cytosine to
uracil at 100 oC and ambient pressure (half-life of 19 days),
compared to geological time scales, has previously been cited, on
the one hand, as a limiting factor in the evolution of life[11] and, on
the other hand, as a driver of evolution.[12]. Moreover, Hazen et al.
posited that the instability of RNA and its components at high
temperatures may be offset by high pressures.[16] Because of
these factors we have examined the effect of pressure and pH on
this hydrolysis, given that the environment around black smokers
is highly acidic, while that around warm haline springs is
moderately alkaline. Moreover, sources of both energy and
molecular building blocks can be found at high hydrostatic
pressures.
Figure 1. (a) Fraction of cytosine remaining, Nf = [cytosine]t/[cytosine]0, plotted
as –ln Nf vs time (t) at 373 K and pH 7.0 for hydrolysis of cytosine at various
pressures (dark blue empty circles 0.1 MPa, blue filled squares 50 MPa, green
filled triangles 100 Pa, orange filled diamonds 150 MPa and red filled circles
200 MPa). The pH is corrected for the effects of pressure. The rate constant for
hydrolysis is the slope of the line. (b) Plot of the rate constant k for the hydrolysis
of cytosine at 373 K vs. pressure p.
The rate constants and associated estimated standard
deviations, derived as the slope of these plots, are tabulated in
Table S1 (Supporting Information), alongside the half-lives, for
hydrolysis of cytosine (or cytidine). The half-life decreases
monotonically as the pressure increases from 0.1 to 200 MPa,
from (19.3 ± 0.7) days at 0.10 MPa to (8.64 ± 0.19) days at 200
The negative activation volumes, ΔV‡, for the hydrolysis of
cytosine and cytidine at 100 °C, respectively, are similar to the
molar volume of a water molecule (18.0 cm3 mol-1) and consistent
with an associative mechanism. A tetrahedral 3,3-hydroxyamine
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intermediate or near-transition-state species had earlier been
deduced from 15N/14N kinetic isotopic effects for the deamination
of cytidine (both uncatalysed and catalysed).[18] Activation
volumes for biochemical processes typically fall within the range
+50 to -50 cm3 mol-1[20] and for the hydrolysis of weakly related
carboxamide species under acid or base catalysis values of -9.4
to -16.9 cm3 mol-1 have been compiled.[21] Our results are
incompatible with the hypothesis that the instability of RNA and its
components at high temperatures may be offset by high
modern life descended from hyperthermophilic survivors of
ocean-boiling asteroid impacts that occurred at the end of the
Hadean epoch ~3.9 Gya.[25]
, although this Late Heavy
Bombardment has been questioned strongly.[28] This leaves open
the question of environment for origin of life. These considerations
and the work presented here do not support a hyperthermophilic,
hyperbaric or acidic environment for an origin of RNA-based life
in the period ~4.2 to 3.9 Gya, at least with cytosine
The possibility has been contemplated for an origin of life that
does not involve cytosine as a nucleobase, where genetic
information is reduced to a two-letter code of just adenine and
uracil, or adenine and inosine, or where the cytosine-guanosine
pair has been substituted for an alternate more stable base pair
(isoguanine and isocytosine, diaminopurine and uracil,
diaminopyrimidine and xanthine [11]). Numerical simulations
indicate that a model containing only adenine and uracil does not
lead to the unique stable folded RNA structures necessary for
catalytic functions [8, 29]. However, this important requirement if
proteins had yet to be used for catalytic function appears to have
been met in a two-nucleotide ribozyme reported by Reader and
Joyce.[30]
pressures in a high-temperature/high-pressure theory[16]
.
The half-life determined at 0.10 MPa and 100 oC in our
measurements of 19.3 ± 0.7 days compares well with that of 19
days determined by Levy and Miller at 0.10 MPa 100 oC for
cytosine at pH 7 under the same conditions (0.05 M phosphate
buffer at an ionic strength of 0.2 M). An average half-life of ~10
days for individual cytosines on a primitive genomic strand of
5000 nucleotides, one quarter of which are cytosine, corresponds
to a spontaneous mutation every couple of hours for each strand.
In the absence of editing and repair mechanism. This is not
conducive to preservation of genomic information in a Model T
Ford of inefficient, slowly reproducing early life forms. At
pressures corresponding to depths in sea water of 3790 m
(current average depth on earth [22]) and 10920 m (maximum
current depth on earth [23]), the half-life of cytosine hydrolysis at
38.2 MPa is calculated to be, respectively, 16.8 days and 12.9
days. Compared to the half-life at 0.1 MPa (atmospheric pressure)
of 19.3 days, the value of the half-life at 3790 m or 38.2 MPa is
only 13% lower. The effects of pressure on the hydrolytic stability
of cytosine in earth’s oceans are significant but modest.
As observed before, the rate of hydrolysis of cytosine at 100°C
is relatively short on the geological time scale. Both cytosine and
cytidine have increasingly faster rates of hydrolysis as pressure is
increased from ambient to 250 MPa. In addition, the rates of
o
hydrolysis of cytosine at 100 C are significantly faster at pH 6
than at pH 7-8, especially at ambient pressure. Our results on the
stability of cytosine point away from a high-temperature/high-
pressure origin-of-life scenario in favour of a low-temperature/low
pressure environment for the emergence of the first RNA-based
life forms.
What still remains unknown is the effect that specific
adjuvants, such as amino acids, short peptides, Mg2+ ions and
molecular crowding may have on the chemical stability of cytosine
and its derivatives while under pressure. There is also the
The pH dependence of the hydrolysis of cytosine is less
marked at 150 MPa than at ambient pressure (Figure 2b). At both
0.1 and 150 MPa, the rate constant for hydrolysis is smallest near
pH 7. At atmospheric pressure this result is to be expected since
the nucleobases are the most stable at pH 7.[9a] It is significant
that this pH dependence has been maintained at high pressure,
provided that pH measured is corrected for the effects of pressure. question of the chemical stability of cytosine within folded
The notably faster rate of hydrolysis at acidic pH, especially
noticeable at 0.1 MPa is not supportive of an acidic environment
for evolution of RNA-based life forms. Our results here are in
apparent conflict with the report by Lewis et al.[12] that the
activation enthalpy remains the same at 23 kcal mol-1 not only
over a wide temperature range but remarkably also at pH 2.4 and
7.0. However, this reported activation enthalpy would strictly
appear to be an internal energy of activation ∆U‡, since
measurements appear to have been made at the essentially
constant volume of sealed quartz tubes. Thus, assuming little
change in the Arrhenius pre-exponential factor over this pH range,
pressure effects on constant volume measurements over a range
of temperature would appear to fortuitously cancel pH effects on
the internal energy of activation.
Both continental crust and oceans were established in
Hadean times (up to ~4.1 bya),[24] and conditions amenable to
chemical synthesis of complex molecules developed remarkably
rapidly,[25], with the average temperature of water being less than
100 oC by 4.2 bya .[26], The sequestration of carbon dioxide from
a dense atmosphere (p(CO2) to ~25 bar for 120 oC and ~30 oC at
1 bar)[27] was also occurring rapidly, leaving a remarkably short
period of just 1-20 My where surface temperatures exceeded 100
oC. Indeed, it is posited that extant hyperthermophiles and all
RNA/DNA molecules under pressure. We will report in due course
on the chemical and physical stability of RNA/DNA molecules.
Experimental Section
High pressure NMR apparatus. A high-pressure NMR cell (zirconia tube,
inner diameter 3 mm, outer diameter 5 mm) was purchased from Daedalus
Innovations and attached to home-designed aluminum manifold,
connected to long stainless steel tube, connected to a remote hand pump.
Pressure is applied to the aqueous sample in the NMR cell via an
immiscible hydraulic fluid (low viscosity paraffin oil). This set up allows the
pressure on the sample to be set at any value between 0.1 and 250 MPa
(measured with HiP Bourdon Gauge). A specially designed rig allowed the
NMR cell to be reproducibly positioned in the spectrometer and to be safely
moved under pressure from an external oil bath at 373 K to the
spectrometer. The cell was interfaced with a Bruker Avance-500 NMR
spectrometer operating at 500.13 MHz for 1H. The sample was positioned
in a commercial Bruker TXI 5 mm 1H-detection inverse probe with a z-field
gradient coil.
Samples. A stock solution of cytosine (or cytidine both from Sigma-
Aldrich), 3-(trimethylsilyl)-2,2',3,3'-tetradeuteropropionicacid (TMSP-d4)
(Merck) and sodium azide was added to a phosphate buffer in H2O/D2O
(90:10 v/v for NMR lock) to give final solution concentrations of 1.00 mM
cytosine, 0.050 M phosphate buffer, 0.020 mM sodium azide (to ensure no
bacterial growth over long time period of measurements) and 0.200 mM
TMSP-d4. The ionic strength of all solutions was adjusted to 0.2 M with
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NaCl. The TMSP-d4 was added to act as both a NMR reference and as an
internal concentration reference. Due to the effect of pressure on pH in a
phosphate buffer, the pH values at atmospheric pressure for the high-
pressure samples were corrected to give the target pH at 298 K while
under pressure[31]. The pH values used are (with values in parentheses at
0.1 MPa): at 0.1 MPa - 6.00, 7.00 and 8.00; at 50 MPa – 7.00 (7.20); at
100 MPa – 7.00 (7.39); at 150 MPa - 6.00 (6.56), 7.00 (7.56) and 8.00
(8.56); at 200 MPa – 7.00 (7.71). Experiments for cytidine were conducted
at 373 K and 0.1 and 150 MPa. As all measurements were conducted at
373 K, no correction to pH for temperature was made.
Hydrolysis measurements. Samples were inserted into the high-
pressure cell and brought up to pressure. A 1H NMR spectrum was
recorded and the sample was then placed in an oil bath at 373 K for a
period of at least 135 hours while maintaining the pressure at the target
value (no leaks were observed over periods of weeks). During this time
further 1H NMR spectra were recorded at 298 K after which the sample
was promptly restored to 373 K. All NMR spectra were recorded with 64
scans with a relaxation time of 10 s with a pre-saturation pulse to suppress
the water peak. The integrals of the NMR peaks corresponding to protons
H5 and H6 for both cytosine and uracil (see Figure 1) were measured for
each spectrum using the TMSP-d4 peak as a reference. Sample spectra
are provided in Supporting Information as Figure S2. From this a plot of
-ln [cytosine]t/[cytosine]t=0 vs. time was used to determine the rate constant
for hydrolysis, k, for each sample. From the pressure dependence of the
rate constant an activation volume was calculated using Equation 1.
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Acknowledgements
We gratefully acknowledge funding from the Marsden Fund of the
Royal Society of NZ (grant 09-MAU-140) for a PhD scholarship to
CPL and the Allan Wilson Centre for Molecular Ecology and
Evolution to purchase the high-pressure/high-temperature NMR
cell. We acknowledge helpful discussions with Associate
Proessor Vyacheslav V Filichev.
Keywords: Cytosine hydrolysis • high pressure • high
temperature • NMR spectroscopy • origin of life
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Title
The relatively low chemical stability of cytosine compared to other nucleobases is a
key concern in origin-of-life scenarios, but the effect of pressure on the rate of
hydrolysis of cytosine to uracil has remained unknown. At high (200 MPa) pressures
o
the half-life of cytosine at 100 C is halved to 8.6 days compared that at ambient
pressure.
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