G Model
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electrochemical reactors for driving the origin of life [18,21]. High
hydrostatic pressure, as one of the important environmental
factors in hydrothermal vents, exerts profound effect on the
physical properties of proteins and some other biomolecules [22–
24]. But the effect of high hydrostatic pressure on the formation of
prebiotic peptides remains unclear. Meanwhile, most of the
submarine hydrothermal vents (e.g., mid-ocean ridge) were found
1–3 kilometers beneath the seafloor [25], where the pressure is up
to 100–300 bar.
In this paper, we experimentally explored the properties of
peptide formation under high hydrostatic pressures (300 bar). To
this purpose, 0.1 mol/L P3m was respectively treated with 0.1 mol/L
amino acids (Phe, Trp, Met, Val, Gly and Ala) under different
pressure and temperature conditions to study the effect of
different pressures on peptide formation.
The dipeptide product (Phe2) was analyzed by HPLC-MS and
identical to authentic dipeptide Phe2 (Scheme 1 and Fig. 1) [26,27].
In order to test effect of substrate concentration and Mg2+ on
the dipeptide formation, reaction mixtures containing varied
concentrations of Phe (10, 30, 50, 80 and 100 mmol/L) and
100 mmol/L P3m were incubated, under 1 bar and 300 bar,
respectively, at room temperature in a aqueous solution with
the pH 10.7 for 7 days. In another set of reactions, 2 mmol/L Mg 2+
was added under the identical conditions. The results showed that
the yield of the dipeptide Phe2 was increased with the increased
concentrations of reactant. However, Mg2+ has no positive effect on
the yields of dipeptide. So Mg2+ was not considered in later
experiments (Fig. S1 in Supporting information).
Based on the above results, we treated 0.1 mol/L P3m with
0.1 mol/L amino acids of Phe, Trp, Met, Val, Gly and Ala,
respectively, at 26 ꢀC under a pressure of 1 bar and 300 bar. The
produced dipeptides were confirmed and quantified through
comparing with the standards by HPLC-MS (Figs. S2–S12 in
Supporting information). The results showed that these amino
acids were converted to corresponding dipeptides except for Val
(Table 1 and Fig. S13 in Supporting information).
Fig. 1. Formation of Phe2 from a reaction mixture of Phe and P3m at alkaline
aqueous solution (pH 10.7). (a) HPLC-MS spectrum of authentic Phe2 (0.48 mmol/L),
(b) HPLC-MS spectrum of the sample, (c) and (d) the standard curve and the formula
for quantifying yields of product Phe2. Ev: Experimental value; Cv: Calculated value.
dipeptide yields of Phe2, Val2, Gly2 and Ala2 showed no significant
differences under these two pressures (Table 1 and Fig. S14 in
Supporting information).
Hence, both temperature and pressure as the deep-sea
environmental conditions should be considered simultaneously.
As mentioned above, the results showed that the yields of
dipeptides increase with raising pressure. Therefore, high hydro-
static pressure could be a much favored factor for prebiotic
dipeptide formation.
Based on density functional theory calculations at the M06-L/6-
311++G(2d, p)// B3LYP/6-31 G(d) level of theory in the gas phase,
free energy profiles of the formation of dipeptides were produced
(Figs. 2 and S15 in Supporting information). The rate-determining
step of the reactions, Phe2 and Gly2, was found with an extremely
high overall barrier of 65.1 kcal/mol and 56.2 kcal/mol, respective-
ly. It suggested that the reactions were thermodynamically and
kinetically unfavorable.
As shown in Table 1 and Fig. S13, for Phe, Trp and Met at 26 ꢀC,
the dipeptide yields under 300 bar were higher than those under
1 bar, which were improved to 2.30, 1.45 and 1.33 times,
respectively. However, the yields of dipeptide were almost the
same for both Gly and Ala under different pressures.
It is worth noting that most of the dipeptide yields were less
than 2% except for Gly and Ala (29.58% and 4.69%, respectively). To
evaluate the effect of high hydrostatic pressure at higher temper-
atures, similar reactions were carried out at 37 ꢀC. Indeed, as the
temperature increased, the dipeptide yields increased for all the
five amino acids (Phe, Trp, Met, Gly and Ala). Meanwhile, for Val,
the dipeptide Val2 was also detected and quantified by comparing
with the Val2 standard using HPLC-MS (Figs. S7 and S8). The
dipeptide yields for Trp2 and Met2 under 300 bar are increased
about 17% and 10% higher than those under 1 bar, respectively. The
These theoretical calculations were consistent with the
experimental results above, i.e., the yields of dipeptide were low
in the aqueous solution. Moreover, from the free energy profiles of
Phe2 and Gly2, it could be found that the energy barriers of all steps
of Phe2 formation were lower than that of the corresponding steps
of Gly2 formation except for the rate-determining step. Perhaps
this explained why the yield of Phe2 under 300 bar was higher than
that under 1 bar at 26 ꢀC, whereas the yield of Gly2 was almost the
same under different pressures. The reason is that the high
hydrostatic pressure of 300 bar reduces the energy barrier of rate-
determining step of Phe2, and the energy barriers of other steps are
relatively lower, resulting in a significant improvement of the final
yield. However, with Gly2 at 300 bar, the energy barriers for other
Scheme 1. Reaction of amino acids with P3m for dipeptide synthesis.
Please cite this article in press as: J. Ying, et al., Effect of high hydrostatic pressure on prebiotic peptide synthesis, Chin. Chem. Lett. (2018),
Ying, Jianxi
