mixture was stirred for 24 h and then analyzed by 31P NMR
spectroscopy.
General Procedure for the Reaction of 1 with an Alcohol.
The alcohol (R-OH, 1 mmol) and disodium salt of 1 (1.89 g,
10 mmol) were dissolved in 10 mL of distilled water. The mix-
ture was stirred for 3 h during which time the pH of the mixture
was maintained at 9 « 0.5 by the addition of 6 M NaOH. After
3 h, the reaction mixture was acidified to pH 4-5 by the addi-
tion of 2 M HCl, and then analyzed by 31P NMR spectroscopy.
The 31P NMR chemical shift of phosphorous acid is 4.08 ppm.
Reaction of 1 and Glyceraldehyde. Glyceraldehyde (0.1
mmol) and disodium salt of 1 (0.19 g, 1 mmol) were dissolved
in 10 mL of distilled water. The mixture was stirred for 3 h
during which time the pH of the mixture was maintained at
9 « 0.5 by the addition of 6 M NaOH. After 3 h, the reaction
mixture was acidified to pH 4-5 by the addition of 2 M HCl,
and then analyzed by 31P NMR spectroscopy.
Calculation of the Conversion Yield.
The yield (%
conversion) was calculated using NMR peak areas as follows.
When the initial molar amount of 1 was 10 mmol, the total
molar amount of phosphorus atom was 20 mmol. Thus, the
molar amount of the product (in mmol) was calculated to be
Figure 1. A) The structure of pyrophosphorous acid (1).
B) Decrease of pH of a 1.0 M aqueous solution of 1,
initially adjusted to pH 9.5, as a function of time.
hydroxy groups of inosine9 and adenosine6 to give the corre-
sponding 2¤ or 3¤-H-phosphonates. Although the roles of H-
phosphonates in chemical evolution has not yet been estab-
lished, it has been hypothesized that dinucleoside H-phospho-
nates were formed by the thermal condensation of nucleosides
and nucleoside H-phosphonates; these are unstable and were
immediately oxidized or thiolated to the stable dinucleoside
phosphate or dinucleoside thiophosphate.10-12 Hence, it is pos-
sible that a variety of H-phosphonate derivatives were inter-
mediates during the formation of biologically important phos-
phates. Although the reactions of 1 with some biomolecules
bearing hydroxy groups, such as nucleosides, L-DOPA,13 and
catechin,14 have been clarified, reactions with other molecules,
such as amino acids and carbohydrates, which are important in
prebiotic chemistry, have not been studied to date.
In this study, we examined the reactions of 1 with a variety
of alcohols, including D-ribose, hydroxy-substituted amino
acids, and glyceraldehyde. We clarified the phosphonylation
chemistry of many types of hydroxy groups, such as 1,2-diols,
primary and secondary alcohols, and phenols in the above-
mentioned biomolecules under mild alkaline conditions. The
results of this study suggest that many types of biomolecules
may have been phosphonylated by 1 in the prebiotic environ-
ment prior to the appearance of phosphorylating enzymes.
The molar amount of product
the peak area of the product ꢀ 20
¼
the sum of all peak areas
Because the initial molar amount of the alcohol was 1 mmol,
the peak area of the product ꢀ 20 ½mmolꢁ
%conversion ¼
the sum of all peak areas
1
ꢀ
1 ½mmolꢁ
3. Results and Discussion
It has been reported that 1 effectively phosphonylates the
2¤ or 3¤-hydroxy group of inosine at initial pH values of 9-11.9
However, phosphorous acid is produced when pyrophospho-
rous acid is hydrolyzed or alcoholyzed, leading to a gradual
decrease in pH, which is problematic. To check its time-
dependency, the pH of a 1.0 M aqueous solution of pyrophos-
phorous acid was measured over a 24 h period after it was
initially adjust to a value of 9.5.
Figure 1B reveals that the pH decreased sharply over the first
2 h, after which it decreased more gently to a value of around
6.5. The 31P NMR spectrum after 24 h is shown in Figure 2.
The doublet of triplets observed at ¹3.92 ppm, (J1PH = 667 Hz;
J2PP = 7.6 Hz) corresponds to 1, while the doublet observed
at 4.08 ppm (J1PH = 577 Hz) corresponds to phosphorous acid.
The final ratio of phosphorous acid to 1 was determined to be
1:4.8 by peak-area integration, and the ratio did not change
afterward. Consequently, in order to study the reactivity of 1
under alkaline conditions, the pH needs to be controlled to avoid
decreases in pH. Therefore, the pH of the reaction mixture was
maintained at 9.0 « 0.5 in the experiments described below
through the continuous addition of 6 M NaOH.
2. Experimental
31P NMR spectra were acquired in D2O-H2O (1:5, v/v) on a
Varian AS500 or a Bruker BioSpin AVANCE III HD500 spec-
trometer at 202 MHz. Disodium salt of 1 was prepared follow-
ing a literature procedure.15 Reverse-phase HPLC was per-
formed using an XBridgeTM Prep C18 (4.6 © 150 mm, 5 ¯m)
column at 30 °C with 30 mM ammonium acetate as the eluent at
¹1
a flow rate of 3 mL min
.
The Degradation Rate of Disodium Pyrophosphate Solu-
tion under Basic Conditions. Disodium salt of 1 (1.89 g,
10 mmol) was dissolved in 10 mL of distilled water. The initial
pH of the mixture was adjusted to 9.5 with 6 M NaOH. The
Under the conditions described above, a variety of com-
pounds bearing hydroxyl groups were reacted with 1 for 3 h, as
depicted in Scheme 1, and the products were analyzed by
31P NMR spectroscopy after acidification of the reaction mix-
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