Properties of dihydroasparagusic acid and its use as an antidote against mercury(II) poisoning

Article abstract of DOI:10.1007/s00706-013-1095-3

Dihydroasparagusic acid is the first naturally occurring dimercaptanic compound that was isolated in 1948 from Asparagus concentrate. Although several synthetic procedures were proposed in the past decades for this natural substance, most of its chemical properties remain unstudied. In this work the capacity of the acid to act as an antidote against mercury(II) toxicity was evaluated in a simple biological model system, Saccharomyces cerevisiae, and is explained by the formation of a precipitate between mercury(II) and dihydroasparagusic acid. The precipitate was analyzed and studied. The solubility was determined by measuring in equilibrated solutions either the concentration of the total mercury(II) present in solution or the free concentration of hydrogen ions. The protonation constants were determined at 25 C and in 1.00 M NaCl, as constant ionic medium, by means of potential difference measurements of a galvanic cell with a glass electrode. The experimental data are explained by proposing the chemical composition of the precipitate and the value of its solubility product. As the solubility of the precipitate increases by increasing the concentration of dihydroasparagusic acid, the further formation of a complex between mercury(II) and dihydroasparagusic acid is assumed. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-013-1095-3

Monatsh Chem (2013) 144:1767–1773
DOI 10.1007/s00706-013-1095-3
ORIGINAL PAPER
Properties of dihydroasparagusic acid and its use as an antidote
against mercury(II) poisoning
•
Armandodoriano Bianco Emilio Bottari Maria Rosa Festa
•
•
•
Lorella Gentile Anna Maria Serrilli Alessandro Venditti
•
Received: 12 July 2013 / Accepted: 23 September 2013 / Published online: 19 October 2013
Ó Springer-Verlag Wien 2013
Abstract Dihydroasparagusic acid is the first naturally
occurring dimercaptanic compound that was isolated in
1948 from Asparagus concentrate. Although several syn-
thetic procedures were proposed in the past decades for this
natural substance, most of its chemical properties remain
unstudied. In this work the capacity of the acid to act as an
antidote against mercury(II) toxicity was evaluated in a
simple biological model system, Saccharomyces cerevisi-
ae, and is explained by the formation of a precipitate
between mercury(II) and dihydroasparagusic acid. The
precipitate was analyzed and studied. The solubility was
determined by measuring in equilibrated solutions either
the concentration of the total mercury(II) present in solu-
tion or the free concentration of hydrogen ions. The
protonation constants were determined at 25 °C and in
1.00 M NaCl, as constant ionic medium, by means of
potential difference measurements of a galvanic cell with a
glass electrode. The experimental data are explained by
proposing the chemical composition of the precipitate and
the value of its solubility product. As the solubility of the
precipitate increases by increasing the concentration of
dihydroasparagusic acid, the further formation of a com-
plex between mercury(II) and dihydroasparagusic acid is
assumed.
Keywords Dihydroasparagusic acid ꢀ
Antidote towards mercury(II) ꢀ Solubility product ꢀ
Coordination chemistry ꢀ Protonation complexes ꢀ
Stability constant
Introduction
Dihydroasparagusic acid (Fig. 1), the first dimercaptanic
compound of natural origin, was first isolated from an
Asparagus concentrate by Jansen [1]. Several investiga-
tions were performed on this product, but many of its
properties remain unstudied. The presence of two mer-
capto groups allows one to hypothesize that H3DA might
display antidote properties. The search for such mercapto-
based chemical species as suitable antidotes against mer-
cury(II) toxicity has been underway since 1980 or earlier
[2]. From a casual survey, Casas and Jones [2] found
several anomalies in the behavior of compounds contain-
ing sulfhydryl groups as ligands of mercury(II). Many
values of constants are cited, but it is not clear whether
they refer to complexes with mercury(II) or to protonation
constants of the studied compounds [2–7]. Values of
constants can be compared when experimental conditions
(i.e., temperature and ionic medium) are known. The
comparison can take place if the protonation constants of
the potential ligands are known.
The behavior of compounds containing sulfhydryl
groups was studied in our laboratory to determine the
protonation constants and to study their properties as
ligands [8–11]. Cysteine [8], cystine [9, 10], and penicil-
lamine disulfide [11] were studied at 25 °C and in constant
ionic media. Their protonation constants were determined
and in particular the complex formation between cysteine
and cadmium(II) was studied at 25 °C and in 1.00 M NaCl
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-013-1095-3) contains supplementary
material, which is available to authorized users.
A. Bianco ꢀ E. Bottari ꢀ M. R. Festa (&) ꢀ L. Gentile ꢀ
A. M. Serrilli ꢀ A. Venditti
`
Dipartimento di Chimica, Universita di Roma ‘‘La Sapienza’’,
P.le A. Moro 5, 00185 Rome, Italy
e-mail: mariarosa.festa@uniroma1.it
123

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A. Bianco et al.
Fig. 1 Structure of
dihydroasparagusic acid
(H3DA)
oxygen in solution so that its concentration approaches the
initial oxygen saturation level. On the contrary, the
simultaneous addition of HgCl2 and H3DA drastically
limits the increase of the oxygen concentration in the
culture medium. The resulting increase or decrease of
oxygen concentration and its dependence on the addition of
poisonous HgCl2 and on H3DA, respectively, will be
explained in the ‘‘Results’’ section.
O
HS
HS
OH
as constant ionic medium in a wide range of hydrogen ion
concentrations.
Basinger et al. [12], by thinking about possible antidotes
for mercury(II) poisoning, which was based upon earlier
studies on arsenic, studied 2,3-dimercaptopropanol-1
(BAL) [13] as a ligand of mercury(II). However, the study
was thus undertaken in order to develop a consistent
understanding of the relationship between the coordination
of mercury(II) and the structural requirements necessary if
a molecule is to be an antidote for acute mercury(II)
chloride intoxication. The study was carried out potentio-
metrically; however, only in few cases could the
measurements be considered reversible. Furthermore, the
ligand BAL was sparingly soluble in aqueous solution.
For the above reasons, dihydroasparagusic acid (H3DA),
the synthesis of which we recently reported [14], is pre-
sented and studied as an antidote against mercury(II) here.
The presence of the carboxylic group increases the aqueous
solubility of this compound compared with BAL. Its sol-
ubility depends on the hydrogen ion concentration of the
solution. To study the behavior of H3DA as a ligand
towards mercury, it is necessary to investigate a wide range
of concentration of the reagents. To minimize the variation
of the activity coefficients of the reagents in spite of the
variation of their concentration, the constant ionic medium
Figure 1 shows that dihydroasparagusic acid is a trip-
rotic acid (H3DA) with two hydrogen atoms bonded to two
sulfur atoms and the third hydrogen atom bonded to the
carboxyl group. The protonation constants of H3DA can be
defined by the following expression:
½HnDAꢁ ¼ Kn½Hnꢂ1DAꢁh
ð1Þ
where n can be 1, 2, or 3 and h is the free concentration of
the hydrogen ion. Charges are omitted for simplicity. The
constants K1, K2, and K3 are determined by measuring the
potential difference (pd) of the following galvanic cell:
RE=solution T/GE
ðIÞ
where RE and GE are reference and glass electrode,
respectively, and the solution T has the following general
composition:
H M in Hþ; ð1 ꢂ HÞ M in Naþ; 1:00 M in Clꢂ:
ð2Þ
In Eq. (2), H is the analytical excess of hydrogen ions
with respect to the ionic medium. At 25 °C, in a constant
ionic medium and in millivolt units, the pd of cell (I) can
be expressed as follows:
EI ¼ Eꢃ þ 59:16 log h þ Ej
ð3Þ
`
method proposed by Biedermann and Sillen [15] was
I
adopted. All measurements were carried out at 25 °C and
in 1.00 M NaCl as constant ionic medium.
E°I, a constant, and Ej, the liquid junction potential [15]
which is dependent on h, are determined in the first part of
each measurement when H = h, i.e., in the absence of
HnDA. To determine all three constants, an investigation in
a wide range (1 B -log h B 12) of hydrogen ion con-
centrations is necessary. In alkaline solution Ej’ is
dependent on OH- and it is determined together with the
ionic product of water, -log Kw.
The aim of this investigation was to verify the efficacy
of H3DA as an antidote against mercury(II), to evidence the
complex formation between H3DA and mercury(II), and to
determine the protonation constants of H3DA and the
thermodynamic parameters of the compounds formed
between H3DA and mercury(II).
For the first purpose, an oxygen electrode was dipped in
10 cm3 of 1.0 M glucose solution magnetically stirred at
constant speed. A measured volume of Saccharomyces
cerevisiae (0.25 cm3) suspension was introduced into the
glucose solution (1.0 M) very slowly to minimize the
fluctuation of concentration of oxygen present in the
solution. The presence of oxygen in solution decreases
owing to the metabolic activity of S. cerevisiae. However,
after about 1 h, the oxygen concentration reaches a quite
constant value because the speed of oxygen consumption
by the yeast and that of dissolution of oxygen coming from
the atmosphere are the same.
In the second part of the measurement, a known con-
centration of HnDA is added to the solution T, and H is
gradually varied by keeping [HnDA]tot (total concentration
of HnDA) constant. From this approach, the analytical
concentrations of H and HnDA, and the h value, experi-
mentally obtained from the measurements using cell (I)
constitute the basis of the following elaboration.
The reaction between HgCl2 and HnDA is studied with
different approaches. As by adding HgCl2 to HnDA the
formation of a precipitate takes place, the determination of
its solubility, S, is carried out. According to a first proce-
dure, the solid is brought in contact with the ionic medium
at known H, and according to a second one it is in contact
The addition of the poisonous compound HgCl2
(6 mg dm-3) produces an immediate increase of the
123

Properties of dihydroasparagusic acid
1769
with a solution at known concentration of total HnDA and
known H. In both cases, suspensions are shaken until they
reach equilibrium. After several tests, it could be deduced
that in both cases 5 h is sufficient and necessary to reach
equilibrium. The equilibrated suspensions are analyzed for
-log h and for S. The former is determined by direct po-
tentiometry and the latter by determining the Hg(II)
concentration present in the filtered solutions by means of
an atomic absorption spectrophotometer connected to a
hydride kit. The values of H, analytically known, S and h,
experimentally measured, are the basis of the elaboration
carried out to obtain the results.
The comparison between the second and the third
experiment is interesting. In all cases, the O2 concentration
is measured as a function of time. The first part of each
experiment shows the same plateau. In the second experi-
ment, the addition of yeast produces a decrease of oxygen,
similar to that observed in the first one. By adding Hg(II)
(with a concentration of 6 9 10-3 g dm-3) an immediate
and remarkable increase of O2 concentration was observed.
The O2 concentration increase is observed because the
yeast no longer consumes oxygen. The O2 concentration
increase is attributed to the mercury(II) which develops its
poisonous effect on S. cerevisiae, with the inhibition of the
yeast.
Vice versa, when, in the last experiment, mercury(II)
was added together with dihydroasparagusic acid, with a
mass ratio 1:600, the increase of O2 concentration is rela-
tively low. This observation shows that the yeast was little
inhibited because the effect of the presence of free mer-
cury(II) is very small. The simultaneously present
dihydroasparagusic acid reacts with mercury(II) seques-
trating it from the solution and therefore preventing its
poisonous effect. As a consequence, the yeast’s vitality was
less affected by the presence of mercury.
Results and discussion
H3DA as an antidote
The capability of H3DA as antidote versus mercury(II) was
evaluated on a simple biologic system, S. cerevisiae, a
yeast very sensitive to mercury, by measuring the oxygen
concentration in a suspension with S. cerevisiae, mer-
cury(II), and H3DA. The oxygen concentration (as
milligrams per cubic decimeter of O2) in 1.0 M glucose
solution at equilibrium with atmospheric oxygen was
measured as a function of time. Three experiments were
carried out by starting from the glucose solution. In the first
one, a measured amount of S. cerevisiae was introduced
and, after about 1 h, another solution of H3DA was added.
In the second experiment after the addition of S. cerevisiae,
a solution of HgCl2 was added. Finally, in the last exper-
iment, after the initial S. cerevisiae addition, solutions of
HgCl2 together with H3DA were added to the glucose
solution.
The percentage of inhibition can be evaluated by using
an internal normalization, because the yeast activity is
related to atmospheric conditions, mainly the room tem-
perature. It is assumed that the difference between the
initial oxygen concentration and the oxygen concentration
reached at the plateau corresponding to the addition of the
yeast (second plateau in the first experiment) corresponds
to 100 % vitality. The ratio of inhibition/toxicity may be
calculated as follows.
From the above experiments, the oxygen consumption
can be obtained from the decrease of oxygen level due to
the presence of S. cerevisiae (D1), whereas the increase of
oxygen can be calculated from the increase of oxygen
corresponding to the introduction of mercury(II) (D2) or
dihydroasparagusic acid (D3), respectively. The ratio (D2/
D1) 9 100 = 97 % is evaluated for the inhibition of
mercury(II), whereas the ratio (D3/D1) 9 100 = 29 %
corresponds to the inhibition in the presence of mercury,
provided that dihydroasparagusic acid is present as well.
The effect of dihydroasparagusic acid as an antidote is
evident.
In the first experiment, an initial plateau (ppm O2 versus
time) is observed before the introduction of the yeast. The
presence of S. cerevisiae in the solution consumes oxygen
thereby decreasing its concentration. After the test solution
is in contact with air for about 1 h, the speed of oxygen
consumption due to the yeast is equal to that of dissolution
of oxygen from air and consequently a new plateau is again
observed. At this point, to evaluate the eventual toxicity of
H3DA, H3DA (with a concentration of 0.120 g dm-3) was
introduced and no remarkable modification was observed,
showing a quite negligible toxicity. In fact, a little increase
of oxygen concentration observed corresponding to the
H3DA addition was due mainly to the oxygen present in
this last solution already in equilibrium with the atmo-
spheric oxygen. The previous tendency to decrease the
oxygen concentration in the medium culture was promptly
restored by the yeast. This result, showing the negligible
toxicity of H3DA, allows one to use a relatively large
amount of this compound as an antidote.
Protonation constants
To obtain the Kn values, H and HnDA, analytical concen-
trations and h, experimentally obtained from the pd
measurements of cell (I) are elaborated. The material bal-
ance of analytical excess of hydrogen ion, H, by taking into
account the mass action law and in a constant ionic med-
ium, can be expressed as follows:
123

1770
A. Bianco et al.
ꢀ
ꢁ
ꢀ
ꢁ
H ¼ h þ K1h DA3ꢂ þ 2K1K2h2 DA3ꢂ
the same value for the protonation constants is obtained. In
Table 1 the proposed values are collected.
ꢀ
ꢁ
þ 3K1K2K3h3 DA3ꢂ
:
ð4Þ
From Eq. (4) and from the total concentration of HnDA,
the protonation function p of DA3- (as the average number
of protons bonded to DA3-) can be calculated as follows:
Reaction between HgCl2 and HnDA and precipitation
To explain the trend of the reaction taking place between
mercury(II) and dihydroasparagusic acid, the data of sol-
ubility, S, and of the free hydrogen ion concentration, -log
h, are elaborated. Experimental data of S at different H and
[HnDA]tot were obtained. The reaction between mer-
cury(II) and DA3- in aqueous solution can be written as
follows:
ꢂ
ꢃꢂ1
p ¼ ðH ꢂ hÞ ½HnDAꢁtot ¼ RnKnhnð1 þ RKnhnÞꢂ1
:
ð5aÞ
In Fig. 2 the protonation function p is plotted versus
-log h. As experimental points obtained at different
concentration of [HnDA]tot fall on the same curve, the
protonation function p is not a function of the dihydroas-
paragusic acid concentration, so that polynuclear species
are not present and Eq. (4) is correct.
Hg2þ þ pHþ þ rDA3ꢂ $ Hg HpðDAÞr
defined by the constant
ꢀ
ꢁ
b1;p;r ¼ Hg HpðDAÞ ½Hgꢁꢂ1hꢂp½DAꢁꢂr
:
ð6Þ
In a first approach the graphical method of normalized
`
curves proposed by Sillen [16] is applied. Equation (5a)
r
In the reaction and in Eq. (6) most of the charges are
omitted for simplicity and the square bracketsindicatethe free
concentration (at equilibrium) of the species. Equation (6)
formulates the hypothesis is that mononuclear species with
mercury(II) are formed.
can be normalized with the following:
ꢂ
ꢃ ꢂ
ꢃꢂ1
p ¼ Ru þ 2u2 þ 3R0u3 1 þ Ru þ u2 þ R0u3
ð5bÞ
where log u = 0.5 log K1K2 ? log h, log K1 = log
R ? 0.5 log K1K2, and log K3 = log R0 ? 0.5 log K1K2.
The family of normalized curves of Eq. (5b) were super-
imposed onto the experimental points of Fig. 2 and the two
plots were moved parallel to the abscissa until the best fit
was reached. In this position the -log h value was read as a
function of log u and, on the basis of the mathematical
position proposed above, the values of the protonation
constants K1, K2, and K3 could be obtained.
The solubility S can be expressed by the following
relation:
ꢀ
ꢁ
ꢀ
ꢁ
ꢀ
ꢁ
r
S ¼ Hg2þ þ RRb1;p;r Hg2þ hp DA3ꢂ
ꢀ
ꢁ ꢂ
ꢀ
ꢁ ꢃ
r
¼ Hg2þ 1 þ RRb1;p;rhp DA3ꢂ
:
ð7Þ
Since the analysis of the precipitate provided the
formula Hg(H2DA)2 and the solubility data are obtained
at -log h B 7, it seems reasonable to assume that in this
range the dihydroasparagusic acid is present in the form
H2DA- and then Eq. (7) can be written as follows:
By treating the experimental points independently using
a graphical program [17] and a PC program BSTAC [18]
ꢀ
ꢁ
ꢀ
ꢁ
w
S ¼ Hg2þ þ Rb1;w Hg2þ ½H2DAꢂꢁ
ꢀ
ꢁ ꢂ
ꢃ
w
¼ Hg2þ 1 þ Rb1;w½H2DAꢂꢁ
which can be transformed into the following:
n
o
n
o
ꢀ
ꢁ
2
2
log S½H2DAꢂꢁ ¼ log Hg2þ ½H2DAꢂꢁ
ꢄ
ꢅ
w
þ log 1 þ Rb1;w½H2DAꢂꢁ
ð8Þ
ð9Þ
and also:
n
o
ꢄ
ꢅ
2
w
log S½H2DAꢂꢁ ¼ logKs þ log 1 þ Rb1;w½H2DAꢂꢁ
Fig. 2 Protonation function of dihydroasparagusate versus -log
h. The curve is the normalized one in the position of best fit
Table 1 Protonation constants of DA3- at 25 °C and in 1.00 M NaCl as ionic medium
Species
log K1
log K2
log K3
log K1K2
log K1K2K3
HDA2-
H2DA-
H3DA
11.30 0.05
9.75 0.05
21.05 0.05
3.50 0.08
24.55 0.10
The error limits correspond to the maximum possible shift between points and curves that still retains an acceptable agreement
123

Properties of dihydroasparagusic acid
1771
reason it was seldom investigated in the past. In light of its
low abundance in the plant, it was recently synthesized by
an improved procedure [14].
The validity of dihydroasparagusic acid as an antidote
against mercury(II) poisoning is supported by the different
oxygen consumption of the yeast (S. cerevisiae) in the
absence and in the presence of H3DA. It can be assumed
that dihydroasparagusic acid, which also contains a car-
boxylic group, is more soluble than BAL and it is a
triprotic acid. The three protonation constants are deter-
mined. As expected, the two hydrogen atoms are strongly
bonded to sulfur and their dissociation as ions can take
place at -log h C 9. This means that, in acid or neutral (-
log h B 7) solution, the compound is present in the form
H2DA-.
Fig. 3 Dependence of solubility on the concentration of -log
[H2DA-]
The analysis of the precipitate and the elaboration of the
solubility data give consistent results. The value of the
solubility product could appear relatively high, but it is
calculated by considering mercury(II) and the compound in
the form H2DA-. The concentration of this form is
obtained from the product K1K2h2 [DA3-]. On the basis of
this calculation, the value of log Ks becomes -log
Ks = log {[Hg2?] (h2 [DA3-])2} = 36.08, which repre-
sents a low value. Furthermore, the value of -log Ks
proposed here is determined in 1.00 M of NaCl as the ionic
medium.
where Ks indicates the solubility product of the precipitate
formed between mercury(II) and dihydroasparagusic acid
and the constant b1,w = [Hg (H2DA-)w] [Hg]-1 [H2DA-]-w
is the stability constant of final complexes formed between
the reagents.
According to Eq. (9), the points are plotted in Fig. 3 in
the form log {S [H2DA-]2} versus -log [H2DA-]. As
points obtained at different H and at different concentration
of H3DA fall on the same curve, the validity of Eq. (9) can
be deduced and Ks as well as the constant of complex
formation can be obtained.
The high stability of the species HgCl- and HgCl2 found
by Ciavatta and Grimaldi [19] and the ionic medium
adopted in this investigation (1.00 M NaCl) must be con-
sidered for a correct thermodynamic analysis of the
solubility data and the proposed complex. The solubility
product and the stability constant are valid under the
selected experimental conditions and in the selected ionic
medium. However, NaCl cannot be considered inert
towards Hg(II). If the high stability of HgCl- and HgCl2 is
taken into account, the solubility product (-log Ks) and the
stability constant of the complex would be still higher than
the proposed value. By taking into account the large excess
of NaCl as the ionic medium, it seems reasonable to
evaluate the presence of HgCl2 with a constant log
b2 = 13.2. This consideration gives more validity to the
antidote property of H2DA-. Really, in the hypothesis of
the absence of the ionic medium, the numerical values of
-log Ks of Hg[H2DA]2 and of log b1,1 of Hg(H2DA)
should be higher by a factor of approximately 13.2.
Although potentiometric measurements and analytical
data are not able to provide information about the structure
of the formed species, but taking into account the investi-
gated concentration ranges and the literature data, some
hypotheses regarding the mechanism of formation of the
assumed compounds can be proposed.
At first the method of normalized curve proposed by
`
Sillen [16] is applied and the formation of only one com-
plex is assumed. A normalized curve can be obtained with
the following equation:
y ¼ logð1 þ vÞ
By comparing the normalized curve with Eq. (9), the
n
o
2
following
positions
log S½H2DAꢂꢁ ꢂ y ¼ log Ks
and log v ¼ log b1;1 þ log½H2DAꢂꢁare assumed.
The normalized curve was superimposed onto the
experimental points and the two plots were moved parallel
to the two axes until the best fit was obtained. In this
position and on the basis of the mathematical positions, the
values of log Ks = 14.72 0.05 and log b1,1
=
4.78 0.05 are obtained. The error limits correspond to
the maximum possible shift between points and curves that
still retains an acceptable agreement.
Conclusions
This paper is a complete investigation of the properties of
dihydroasparagusic acid. No literature data can be found
about this subject because this natural product is present in
very small quantities in its natural source and for this
The presence of the compound acting as H2DA-
explains the composition of the precipitate. The formation
123

1772
A. Bianco et al.
milligrams per kilogram of the fresh plant. In order to
obtain a good yield of this acid, in a few steps and utilizing
commercial reagents, we revised some existing syntheses
[22–25]. The revised synthetic procedure for the prepara-
tion of dihydroasparagusic acid (H3DA) was recently
described [14] and is summarized as follows (Scheme 1):
diethyl bis(hydroxymethyl)malonate was refluxed with
concentrated hydroiodic acid to obtain a mixture of b,b0-
diiodoisobutyric acid and iodomethylacrylic acid. After
salification with 0.8 M KOH the solution was added with a
solution containing thioacetic acid and potassium thioace-
tate. Under these conditions, in the presence of thioacetate
anion and free thioacetic acid, two different reactions occur
simultaneously: the substitution of iodide by thioacetate
anion and the addition of free thioacetic acid to the double
bond of the acrylic by-product, leading to the desired
bis(thioacetyl) intermediate. The final alkaline hydrolysis
under mild conditions allows one to obtain the final
product.
HS
HS
SH
SH
COO
Hg
OOC
Fig. 4 Proposed structure of the Hg[H2DA]2 precipitate
of a chelate should be excluded because the form H2DA-
can work as a monodentate compound and because the
structure of mercury(II) is compatible only with an angle of
180°. The remaining possibility is the bond between mer-
cury(II) and two H2DA- through the two carboxyl groups.
Furthermore, the formation of the complex with H2DA-
can be seen as a bond through the carboxylic group because
the dihydroasparagusic acid, in the investigated -log
h range, has the two protonated sulfur atoms and the car-
boxyl free to bind mercury.
This hypothesis is supported by the literature investi-
gation on the formation and the stability of complexes of
mercury(II) with acetate and chloride, respectively. Ross-
otti and Whewell [20] explained the experimental data
obtained for the system mercury(II) acetate with the for-
mation of two complexes with similar stability constants
(log b1 = 4.22 and log b2 = 4.23). The values proposed in
this paper for the complex Hg(H2DA) are similar to
Rossotti’s. Further support is provided by the investigation
of the system Hg(II) Cl-, studied by Ciavatta and Grimaldi
[19]. The experimental data of this paper were explained
by assuming the presence of two very stable complexes
HgCl? and HgCl2 (log K1 = 6.72 and log K2 = 6.51), the
A preliminary investigation of the reaction between
H3DA and HgCl2 was performed by means of cyclic vol-
tammetry [26]. A solution of 10-3 M of HgCl2 was mixed
with H3DA in ratios of 1:4 or 1:8. The solution was buf-
fered at pH 7.4 by adding phosphate. The shift of the
potential to more positive values was explained by
assuming the formation of a 2:1 complex between H3DA
and HgCl2.
To evaluate the dihydroasparagusic acid capacity of the
antidote against mercury(II), a suspension of S. cerevisiae
was prepared by mixing 2.5 g of S. cerevisiae with 50 cm3
of distilled water. The resulting suspension was kept at
room temperature for at least 2 h before use. A volume of
0.25 cm3 of this suspension, shaken before use, was
introduced into the culture medium. A 1 M solution of
glucose (10 cm3) was used as the medium.
-
formation of HgCl3 with low stability (log K3 = 1.00),
and the formation of HgCl42- which is still less stable (log
K4 = 0.97). The presence of the ionic medium does not
interfere with the composition of the precipitate, because
the analysis showed that its composition was Hg[H2DA]2
and no chloride was present in the precipitate.
By taking into account all these literature data and the
mercury(II) properties, the structure shown in Fig. 4 is
proposed for the precipitate. No chelate structure seems to
be probable for the precipitate of Hg[H2DA]2.
By adding a moderate excess of HgCl2 to a solution of
dihydroasparagusic sodium salt, a precipitate formed. The
solid was filtered and washed several times to eliminate
chloride ions. The solid was dried in an oven at 80 °C and
analyzed according to Schwarzenbach and Flaschka’s
method [27] to obtain the percentage of mercury(II). The
analysis provided a molecular mass of 501 with respect to
the theoretical value of 500.61 corresponding to the for-
mula Hg(H2DA)2.
The behavior of the dihydroasparagusic acid as an
antidote against mercury(II) is confirmed by the very low
value of the solubility product of the precipitate.
Experimental
Mercury(II) chloride (RPE product, C. Erba), was recrys-
tallized twice from water and dried over H2SO4 in a
vacuum desiccator for a week, as described by Ciavatta and
Grimaldi [21]. Hydrochloric acid, sodium chloride, and
sodium hydroxide were prepared and analyzed as described
previously [8]. Dihydroasparagusic acid (H3DA) is the first
dimercapto compound isolated from a natural source [1]. It
is contained in Asparagus species in the order of a few
Experimental apparatus
All the measurements were performed in a thermostat room
at 25 0.5 °C and in a thermostat at 25 0.05 °C. A
stream of nitrogen (99.999 %) further purified by passing
through 10 % NaOH, 10 % H2SO4, and the ionic medium,
was bubbled through all the solutions during the
measurements.
123

Properties of dihydroasparagusic acid
1773
Scheme 1
O
O
O
O
HI 57%
HO
HO
O
O
I
I
I
+
OH
OH
O
O
O
1. KOH
aq. KOH
N2
S
HS
HS
S
2. CH3COSH /
CH3COSK
OH
OH
O
H3DA
4. Lenz GR, Martell AE (1964) Biochemistry 3:745
The capability of H3DA to act as an antidote was
evaluated by measuring the oxygen present in the S. ce-
revisiae mixtures both in the presence and absence of
mercury(II) and H3DA by means of an oxygen electrode
(model 97-08, Orion Research). The O2 electrode provides
its measurements in milligrams per cubic decimeter of
oxygen present. The solubility of the precipitate, S, i.e., the
mercury(II) concentration, was determined by means of an
atomic absorption spectrophotometer ( model Solaar AA,
Thermo Fisher), equipped with a hydride kit, able to
5. Van der Linden WE, Beers C (1974) Anal Chim Acta 68:143
6. Kuchinskas EJ, Rosen Y (1962) Arch Biochem Biophys 97:370
7. Sugiura Y, Yokoyama A, Tanaka H (1970) Chem Pharm Bull
18:693
8. Bottari E, Festa MR (1997) Talanta 44:1705
9. Apruzzese F, Bottari E, Festa MR (2002) Talanta 56:459
10. Apruzzese F, Bottari E, Festa MR (2003) J Sol Chem 32:65
11. Apruzzese F, Bottari E, Festa MR (2004) Ann Chim (Rome)
94:45
12. Basinger MM, Casas JS, Jones MM, Weaver AD, Weinstein NH
(1981) J Inorg Nucl Chem 43:1419
13. Peters RA, Stocken LA, Thompson RHS (1945) Nature 156:616
14. Venditti A, Mandrone M, Serrilli AM, Bianco A, Iannello C,
Antognoni F, Poli F (2013) J Agric Food Chem 61:6848
determine mercury(II) up to 0.1 lg dm-3
.
The free hydrogen ion concentration, h, was obtained by
potentiometric measurements of a galvanic cell containing
a glass electrode (GE), by previous calibration in units of -
log h with at least two or three solutions of known
hydrogen ion concentration in the selected ionic medium.
All the pd measurements were carried out by a Metrohm
model 654 pH meter equipped with a glass electrode No.
6.0102.000 from the same firm. The reference electrode
(RE) was prepared according to Brown’s method [28]
(RE = Ag, AgCl/1.0 mol dm-3 NaCl, saturated with
AgCl/1.0 M NaCl). Constant values of galvanic cell mea-
surements were obtained within a few minutes. The values
were reproducible within 0.2 mV. The GE response
agreed with that of a hydrogen electrode until -log
h B 10. Beyond this limit, the values obtained from GE
were adjusted assuming the response provided by the
hydrogen electrode as correct.
`
15. Biedermann G, Sillen LG (1953) Ark Kemi 5:425
16. Sillen LG (1956) Acta Chem Scand 10:186
17. Bottari E, Coccitto T, Curzio G, Festa MR, Jasionowska R (1988)
Ann Chim (Rome) 78:635
18. Di Stefano C, Mineo P, Rigano C, Sammartano S (1993) Ann
Chim (Rome) 83:343
19. Ciavatta L, Grimaldi M (1968) J Inorg Nucl Chem 30:197
20. Rossotti F, Whewell R (1977) J Chem Soc Dalton Trans Inorg
Chem (12):1223
`
21. Ciavatta L, Grimaldi M (1968) J Inorg Nucl Chem 30:563
22. Yanagawa H, Kato T, Sagami H, Kitahara Y (1973) Synthesis
10:607
¨
23. Schotte L, Strom H (1956) Acta Chem Scand 10:687
24. Singh R, Whitesides GM (1990) J Am Chem Soc 112:1190
25. Zervas L, Photarki I, Ghelis N (1963) J Am Chem Soc 85:1337
26. Venditti A, Serrilli AM, Campanella L, Bianco A (2010) In:
Proceedings of the 24th international symposium on organic
chemistry of sulfur (ISOCS-24), Firenze, 25–30 July 2010
27. Schwarzenbach G, Flaschka H (1965) Die Komplexometrische
titration. Ferdinand Enke Verlag, Stuttgart
28. Brown AS (1934) J Am Chem Soc 56:646
As the measurements obtained from solutions prepared
with different procedures agreed well, this proved that the
measurements correspond to the real equilibrium.
References
1. Jansen EF (1948) J Biol Chem 176:657
2. Casas JS, Jones MM (1980) J Inorg Nucl Chem 42:99
3. Coates RL, Jones M (1978) J Inorg Nucl Chem 39:677
123