Synthesis, Characterisation and Reactivity of 3-Mercaptopyruvic Acid

Article abstract of DOI:10.1002/cbic.201800199

The synthesis, isolation and spectroscopic characterisation of the sulfur metabolic compound 3-mercaptopyruvic acid (3-MPH) is reported, for the first time. The compound is isolated without tedious workup, with a purity of 97 %, as indicated by chemical and biochemical analyses. Detailed kinetic and thermodynamic studies of its complex behaviour in solution are discussed. 3-MPH is stable in the enol form in non-polar solvent. In polar solvent, a fast equilibrium between the α-ketoacid and a cyclic dimer dithiane is observed. The formation of the dimer confers increased stability to 3-MPH towards hydrogen peroxide, in comparison with cysteine.

Full text of DOI:10.1002/cbic.201800199

Accepted Article
Title: Synthesis, characterization and reactivity of 3-mercaptopyruvic
acid.
Authors: Erwan Galardon and Jean-Chrstophe Lec
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10.1002/cbic.201800199
ChemBioChem
COMMUNICATION
Synthesis, characterization and reactivity of 3-mercaptopyruvic acid.
Dr. Erwan Galardon[a]* and Dr. Jean-Christophe Lec [b]
Abstract:
A
synthesis of the sulfur metabolic compound 3-
solution. In non-polar solvents, 3-MPH is stable and in its enol
form. In polar solvents, 3-MPH is in rapid equilibrium with the
dimer 2,5-dihydroxy-1-4-dithiane-2,5-dicarboxylic acid. At
physiological pH, the equilibrium is strongly shifted towards the
dimeric moiety at high, non-physiological concentrations.
However, at more relevant concentration, the amount of 3-MPH
becomes more significant.
mercaptopyruvic acid is reported. It allowed its isolation and thorough
spectroscopic characterization for the first time. The compound is
isolated without tedious work-up, with a purity of 97%, as indicated by
chemical and biochemical analyses. Detailed kinetic, and
thermodynamic studies of its complex behaviour in solution are
discussed. 3-MPH is stable under its enol form in non-polar solvent,
and in polar solvent a fast equilibrium between the -ketoacid and a
cyclic dimer dithiane is observed. The formation of the dimer confers
increased stability towards hydrogen peroxide to 3-3-mercaptopyruvic
acid when compared to cysteine.
Based on previous work on 3-MP,[1,14] we anticipated that
acidic conditions and non-polar solvents would facilitate the
isolation of monomeric 3-MPH. We therefore designed the
synthesis presented in Scheme 1, in which the sulfur atom is first
incorporated in its trityl-protected form. Deprotection in the
presence of trifluoroacetic acid and the trityl cation quencher
triisopropylsilane leads, after simple washing with pentane, to 3-
MPH with an overall yield of 54%.
3-mercaptopyruvic acid is a sulfur metabolic compound
formed during the catabolism of cysteine.[1] It has regained
considerable interest in the last decade, because it is the
substrate of the enzyme 3-mercaptopyruvatesulfurtransferase (3-
MST). As such, it is involved in the production of reactive sulfur
species taking part in redox signalling[2] (hydrogen sulfide,[3-4]
persulfides,[5] polysulfides)[4,6]
,
as well as in cyanide
detoxification.[7-8] So far, the main synthetic route to 3-
mercaptupyruvic acid proposed in the literature involves the
reaction of sodium hydrosulfide with bromopyruvic acid[9-11]
However, a recent study proposed that the product is not 3-
mercaptopyruvate, but the cyclic dimer 2,5-dihydroxy-1-4-
dithiane-2,5-dicarboxylate.[8] Although the use of the dimer is
under most circumstances not problematic per se for chemical,
biochemical or biological studies (see below), several major
drawbacks are associated with this synthesis: i) a tedious and
harmful purification of sodium hydrogen sulfide is required prior to
the reaction to avoid erratic yield and purity ii) a second
purification step is necessary prior to certain biochemical
experiments to remove the traces of hydrosulfide which could
affect the data.[12] Last year, an alternative synthesis was
proposed,[13] but the resulting product was barely characterized.‡‡
Here, we present a synthetic route unambiguously leading to 3-
mercaptopyruvic acid (from now on in the manuscript, we will use
3-MPH to refer to 3-mercaptopyruvic acid synthesized in this work,
and 3-MP for the commercially available "sodium 3-
mercaptopyruvate" or for the product derived from the synthesis
using sodium hydrosulfide and bromopyruvic acid, regardless of
the protonation state of the compounds in solution) with purity
greater than 97%, after simple purification steps. The isolation of
3-MPH allowed its first characterization by standard
spectroscopic techniques, and permitted to study its reactivity in
Scheme 1. Synthetic route towards 3-MPH..
Since the spectroscopic data reported for 3-MP in the
literature are probably ascribable to the dimeric compound 2,5-
dihydroxy-1-4-dithiane-2,5-dicarboxylate (Scheme 2),[1,8] we
undertook the characterization of 3-MPH. The product isolated
from the reaction described in Scheme 1 is the enol tautomer, as
1
indicated by H and 13C NMR spectra recorded in CDCl3. Thus,
the 1H-13C HSQC spectrum shows a resonance of a single proton
at  = 6.49 ppm, attached to a carbon atom with a chemical shift
at  = 108.4 ppm (Figure S1). The enolic form of 3-MPH is further
supported by FT-IR (Figure S2) and UV-Vis spectroscopy (Figure
S3). The FT-IR spectrum of 3-MPH strongly differs from that
recorded with 3-MP[15]. Hence, the spectrum of 3-MP (Figure S2B)
exhibits a single strong band in the carbonyl region at 1714 cm-1,
while the spectrum of 3-MPH (Figure S2A) displays two strong
bands at 1703 and 1652 cm-1, which are attributed to the acid and
enol moieties, respectively. In addition, 3-MPH also displays an
absorbance around 2550 cm-1 (S-H stretching) and at 3091 cm-
a.
b.
UMR 8601, LCBPT, CNRS-Université Paris Descartes, Sorbonne
Paris Cité, 45 rue des Sts Pères, 75006 Paris, France. *
erwan.galardon@parisdescartes.fr
Laboratoire IMoPA UMR 7365 9 Avenue de la forêt de Haye
Bâtiment Biopole 54505 Vandoeuvre-les-Nacy, France
1
(C-H alkene stretching). The UV-visible spectrum recorded in
chloroform also confirms the enolic form, with a strong absorption
(= 9500 L.M-1.cm-1) at 274 nm (Figure S3) which is absent in
the spectrum of 3-MP.
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
1
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10.1002/cbic.201800199
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To assess the purity of 3-MPH, we used physicochemical
and biochemical techniques. When commercial 3-MP is
essentially sold with a purity of >90%, elemental analyses for 3-
MPH indicate a purity of over 99% for 3-MPH, with the remaining
1% being triphenylmethane.
solution clearly indicates that there is no incorporation of
deuterium into the enol over the course of the reaction. This
suggest that no equilibrium takes place between the -ketoacid
and the enol forms of 3-MPH under these conditions, and that the
former is rapidly converted into the dimer. The reaction rate
constant obtained from the NMR data presented in Figure 2
(ktautom = (1.71 ± 0.02)x10s) is similar to that obtained by
monitoring the decrease of the enol absorbance at 262 nm by UV-
vis (Figure S7) (ktautom  (1.12 ± 0.04)x10s).
To confirm this result, we explored the stoichiometry of the
reaction between E.Coli 3-MST and 3-MPH in the presence of the
recycling system thioredoxin (Trx) / NADPH-dependant Trx
reductase (TrxR) (see the Supplementary Information). Under
these conditions, the variation in NADPH concentration monitored
by UV-vis spectroscopy at 340 nm allows a biochemical
determination of the purity of the 3-MPH. The curve displayed in
Figure 1 confirms that 3-MPH is at least 97% pure (vs 89% for the
commercial sample). Additional experiments carried out with 3-
MST under either single turnover or steady-state conditions give
identical enzymatic parameters for both the commercial 3-MP and
the synthetic 3-MPH (Figure S4 and S6).
The conversion of 3-MPH to the cyclic dithiane is pH
dependent, and at physiological pH (7.4) the reaction is too fast
to be monitored by 1H NMR spectroscopy. Indeed, only the
isomers of the dithiane are present in solution after the
preparation of a 10 mM solution of 3-MPH (Figure S8).
Figure 2. Evolution of the 1H NMR spectrum of 3-MPH (10 mM) in D2O (pH~
2.5, left, t=0 at the bottom, t= 50 min at the top, X-axis units are chemical shifts
(ppm)) and of the integration of the signal at 6.59 ppm over time (arbitrary
reference: solvent residual peak) (right) with the resulting curve fit obtained
using a single exponential equation.
However, monitoring the absorbance at 262 nm by stopped-flow
experiments (Figure S9) allows the determination of rate
constants of ktautom  5.02x10.sand 4.82x10.s for solutions
at 25 and 12.5 µM, respectively. Together, these data clearly
indicate that the conversion of the enol to the corresponding -
ketoacid is the limiting step of the reaction leading to the formation
of 2,5-dihydroxy-1-4-dithiane-2,5-dicarboxylic acid from 3-MPH.
Figure 1. Determination of the purity of 3-MPH by monitoring its reaction with
3-MST / Trx-regenerating system. Reaction conditions: Tris‡ buffer, E. coli 3-
MST: 10 µM, Trx: 250 µM, TrxR: 1.25 µM, NADPH: 0.3 mM, 3-MP (Santacruz
biotechnology, > 90%, as a reference (___)) or 3-MPH (___): 145 µM. The
theoretical variation in absorbance is 0.91.
Although neither we nor others have evidence for the
presence of monomeric 3-MPH at NMR concentrations,[1,14] the
full conversion of a 0.5 µM solution of reduced 3-MST to its
persulfide form within 0.1s in the presence of a 5 µM solution of
3-MPH (Figure S4A) seems to indicate that either a substantial
amount of 3-MPH is present in solution at this concentration, or
that the conversion of the dimer to 3-MPH is very fast on the
experimental time-scale. However, monitoring the incorporation
Scheme 2. Simplified equilibria taking place upon dissolution of 3-MPH, and
thermodynamic and kinetic constants experimentally determined in this work.
3-mercaptopyruvic acid has not been unambiguously isolated
before, due to its conversion to the corresponding cyclic dithiane
(Scheme 2) in polar solvents.[1] Indeed, while 3-MPH is stable for
1
of deuterium at the -carbon position of the dithiane by H NMR
previously gave t1/2 values of 2 hours for the ring opening.14 To
further investigate the equilibrium Kdimer and the related kinetics,
we studied the reactivity of solutions containing various initial
concentrations of 3-MPH, at pH 7.4, with 5,5-dithio-bis-(2-
nitrobenzoic acid) (DTNB) by absorbance stopped-flow
experiments. Only the open forms of the 2,5-dihydroxy-1-4-
dithiane-2,5-dicarboxylic acid and 3-MPH ("free thiols" in Scheme
S1) can react with the thiophilic reagent, allowing the
determination of their concentration in solution. A typical kinetic
1
hours in chloroform, its typical H NMR signature (= 6.59 ppm)
is slowly converted to new peaks over time upon dissolution in
D2O (pH approximatively 2.5). These new peaks (Figure 2) are
found at similar chemical shifts, and display the same features as
those reported for the diastereoisomers of the cyclic dimer 2,5-
dihydroxy-1-4-dithiane-2,5-dicarboxylic acid.[14] During the course
of the reaction, we did not detect any intermediate. In addition, the
1
comparative integration of the H NMR signals of the species in
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10.1002/cbic.201800199
ChemBioChem
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sensitive to oxidation by hydrogen peroxide than the related
amino-acid cysteine (Figure 4).
experimental trace is shown in Figure 3 (inset). The fitting of the
data at each concentration with the biphasic first order equation
(1) (Experimental Section) gives observed rate constants kobs1
=
3.79 ± 0.04 s-1 and kobs2 = 0.16 ± 0.01 s-1 (Figures S5). The total
variation in absorbance for each experiment corresponds to the
theoretical value expected for a full conversion of [3-MPH]0 to the
mixed disulfide 3-MPH-TNB. The fast phase can be
unambiguously attributed to the reaction of "free thiols" with DTNB
(a similar rate of 3.24 ± 0.01 s-1 is obtained with cysteine under
identical conditions, data not shown). The slower one most likely
represents the rate limiting conversion of the dithiane into 3-MPH
(along with the conversion of the open dimer-TNB adduct to
3MPH-TNB and 3-MPH), that is k-1dimer. The rate constant thus
obtained is 3 orders of magnitude higher than the one determined
In conclusion, we report
a simple synthesis of 3-
mercaptopyruvic acid, allowing detailled spectroscopic
characterization of this biologically important derivative, and the
study of its behaviour in solution. Our data indicate that
commercially available 3-MP is not sodium 3-mercaptopyruvate
(CAS 10255-67-1), but its cyclic dimer disodium 2,5-dihydroxy1-
4-dithiane-2,5-dicarboxylate (CAS 1309654-46-3 / 1001081-70-4).
The dimer formation may be relevant in biological systems.
by H NMR (k = 9.6x10.s),[14] probably reflecting the strongly
1
unfavourable formation of the enol tautomer[1] required for
deuterium incorporation (Scheme S1).
When assuming that only 3-MPH and 5-dihydroxy-1-4-
dithiane-2,5-dicarboxylic acid are present in solution, and that the
variation in the absorbance during the first phase is directly
related to the concentration of 3-MPH, a Kdimer value of (3.64 ±
0.19)x10 M-1 and a corresponding kdimer value of (6.3 ± 2.0)x10
M-1.s-1 are obtained by fitting the data of Figure 3.
Figure 4. Percentage of remaining thiol (determined by DTNB titration
after quenching the reaction with 500U of catalase) over time upon
reacting 200 µM of Cysteine () or 3-MPH () with 1 mM of H2O2 in
KPi at 25°C.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
E.G. is grateful to Dr D. Over and to Dr D. Padovani for useful
discussions and for critically reading the manuscript.
Keywords: mercaptopyruvate • Hydrogen sulfide • H2S Donor
Figure 3. Plot of the fraction of monomeric 3-MPH (, calculated as described
in the Experimental Section) vs the initial 3-MPH concentration. The data were
fitted with equation (2) (Experimental Section). Inset: Representative kinetic
trace (black line) obtained when reacting 30 µM 3-MPH in KPi with 1.6 mM
DTNB in KPi, and the fit curve (white line over the black line) obtained with
equation (1) (Experimental Section).
‡ We use the following abbreviations in the text: KPi: potassium phosphate
buffer (50 mM, pH 7.4, containing 1 mM diethylenetriaminepentaacetic acid
as chelating agent), Tris: trishydroxyméthylaminomethane buffer (Tris-HCl
50 mM, ethylenediaminetetraacetic acid 2 mM, pH 8.0).
[1]
A. J. L. Cooper, M. T. Haber, A. Meister, J. Biol. Chem. 1982, 257,
816.
The determination of this equilibrium constant allowed the
refinement of the K3-MP value calculated for the sulfur transfer step
from 3-MPH to 3-MST (70 ± 4 µM with 3-MPH and 73 ±3 µM with
commercial 3-MP, previously 160 ± 30 µM)12 (Figure S4). The
parameters obtained under steady state conditions remain
[2]
[3]
[4]
H. Kimura, Antioxid. Redox Signal. 2015, 22, 362.
M. H. Stipanuk, P. W. Beck, Biochem. J. 1982, 206, 267;
N. Shibuya, M. Tanaka, M. Yoshida, Y. Ogasawara, T. Togawa,
K. Ishii, H. Kimura, Antioxid. Redox Signal. 2009, 11, 703.
Y. Kimura, S. Koike, N. Shibuya, D. Lefer, Y. Ogasawara, H.
Kimura, Sci Rep 2017, 7, 10459.
J. W. Hylin, J. L. Wood, J. Biol. Chem. 1959, 234, 2141.
E. Kun, D. W. Fanshier, Biochem. Biophys. Acta 1959, 33, 26.
H. T. Nagasawa, D. J. Goon, D. L. Crankshaw, R. Vince, S. E.
Patterson, J.Med. Chem. 2007, 50, 6462.
[5]
[6]
[7]
[8]
unchanged, since the conversion of the dithiane to 3-MPH (t1/2
=
4s) is fast on the experiment time scale. Similarly, 3-MPH or 2,5-
dihydroxy-1-4-dithiane-2,5-dicarboxylate can indiscriminately be
used for biological experiments such as the study of 3-
mercaptopyruvate analogues as antidote for cyanide
poisoning.[8,14]
[9]
[10]
[11]
J. Parrod, Compt. rend. 1942, 215, 146
J. F. Jackson, T. M. Smith, Biochim. Biophys. Acta 1966, 121, 159.
S. Tanabe, Y. Ogasawara, M. Nawata, K. Kawanabe, Chem.
Pharm. Bull. 1989, 37, 2843
J.-C. Lec, S. Boutserin, H. Mazon, G. Mulliert, S. Boschi-Muller, F.
Talfournier, ACS Catal. 2018, 8, 2049-2059.
S. Mori, A. Garzan, O. V. Tsodikov, S. Gameau-Tsodikova,
Biochemistry, 2017, 56 6087.
S. E. Patterson, A. R. Monteil, J. F. Cohen, D. L. Crankshaw, R.
Vince, H. T. Nagasawa, J. Med. Chem., 2013, 56, 1346
W. D. Kumler, E. Kun, Biochem. Biophys. Acta, 1958, 27 464.
[12]
[13]
[14]
[15]
A full investigation of the potential physiological significance
of the formation of the dimer is beyond the scope of this study.
However, in this context, it is interesting to note that 3-MPH is less
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10.1002/cbic.201800199
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COMMUNICATION
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10.1002/cbic.201800199
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COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
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COMMUNICATION
A synthesis of the sulfur metabolic
compound 3-mercaptopyruvic acid (3-
MPH) is reported and allowed its
isolation and characterization for the
Erwan Galardon* and Jean-Christophe
Lec
Page No. – Page No.
first
time.
Detailed
kinetic,
thermodynamic and spectroscopic
studies of its complex behaviour in
solution are discussed.
Synthesis,
reactivity of 3-mercaptopyruvic acid..
characterization
and
This article is protected by copyright. All rights reserved.