Detoxification system for inorganic arsenic: Transformation of As 2O3 into TMAO by vitamin B12 derivatives and conversion of TMAO into arsenobetaine

Article abstract of DOI:10.1039/b808937j

A new two-step synthetic pathway developed for the transformation of arsenic trioxide [iAs(iii); As₂O₃] into arsenobetaine (AB; Me₃As⁺CH₂CO₂^-) involves treatment of iAs(iii) with native B₁₂ or biomimetic B₁₂ in the presence of glutathione (GSH) to give TMAO with a high selectivity and a high conversion rate; subsequent treatment of TMAO with iodoacetic acid in the presence of GSH gives arsenobetaine. The Royal Society of Chemistry.

Full text of DOI:10.1039/b808937j

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Detoxification system for inorganic arsenic: transformation of As2O3
into TMAO by vitamin B12 derivatives and conversion of TMAO
into arsenobetainew
Koichiro Nakamura,*a Yoshio Hisaeda,b Ling Panb and Hiroshi Yamauchic
Received (in Cambridge, UK) 27th May 2008, Accepted 26th August 2008
First published as an Advance Article on the web 25th September 2008
DOI: 10.1039/b808937j
A new two-step synthetic pathway developed for the transforma-
tion of arsenic trioxide [iAs(III); As2O3] into arsenobetaine (AB;
Me3As+CH2CO2ꢀ) involves treatment of iAs(III) with native
B12 or biomimetic B12 in the presence of glutathione (GSH) to
give TMAO with a high selectivity and a high conversion rate;
subsequent treatment of TMAO with iodoacetic acid in the
presence of GSH gives arsenobetaine.
one three-hundredth of that of arsenic trioxide [iAs(III);
arsenite; As2O3:LD50, 0.03 g kgꢀ1, mouse, oral].5 Moreover,
as a result of studies on the metabolism and excretion of AB in
humans and animals,6 it is now widely accepted that AB is
chemically stable, has a low tissue affinity, and is rapidly
excreted from the human body. Although AB is produced
by the action of marine organisms, there have been no
successful attempts to synthesize AB artificially in a safe and
cost-effective way.
Inorganic arsenics (iAs) are widely used in non-ferrous metal
refining, the glass industry, and in the production of arsenic
agricultural chemicals, wood preservatives, defoliants, and
arsenic semiconductors. However, chronic poisoning by
arsenicals has occurred through large-scale environmental
pollution,1 and acute poisoning has occurred through the
use of these compounds as a means of suicide or homicide.2
Causal associations between the use of inorganic arsenics and
occurrences of chronic poisoning, lung cancer, skin cancer,
and bladder cancer have been demonstrated.3 With regard to
arsenic trichloride, a raw material for metallic arsenic, an
ingredient of Group III/Group V compound semiconductors
(GaAs), new research has been reported on the synthesis of
chelate compounds through control of their oxidation states.4
Although this research shares with our research a common
goal of synthesizing new arsenic compounds, our research
differs in that our principal goal is to synthesize arsenic
compounds that are nontoxic or which have a level of toxicity
that is as low as possible. The toxicity of arsenic compounds is
markedly dependent on their chemical structure, and some
methylated arsenic compounds have lower toxicities than
those of inorganic arsenic compounds. In particular, arseno-
betaine [AB; Me3As+CH2CO2ꢀ; (trimethylarsonio)acetate]
has a low toxicity (LD50, 10 g kgꢀ1, mouse, oral)5 and is
found in high levels in fishery products.5 The acute toxicity of
AB, as determined from animal experiments, is approximately
The conversion of highly toxic inorganic arsenic compounds
into nontoxic AB (in other words, the elimination of toxicity) is
defined as ‘‘detoxification’’. The detoxification process should
be safe and cost-effective. In marine ecosystems, inorganic
arsenicals are biologically methylated, resulting in accumula-
tion, through the food chain, of arsenic in the form of AB
mainly in fish and shellfish.7 It has been hypothesized that this
biological methylation is promoted by alternating reduction of
As(V) to As(III) and oxidative methylation.8 These biochemical
reactions involve a methyltransferase and a reductase, respec-
tively.9 Although many enzymes have been assumed to be
involved in the conversion of inorganic arsenicals into AB, no
enzyme has been isolated that is directly associated with the
synthesis of AB.10 Although chemical methods have been
proposed for converting arsenic trioxide into trimethylated
arsenic by treatment with alkylaluminium or Grignard
reagents, these methods are both difficult and hazardous to
apply.11 Because the former is a reagent that can only be used
under anhydrous conditions and the latter is inactivated by
moisture, they can only be used for small-scale syntheses, so the
current chemical syntheses of methylated arsenics may have no
contribution to make in reducing future health problems
from exposure to arsenicals or in establishing counter-
measures to existing health problems. In addition, these reac-
tions are unlikely to be useful in safe treatment of inorganic
arsenicals in the industrial sphere.
Recently, a scientific technology called ‘‘biomimetics’’ has
been developed that involves the application of various bio-
chemical reactions that are highly efficient and occur without the
production of undesirable by-products.12 The biochemical
methylation of arsenic involves a methyltransferase and a
reductase. The establishment of a biomimetic system using this
reaction could enable artificial conversion of arsenic trioxide into
AB in a highly efficient and environmentally friendly manner.
Transmethylation is among the enzymatic reactions in
which vitamin B12 is involved. In this study, a system was
a R & D Department, Nippon Sheet Glass Co., Ltd., Chitose-cho 2,
Yokkaichi, Mie, Japan. E-mail: KoichiroNakamura@mail.nsg.co.jp;
Fax: +81-59-353-4707; Tel: +81-59-352-2493
b Department of Chemistry and Biochemistry, Graduate School of
Engineering, Kyushu University, Moto-oka 744, Nishi-ku, Fukuoka
819-0395, Japan. E-mail: yhisatcm@mail.cstm.kyushu-u.ac.jp;
Fax: +81-92-802-2827; Tel: +81-92-802-2826
c School of Allied Health Sciences, Kitasato University, Kitasato 1-15-1,
Sagamihara, Kanagawa, Japan. E-mail: hyama@kitasato-u.ac.jp;
Fax: +81-42-778-8113; Tel: +81-42-778-8113
w Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/b808937j
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Table 2 Arsenic carboxymethylation of trimethylarsine oxide into
arsenobetainea
Absolute yield (%)b
iAs
0
MMA
0
DMA
0
TMAO
0
AB
99
Carboxymethyl donor
ICH2CO2H
a
Carboxymethylation reaction were carried out in 100 mM phospho-
ric acid–citric acid buffer (pH 5) at 37 1C for 2 h. Initial concentrations
were as follows. TMAO: 2.0 ꢂ 10ꢀ7 M; GSH: 5.0 ꢂ 10ꢀ3 M;
b
iodoacetic acid: 5.0
HPLC-ICP-MS.
ꢂ
10ꢀ3 M. Products were assayed by
TMAO (0.2 mM) was also quantitatively converted into AB
(99%) in the presence of GSH (5 mM) and iodoacetic acid
(5 mM) in 100 mM phosphoric acid–citric acid buffer
(pH 5, 37 1C) (Table 2).
Scheme 1 Detoxification system for inorganic arsenic.
For ‘‘biomimetic B12’’ (methylcobyric acid), there is a pre-
vious report of a synthesis of methylcobyric acid from mono-
cyano monoaquo cobyrinate,13 but no details of the
experimental procedures are given. We first synthesized hydro-
phobic B12 [(methyl)(aquo)cobyrinate heptamethyl ester
perchlorate] from naturally occurring vitamin B12 (cyano-
cobalamin). Seven of the methyl ester groups located near
the corrin ring of the hydrophobic B12 were then hydrolyzed
with sodium hydroxide to give ‘‘biomimetic B12’’.z This is a
new synthetic method. In addition, the previous report13 states
that the authors synthesized methylcobyric acid with the aim
of using it as a standard substance for the identification of
methylated corrinoids biosynthesized by microbial cells. They
did not assess the reactivity of methylcobyric acid as a methyl
donor. Our study is the first, therefore, to report on the
reactivity of methylcobyric acid in a transmethylation reac-
tion. It follows, therefore, that this is the first study to report
on the methylation of arsenic by ‘‘biomimetic B12’’.
constructed that consists of ‘‘biomimetic vitamin B12’’ [sodium
(methyl)(aquo)cobyrinate perchlorate],z which mimics natu-
rally occurring methylated vitamin B12 (methylcobalamin),
together with a B12 coenzyme; these serve as a model for
methyltransferase (Scheme 1), whereas the reduced form of
glutathione (GSH) serves as a reductase model. We found that
this system effected a highly efficient conversion of arsenic
trioxide into trimethylarsine oxide (TMAO; Me3AsQO)
(Table 1).
When naturally occurring coenzyme B12 was used as a
source of methyl groups, the absolute yields of the products
were as follows: inorganic arsenics (iAs) 0%, methylarsonic
acid [MMA; MeAs(O)(OH)2] 0%, dimethylarsonic acid
[DMA; Me2As(O)(OH)] 0%, TMAO 97%, tetramethylarso-
nium (TeMA; Me4As+) 1% (Table 1). When the biomimetic
coenzyme B12 was used as the source of methyl groups, the
absolute yields of the products were as follows: iAs 0%, MMA
0%, DMA 0%, TMAO 99%, and TeMA 1% (Table 1).
Therefore, the biomimetic coenzyme system produced the
same level of selectivity in conversion as that of the naturally
occurring coenzyme. There have been no previous reports on
the selective conversion of arsenic trioxide into TMAO by a
naturally occurring coenzyme with an efficiency as high as
97%, and no reports of any such conversion by using a
biomimetic coenzyme.
Our literature searches have failed to find any reports of
studies on the conversion of arsenic trioxide into TMAO in the
presence of methylcobalamin. One paper14 reports the con-
version of arsenic trioxide into MMA in the presence of
methylcobalamin; however, this does not give a description
of TMAO synthesis. Another study15 reports that both MMA
as the main product and DMA as a by-product were obtained
in the presence of methylcobalamin and a reducing agent;
however, this does not give a description of the synthesis of
TMAO. Likewise, a third study16 reports that both MMA as
the main product and DMA as a by-product were obtained;
however, that does not report a confirmation of the synthesis
of TMAO. On the basis of the above information, it could be
deduced that methylation of arsenic trioxide in the presence of
methylcobalamin proceeds only as far as the synthesis of
MMA and DMA. Our report is the first to describe the
conversion of arsenic trioxide into TMAO in the presence of
methylcobalamin, with an absolute yield of 97%.
We also found that AB was quantitatively generated from
TMAO and haloacetic acid in the presence of GSH in a
neutral aqueous solution under mild conditions (Table 2).
Table 1 Arsenic methylation mediated by methyl B12 derivativesa
Absolute yield (%)b
iAsc MMA DMA TMAO TeMA
Entry Methyl donor
1
2
a
Native B12
Biomimetic B12
0
0
0
0
0
0
97
99
1
1
TMAO is an arsenic compound of low toxicity [LD50,
10 600 mg kgꢀ1],17 and it is known, from an oral and intraper-
itoneal administration study in hamsters, that under in vivo
conditions TMAO is reduced to trimethylarsine (TMA), part
of which is excreted in exhaled breath. TMA is also an arsenic
compound of low toxicity (LD50, 7870 mg kgꢀ1), but a study
Methylation reactions were carried out in Tris-HCl buffer (pH 8) at
100 1C for 2 h. Initial concentrations were as follows. Native B12
(methylcobalamin) or biomimetic B12, sodium (methyl)(aquo)cobyrinate
perchlorate, [(CH3)(H2O)Cob(III)(CO2Na)7]+ClO4ꢀ: 7.4 ꢂ 10 ꢀ2M;
b
GSH: 1.3 M; iAs: 5.4
c
ꢂ
10ꢀ5 M. Products were assayed by
HPLC-ICP-MS. iAs includes arsenite [iAs(III)] and arsenate [iAs(V)].
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in animals has shown that it may cause mild transient hemo-
lysis.18–19 On the other hand, it is known that AB is stable in
vivo, has low affinity for body tissues, and is rapidly excreted.6
Thus, it can be inferred that desirable detoxification of arsenic
trioxide should proceed from conversion of arsenic trioxide
into TMAO with subsequent conversion into AB.
arsenicals generated in the process of producing chemical
agents.20
This research was supported in part by a grant program
‘‘Collaborative Development of Innovative Seeds’’ from the
Japan Science and Technology Agency (JST) and a Grant-
in-Aid for Scientific Research on Priority Areas (460) from the
Ministry of Education, Culture, Sports, Science and Technol-
ogy (MEXT) of Japan.
There is a report on a method for synthesizing AB from
TMA with halogenated acetate ester in an organic solvent to
form an AB ester that is subsequently hydrolyzed to produce
AB.11 However, there is no report describing the synthesis of
AB from TMAO in an aqueous solution under mild condi-
tions; our report is the first to describe such a synthesis. The
reaction does not require an organic solvent and it proceeds in
aqueous solution under mild conditions, and it can therefore
be regarded as an economical and eco-friendly process.
In summary, [i] we propose a new treatment system for
toxicants, i.e. ‘‘detoxification of arsenic’’ in which arsenic
trioxide is transformed into AB. [ii] To accomplish this
detoxification (iAs(III) - AB), we designed a two-step synth-
esis process [iAs(III) - TMAO - AB] involving reactions
under mild aqueous conditions. [iii] The first step in this two-
step process is the trimethylation of arsenic trioxide, which is
very difficult to accomplish selectively. In other words, the
following six types of arsenic compound may be produced
during methylation of arsenic trioxide: unchanged iAs(III),
iAs(V), MMA, DMA, TMAO, and TeMA. We succeeded,
for the first time, in selectively synthesizing TMAO from
arsenic trioxide by using naturally occurring vitamin B12
(methylcobalamin). [iv] To reproduce, in our biomimetic
system, the high selectivity shown by naturally occurring
vitamin B12, we successfully synthesized ‘‘biomimetic B12’’
by using a new synthesis method. [v] By using this ‘‘biomimetic
B12’’, we succeeded, in selectively synthesizing TMAO exclu-
sively from arsenic trioxide in a similar manner to the reaction
of naturally occurring vitamin B12; this is the first report of
such a synthesis. [vi] The second reaction (TMAO - AB) in
the two-step synthetic pathway is the first reported case in
which TMAO has been directly converted into AB in
an aqueous solution under mild conditions without the use
of organic solvents or the use of highly volatile TMA as
reactant. As described above, we propose a detoxification
system for converting a toxicant (arsenic trioxide) into a
nontoxic compound (AB) without the production of toxic
by-products, and our experiments have demonstrated the
feasibility of this process.
Notes and references
z Biomimetic B12 was prepared as follows. First, 4 M aqueous NaOH
(20 mL) was added to a solution of hydrophobic vitamin B12 (5 mg) in
methanol (10 mL).21 The mixture was stirred in a screw-capped
polypropylene tube and incubated in a thermostated bath at 30 1C
for 20 h. The solution was then neutralized with 6 M aqueous HCl and
diluted with ultra-pure water (10 mL).
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The vitamin B12 derivatives and the GSH that are used in
the detoxification method developed in this study are safe and
environmentally friendly materials. Therefore, our detoxifica-
tion process is also safe and environmentally friendly. Our
method for detoxification of inorganic arsenics could be
applied in the prevention of poisoning by inorganic arsenical
compounds that are present in well water, which has been a
widespread cause of chronic poisoning by arsenic. In addition,
the method could be used to detoxify surplus inorganic
arsenicals in industry, which currently present considerable
difficulties in terms of their safe disposal. Finally, a more-
specific application would be in the detoxification of inorganic
21 Y. Murakami and Y. Hisaeda, Bull. Chem. Soc. Jpn., 1985, 58,
2652–2658.
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