Diastereoisomerism of thiol complexes of arsenic acids and pseudoasymmetry of arsenic: A 1H and 13C NMR study

Article abstract of DOI:10.1002/mrc.1739

Diastereoisomeric complexes of methylphenylarsinic acid and (L)-glutathione could be partially separated by HPLC, but the separated compounds rapidly racemized, presumably by pyramidal inversion at the arsenic atom. Hydrolysis of the diastereoisomeric com

Full text of DOI:10.1002/mrc.1739

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2006; 44: 151–162
Published online 15 December 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1739
Diastereoisomerism of thiol complexes of arsenic acids
and pseudoasymmetry of arsenic: a 1H and 13C NMR
study
John S. Edmonds,∗ Takashi Nakayama, Takuya Kondo and Masatoshi Morita
Endocrine Disrupter Research Laboratory, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
Received 11 August 2005; Revised 06 October 2005; Accepted 11 October 2005
Diastereoisomeric complexes of methylphenylarsinic acid and (L)-glutathione could be partially separated
by HPLC, but the separated compounds rapidly racemized, presumably by pyramidal inversion at
the arsenic atom. Hydrolysis of the diastereoisomeric complexes yielded methylphenylarsinous acid
as a pair of enantiomers revealed by a 1H NMR study with an asymmetric lanthanide shift reagent.
Methylphenylarsinous acid was also synthesized as an enantiomeric pair, shown by an asymmetric shift
1
reagent experiment, by the hydrolysis of iodomethylphenylarsine. H and 13C NMR spectroscopy were
used to demonstrate that complexing of phenylarsonic acid with (R,S)-3-mercapto-1,2-propanediol and
with (R,S)-1-mercapto-2-propanol yielded, in each case, a pair of enantiomers, PhAs[(R)-ligand)]2, PhAs[(S)-
ligand)]2, in which the homomorphic ligands were diastereotopic, and a pair of diastereoisomers, PhAs[(R)-
ligand][(S)-ligand], which differed from each other in the configuration about the pseudoasymmetric
arsenic atom. Copyright  2005 John Wiley & Sons, Ltd.
KEYWORDS: 1H NMR; 13C NMR; COSY; HMQC; methylphenylarsinic acid; glutathione complexes; methylphenylarsinous
acid; asymmetric lanthanide shift reagent; phenylarsonic acid; thiol complexes; pseudoasymmetric arsenic; chemical warfare
agents
be expected to generate a chiral arsenic atom. If the thiol
is also chiral, e.g. (L)-glutathione, the complexes formed
between the reduced arsenic species and the ligand should
exist as a pair of diastereoisomers (e.g. 1d). Subsequent
hydrolysis of the complexes might be expected to yield the
enantiomeric pair of methylphenylarsinous acids (1e). We
investigated these possibilities in the 1H and 13C NMR study
reported here.
INTRODUCTION
Diphenylarsinic acid (Fig. 1, 1a) and phenylarsonic acid (1b)
have been found in ground water at Kamisu in Ibaraki
Prefecture in eastern Japan and their presence is believed to
result from the inappropriate disposal of warfare agents
or industrial wastes.1 The toxicity of these compounds,
at least in part, must result from their readiness to be
reduced by, and complexed with, cellular thiols such as
glutathione.2 We examined the stereochemistry of complexes
of these acids with (L)-glutathione [N-(N-(S)-ꢀ-glutamyl-(R)-
cysteinyl)glycine] and with (R)-cysteine by 1H and 13C NMR
spectroscopy and demonstrated the diastereotopic nature
of the pair of homomorphic ligands [(L)-glutathione or (R)-
cysteine] bound to trivalent arsenic when phenylarsonic
acid was allowed to react with the thiol ligand, and the
diastereotopicity of the phenyl groups when diphenylarsinic
acid was reduced and complexed by the chiral ligand (L)-
glutathione.3
In addition, we used NMR spectroscopy to explore the
stereochemistry of complexes of phenylarsonic acid (1b) with
(R,S)-mixtures of chiral thiol ligands. The reduction of pheny-
larsonic acid (1b) and its bonding to enantiomeric ligands
gives the possibility for the formation of PhAs[ꢁRꢂ-ligand]2,
PhAs[ꢁSꢂ-ligand]2, and PhAs[(R)-ligand][(S)-ligand]. The
first two of these possibilities (those with homomorphic
ligands) are, of course, enantiomeric and will be identical
to each other in all but chiroptical properties. However, the
ligands in each case will be diastereotopic and would be
expected to be magnetically nonequivalent. Configuration
at the arsenic atom will be of no consequence. In the case
where the ligands are of different configuration, (R) and
(S), two diastereoisomers are possible that differ from each
other in the configuration of the arsenic atom, i.e. the arsenic
atom would be expected to be a pseudoasymmetric centre by
Subsequently, methylphenylarsinic acid (1c) was found
in ground water and rice plants from Kamisu.4 Possibly,
methylphenylarsinic acid (1c) has arisen by microbial
methylation by soil organisms of phenylarsonic acid (1b).
Reduction of methylphenylarsinic acid (1c) by thiols would
1
the definition of Prelog.5,6 We used H and 13C NMR spec-
ŁCorrespondence to: John S. Edmonds, Endocrine Disrupter
Research Laboratory, National Institute for Environmental Studies,
16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan.
E-mail: edmonds.john.s@nies.go.jp
troscopy to investigate whether experimental results were
consistent with predictions made from such stereochemical
considerations.
Copyright  2005 John Wiley & Sons, Ltd.

152
J. S. Edmonds et al.
Figure 1. Some compounds used or referred to in this study. (1a), diphenylarsinic acid; (1b), phenylarsonic acid; (1c),
methylphenylarsinic acid; (1d), methylphenylarsinic acid – (L)-glutathione complexes (stereochemistry is not shown, but both the
arsenic atom and the glutathione residue are chiral, giving rise to a pair of diastereoisomers); (1e), methylphenylarsinous acid (shown
as a pair of enantiomers resulting from chiral arsenic); (1f), europium tris[3-(heptafluoro-propyl-hydroxymethylene)-(C)-camphorate];
(1g), iodomethylphenylarsine; (1h), methylphenylarsinous acid anhydride; (1i), phenylarsonic acid – ꢁ1-mercapto-2-propanolꢂ2
complexes (no stereochemistry is indicated, but see Fig. 5); (1j), phenylarsonic acid – ꢁ3-mercapto-1,2-propanediolꢂ2 complexes
(the stereochemistry will be analogous to that for the phenylarsonic acid – ꢁ1-mercapto-2-propanolꢂ2 complexes shown in Fig. 5).
°
relaxation delay, 1 s; pulse width, 7.6 µs (45 ); number
of scans, 64 to 256. Typical parameters for 13C NMR
were as follows: spectral width, 63 kHz; number of data
points, 32.8K; 1.92 Hz per point 13C digital resolution. i.e.
š4.8 ð 10ꢀ3 ppm; acquisition time 0.52 s; relaxation delay,
EXPERIMENTAL
NMR measurements
NMR spectra were recorded in D2O, buffers made up in
D2O, and CDCl3 on a JEOL ECA 800 spectrometer (JEOL,
Tokyo, Japan) operating at 800 MHz (1H) or 200 MHz (13C).
°
1.4 s; pulse width, 2.38 µs (30 ); number of scans, 1000 to
16 000. Two-dimensional 1H–1H COSY and HMQC spec-
tra were recorded using the pulse programs of the JEOL
1
Shifts are reported relative to external TMS. H NMR spec-
tra were also recorded on some CDCl3 solutions containing
the compound under investigation and the asymmetric lan-
thanide shift reagent europium tris[3-(heptafluoro-propyl-
hydroxymethylene)-(C)-camphorate] (1f). Data processing
was carried out using Delta 4.2.3 software. Typical param-
Delta software library. H–1H COSY data parameters were
1
as follows: spectral width, 15 kHz; relaxation delay, 1.5 s;
acquisition time, 0.107 s; 1024 ð 256 data points; 256 scans.
The HMQC data parameters were as follows: spectral width,
15 kHz; 1024 ð 256 data points; 4 scans. All spectra were
1
eters for H NMR were as follows: spectral width, 15 kHz;
1
°
number of data points, 16.4K; 0.917 Hz per point H digital
resolution, i.e. š5.7 ð 10ꢀ4 ppm; acquisition time, 1.09 s;
recored on solutions at 25 C (probe temperature). Spectra
were recorded for 1 to 10 mM solutions.
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2006; 44: 151–162

Phenylarsenic acid–thiol complexes studied by NMR 153
ODS 3 reversed phase C18 column (250 mm ð 4.6 mm i.d.
3 µm particle size, GL Sciences, Tokyo, Japan), or on to
Atlantis dC18 columns (150 mm ð 4.6 mm i.d. 5 µm particle
size, or 250 mm ð 19 mm i.d. 10 µm particle size, Nihon
Waters, Tokyo, Japan). A similar degree of partial sep-
aration was achieved on all three columns. Flow rates
were 0.5 ml/min for the smaller columns and 9 ml/min
for the larger ones. The individual partially separated
(enriched) diastereoisomers were accumulated indepen-
dently during a single day when the mobile phase con-
tained formate buffer. The methanol and water were
Mass spectrometry
High-resolution FABC MS were measured on A JEOL
JMS-700 GC/MS (JEOL, Tokyo, Japan). Electrospray (ES)
MS were measured on a Quattro Ultima instrument (Micro-
mass, UK).
Compounds
Chemicals
Reduced (L)-glutathione (99%) was purchased from Sigma
Chemicals, Tokyo, Japan, and phenylarsonic acid (97%)
from Acros Organics, Geel, Belgium. (R,S)-1-Mercapto-2-
propanol (95%) and europium tris[3-(heptafluoro-propyl-
hydroxymethylene)-(C)-camphorate] (1f, 98%) were ob-
tained from Aldrich, Tokyo, Japan. Methylphenylarsinic
acid (1c) was synthesized by the methylation of pheny-
larsine oxide with iodomethane by the method of Bertheim.7
Iodomethylphenylarsine (1g) was synthesized by the
method of Burrows and Turner.8 Other chemicals, includ-
ing (R,S)-3-mercapto-1,2-propanediol (thioglycerol, 98%)
and solvents were from Wako Pure Chemicals, Osaka,
Japan.
°
then removed by rotary evaporation (<40 C) and the
remaining solid ammonium formate was removed by
sublimation at room temperature overnight under high
vacuum. The individual partially separated (enriched)
diastereoisomers were examined by 1H NMR and rechro-
matographed under the same conditions on the follow-
ing day.
Synthesis of methylphenylarsinous acid (1e) by the
hydrolysis of iodomethylphenylarsine (1g)
1H and 13C NMR data for iodomethylphenylarsine are given
in Table 2. A portion (5 µl) was dissolved in acetone-d6
(0.75 ml) and an aliquot of this acetone solution (10 µl)
Buffers
Buffers (0.1 M) were made according to the formulas
listed in the literature.9 Buffer of pH value 7.0 was
made with phosphate; of pH 2.0 with KCl/DCl; those
of pH 3.0 to 6.0 with citrate; and that of pH 13.0 with
KCl/NaOH. The same quantities of reagents (weights of
chemicals, volumes of solvent) were used for making
buffers in D2O as if they were being made in water. No
consistent differences were found between readings made
for buffers in D2O and those in water (Piccolo 2 HI 1290
meter with amplified pH electrode (Hanna Instruments,
Portugal) and a ISFET KS723 solid-state meter (Shindengen,
Tokyo, Japan)) and no corrections for isotope effects were
made.
1
was then added to D2O (1 ml). The H NMR spectrum of
the resulting methylphenylarsinous acid was then recorded
(Table 2).
1H NMR examination of methylphenylarsinous acid (1e),
prepared by the hydrolysis of iodomethylphenylarsine
(1g), in the presence of europium tris[3-(heptafluoro-
propyl-hydroxymethylene)-(C)-camphorate] (1f)
A solution of iodomethylphenylarsine in acetone-d6 (2 µl in
250 µl) was added to nitrogen-purged aqueous phosphate
buffer (5 ml, 0.1 M, pH 7) in a separating funnel. Nitrogen
was passed through the solution for 1 h and then it
was extracted with deuterochloroform (3 ð 0.5 ml). The
1H NMR spectrum of this solution showed the presence
of two compounds. One of these was readily identified
as methylphenylarsinous acid by comparison with the
spectrum of the acid produced simply by hydrolysis of
iodomethylphenylarsine in D2O, and the other, which was
apparently present as a pair of diastereoisomers (doubling
of resonances), was tentatively identified as the anhydride
of methylphenylarsinic acid (1h). NMR data are given in
Table 2. ES MS (Cve mode) m/z 185 [M C H]C, 201 [M C
NH3]C (methylphenylarsinous acid), and m/z 351 [M C H]C,
367 [M C NH3]C (methylphenylarsinous acid anhydride).
Portions of a solution of europium tris[3-(heptafluoro-
propyl-hydroxymethylene)-(C)-camphorate] in CDCl3 were
added so that the estimated molar ratio of the shift reagent
to methylphenylarsinous acid ranged from 0.25 : 1 to 1 : 1.
Methylphenylarsinic acid – (L)-glutathione complexes
(1d)
Methylphenylarsinic acid (80 mg, 0.4 mmol) was added
to an aqueous solution (10 ml) of (L)-glutathione (614 mg,
2 mmol) that had been purged with oxygen-free nitrogen.
The solution was allowed to stand at room temperature
overnight and the methylphenylarsinic acid – (L)-glutathione
complexes formed as a white solid that was collected by
centrifugation, washed with water (3 ð 5 ml), and dried;
yield 84%, mp 178 – 182 C (decomp.) 1H NMR and 13C
°
NMR spectral data (Table 1). High-resolution FAB MS
(Cve mode) (M C H) 474.0540 C17H25O6N3SAs requires
474.0680.
HPLC separation of methylphenylarsinic
acid – (L)-glutathione diastereoisomers (1d)
1
The H NMR spectrum of the solution was recorded after
Partial separation (Fig. 4) of the two methylphenylarsinic
acid – (L)-glutathione diastereoisomers was achieved by
HPLC (Shimadzu LC-10ADVP with SPD-10AVP UV–vis
detector set at 260 nm). The mobile phase was 10
or 20 mM citrate (pH 4) or formate buffer (pH 4),
60%; methanol, 40%. A solution of the diastereoisomers
(300 µg/ml, 250 µl or 500 µl) was injected on to an Inertsil
each addition. Protons assigned to methylphenylarsinous
acid underwent marked downfield shifts, but those assigned
to the anhydride were unaffected by the presence of the shift
reagent. The aromatic protons of the methylphenylarsinous
acid showed the clearest shifts unmasked by the resonances
of the shift reagent.
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J. S. Edmonds et al.
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Phenylarsenic acid–thiol complexes studied by NMR 155
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156
J. S. Edmonds et al.
reaction mixture for a further 90 min. The aqueous solu-
tion was then extracted with dichloromethane (3 ð 10 ml),
the combined extracts were dried (anhydrous MgSO4),
and the solvent was evaporated to give phenylarsonic
acid – (R,S)-mercapto-1,2-propanediol)2 complexes (1j) as a
colourless syrup (21 mg, 28%). 13C NMR, CDCl3, υ ppm 33.99,
34.30, 35.16, 35. 48 (4 ð –CH2SH); 64.93, 64.96, 65.03, 65.06
(4 ð –CH2OH) 71.49, 72.25, 72.77, 73.18 (4 ð –CH(OH)–);
128.96, 129.10, 129.27, 130.91, 131.07, 131.12, 131.54, 131.68 (o-
, m-, p- aromatic carbons), 142.17, 142.41, 142.73 (3 ð C-As).
High-resolution FAB MS Cve mode [ꢁM–2Hꢂ C H] 364.9861,
C12H18O4S2As requires 364.9863.
Hydrolysis of methylphenylarsinic acid – (L)-glutathione
complexes (1d) – preparation and 1H NMR examination
of methylphenylarsinous acid (1e) in the presence of
europium tris[3-(heptafluoro-propyl-hydroxymethylene)-
(C)-camphorate] (1f)
Unseparated methylphenylarsinic acid – (L)-glutathione di-
astereoisomers (10.3 mg, 0.02 mmol) were dissolved under
nitrogen, at room temperature, in NaOH–KCl buffer (0.1
M, pH 13, 5 ml) and nitrogen bubbling continued for 1 h.
The solution was then extracted with dichloromethane
(3 ð 3 ml), the combined extracts dried with MgSO4, the
1
solvent evaporated, and the residue examined by H NMR
spectroscopy (CDCl3). As for the extracted hydrolysis prod-
uct of iodomethylphenylarsine, both methylphenylarsinous
acid and its anhydride were present. Portions of a solution
of europium tris[3-(heptafluoro-propyl-hydroxymethylene)-
(C)-camphorate] in CDCl3 were added to the CDCl3 solution
in the NMR tube so that the estimated molar ratio of the
shift reagent to methylphenylarsinous acid ranged from
1 : 1 to 2 : 1. The 1H NMR spectrum of the solution was
recorded after each addition. As for the products of the
hydrolysis of iodomethylphenyl arsine, protons assigned to
methylphenylarsinous acid underwent marked downfield
shifts, but those assigned to the anhydride were unaffected
by the presence of the shift reagent. Again, the aromatic pro-
tons of the methylphenylarsinous acid showed the clearest
shifts unmasked by the resonances of the shift reagent.
RESULTS AND DISCUSSION
Methylphenylarsinic acid – (L)-glutathione complexes
(1d)
Both 1H and 13C NMR spectra of the complex formed between
methylphenylarsinic acid and (L)-glutathione (1d) exhibited
duplication of all resonances (Table 1) and the 1H–1H
COSY spectrum (Fig. 2) clearly showed the connectivities
of the glutamic acid and cysteine residues consistent with
the presence of two separate compounds. As expected,
chirality resulting from the tetrahedral configuration of
trivalent arsenic (with the lone electron pair providing the
fourth arm of the tetrahedron) together with the chirality
of (L)-glutathione yielded a pair of diastereoisomers in a
1 : 1 ratio. The two diastereoisomers revealed in the NMR
spectra were partially separated by HPLC (Fig. 3). However,
after removal of the eluent (methanol and water by rotary
Phenylarsonic acid – (R,S)-1-mercapto-2-propanol)2
complexes (1i)
(R,S)-1-Mercapto-2-propanol (188 mg, 179 µl, 2.04 mmol)
was added to a solution of phenylarsonic acid (41.3 mg,
0.204 mmol) in water (50 ml) at room temperature. The
water had been previously deoxygenated by the passage
of oxygen-free nitrogen. Nitrogen was bubbled though the
reaction mixture for a further 90 min during which time the
phenylarsonic acid – ꢁ1-mercapto-2-propanolꢂ2 complexes
(1i) precipitated as a white solid. The solid was collected
by centrifugation, washed with water (3 ð 10 ml) and dried
°
evaporation with temperature <40 C; formate buffer under
high vacuum), rechromatography in the same system 24 h
later of each partially separated (enriched) diastereoisomer,
independently, showed peaks of approximately equal size
for the two compounds and therefore racemization had
occurred, presumably by pyramidal inversion of the arsenic
atom. The 1H NMR spectra of the separated peaks confirmed
this; corresponding resonances for each compound were of
approximately equal intensity. Thus, at room temperature,
the half-life for the racemization about the arsenic atom was
probably a few hours; long enough to allow partial separation
by HPLC (Fig. 3), but too short to allow the time necessary
for removal of the eluent before rechromatography or NMR
examination. This was unexpected as previous work10–12
suggested that the pyramidal chiral arrangement of ligands
at the arsenic atom would be sufficiently stable for the
separation of the methylphenylarsinic acid – (L)-glutathione
diastereoisomers by HPLC.
The ease with which the configuration about the arsenic
atom in the methylphenylarsinic acid – (L)-glutathione com-
plex (1d) racemized precluded any possibility of resolution
of potentially enantiomeric methylphenylarsinous acids (1e)
by chromatographic separation of the diastereoisomers of
which they were part. However, the presence of the two
enantiomers of methylphenylarsinous acid (1e) in solution
was demonstrated by the use of an asymmetric lanthanide
shift reagent (1f), and their separate existence, at least in the
presence of the shift reagent, on the NMR timescale, was
(56 mg, 82%; mp 129 C). 1H and 13C NMR spectral data
°
are given in Table 3. High-resolution FAB MS Cve mode
[ꢁM ꢀ 2Hꢂ C H] 333.0021, C12H18O2S2As requires 332.9964.
1H NMR spectra of phenylarsonic
acid – (R,S)-1-mercapto-2-propanol)2 complexes (1i) in
the presence of europium tris[3-(heptafluoro-propyl-
hydroxymethylene)-(C)-camphorate] (1f)
The 1H NMR spectrum of the phenylarsonic acid – (R,S)-
1-mercapto-2-propanol)2 complexes in CDCl3 solution
(0.0045 mmol, 0.5 ml) was recorded with 0, 0.5, 1.0, 1.5, and
2.0 molar equivalents of the lanthanide shift reagent.
Phenylarsonic acid – (R,S)-3-mercapto-1,2-propanediol)2
complexes (1j)
(R,S)-3-Mercapto-1,2-propanediol (220 mg, 176 µl, 2.04
mmol) was added to a solution of phenylarsonic acid
(41.3 mg, 0.204 mmol) in water (50 ml) at room temperature.
The water had been previously deoxygenated by the passage
of oxygen-free nitrogen. Nitrogen was bubbled though the
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Phenylarsenic acid–thiol complexes studied by NMR 157
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158
J. S. Edmonds et al.
acid – glutathione complex (1d) and by hydrolysis of
iodomethylphenylarsine (1g). The first method involved dis-
solving the glutathione complex of the arsinic acid in aqueous
buffer at pH 13 (or the equivalent buffer made up in D2O),
the second involved the hydrolysis of iodophenylmethylar-
sine (1g) by introducing a small amount of the compound,
dissolved in acetone, into water. Experiments with the asym-
metric shift reagent were carried out on the methylpheny-
larsinous acid (1e) obtained by both methods (Fig. 4). In
each case, the synthesis of methylphenylarsinous acid (1e)
involved the extraction of the aqueous solution in which
hydrolysis had taken place with either dichloromethane or
deuterochloroform. The dichloromethane was dried with
anhydrous magnesium sulfate and evaporated, and the
residue dissolved in deuterochloroform for NMR examina-
tion. If deuterochloroform was used as the extracting solvent,
NMR spectra were taken directly in this solution, both dried
and wet. In all cases, another compound in addition to
methylphenylarsinous acid (1e) was revealed in both 13C and
proton NMR spectra. The NMR spectra and ES MS indicated
that this compound was the anhydride of methylphenylarsi-
nous acid (1h). Also, it was known that similar treatment
of the glutathione complex of diphenylarsinic acid (hydrol-
ysis at pH 13 and extraction into dichloromethane) gave
the anhydride of diphenylarsinous acid (Kondo, Edmonds;
unpublished results). The amount of methylphenylarsinous
acid anhydride (1h) relative to the acid depended on the
initial concentation of the acid in aqueous solution (the more
dilute it was, the higher was the proportion of the acid over
the anhydride) and, when deuterochloroform was used as
the extracting solvent, on whether it was dried (magnesium
sulfate) or not (drying tended to increase the proportion
of the anhydride). NMR spectra of the anhydride showed
the duplication of peaks (1 : 1 ratio) expected for a pair of
diastereoisomers in this case also. There was no evidence
of racemization on the NMR timescale and peaks presum-
ably corresponding to (R,S)- and ꢁR, Rꢂ/ꢁS, Sꢂ-forms were
apparent. All signals attributed to these diastereoisomeric
ppm
2.0
3.0
4.0
5.0
5.0
4.0
3.0
2.0
ppm
Figure 2. 1H–1H COSY spectrum of methylphenylarsinic
acid – (L)-glutathione complexes. Connectivities of cysteine
and glutamic acid residues consistent with the presence of two
diastereoisomers are indicated.
confirmed (Fig. 4). The results of the shift reagent experi-
ment were most clearly seen with the aromatic protons of
the methylphenylarsinous acid (1e); the methyl signal was
masked by resonances of the shift reagent itself. Of the
aromatic protons, those ortho to the arsenic atom showed
the greatest shifts with high-frequency displacement of the
resonances of about 1.7 ppm for the addition of only 0.25
equivalents of the shift reagent (Fig. 4(c)). The signals for the
other aromatic protons were rather less mobile with high-
frequency shifts of approximately 1.4 ppm and 1.3 ppm for
meta- and para-protons respectively, for the addition of two
equivalents of the shift reagent (Fig. 4(i)).
Methylphenylarsinous acid (1e) was generated in
two ways: by hydrolysis of the methylphenylarsinic
0.15
0.10
0.05
0.00
0
10
20
30
40
50
Retention Time (min)
Figure 3. HPLC trace (absorbance at 260 nm) showing partial separation of methylphenylarsinic acid – (L)-glutathione complexes.
(inertsil ODS3 reversed phase C18 column, 250 mm ð 4.6 mm; injection volume, 250 µl at 300 µg/ml; mobile phase, 10-mM formate
buffer pH 4 60%, methanol 40%).
Copyright  2005 John Wiley & Sons, Ltd.
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Phenylarsenic acid–thiol complexes studied by NMR 159
Figure 4. Partial 1H NMR spectra of methylphenylarsinous acid. Only resonances of aromatic protons are shown. a,
methylphenylarsinous acid from hydrolysis of iodomethylphenylarsine; b, methylphenylarsinous acid from hydrolysis
methylphenylarsinic acid – (L)-glutathione complexes; c to f, methylphenylarsinous acid, derived from iodomethylphenylarsine, in the
presence of increasing quantities of the asymmetric lanthanide shift reagent (1f). Resonances attributed to (R)- and (S)-forms show
increasingly different chemical shifts in the asymmetric environment of the shift reagent. c, 0.25 equivalents of shift reagent; d, 0.5
equivalents; e, 0.75 equivalents; f, 1 equivalent. The ortho-protons show the greatest shifts (υ 1.7 ppm with only 0.25 equivalents of
shift reagent, as shown in c). The meta- and para-protons of the two enantiomers are clearly separated with the addition of 0.5
equivalents of shift reagent (d). g to i, methylphenylarsinous acid derived from methylphenylarsinic acid – (L)-glutathione complexes
in the presence of 1, 1.5 and 2 equivalents of shift reagent, respectively.
anhydrides (1h) were unaffected by the presence of the asym-
metric shift reagent (1f). Possibly the anhydride molecule,
lacking the acidic OH group of the methylphenylarsinous
acid, was insufficiently polar to effectively complex the shift
reagent. The methylphenylarsinous acid (1e) generated in
aqueous solution from either starting material showed no
tendency to form the anhydride (1h), although it slowly oxi-
dized to the arsinic acid (1c) if oxygen was not excluded.
Thus, it was the extraction (and drying/evaporation of sol-
vent) of the methylphenylarsinous acid (1e) that was shown
to result in formation of the anhydride (1h).
formed between phenylarsonic acid (1b) and (R,S)-cysteine,
but the products were difficult to isolate.
In the present study, we reduced and complexed
phenylarsonic acid with the racemic chiral sulfur ligands
(R,S)-1-mercapto-2-propanol (1i) and (R,S)-3-mercapto-1,2-
propanediol (1j). These proved easier to handle; the thioglyc-
erol complex (1j) could be extracted into dichloromethane
from the aqueous reaction solution, and the mercapto-
propanol complex (1i) precipated and could be filtered off.
With racemic thiol ligands; one configuration of the
ligand would afford one of a possible pair of enantiomers
[say, (R,R), Fig. 5(a)]; the other configuration would give
the other [(S,S), Fig. 5(b)]. The two enantiomers would, of
course, be identical to each other in all aspects (including
NMR spectra) except chiroptical ones. Furthermore, if for
each enantiomer the positions of attachment of the phenyl
group and the lone pair on the tetrahedral arsenic atom were
reversed, there would be no overall change to any structure.
Thus, whether arsenic pyramidally inverts or not in this
case is irrelevant. In both cases [(R,R)- and (S,S)-ligands], the
ligands would be diastereotopic and would be expected to be
nonequivalent in the NMR spectra, i.e. the (R,R)- and (S,S)-
compounds would give identical spectra, but the ligands in
Phenylarsonic acid with sulfur ligands. Complexes of
phenylarsonic acid (1b) with thioglycerol and with
mercaptopropanol
We previously demonstrated the diastereotopic nature of
configurationally identical ligands [(L)-glutathione] attached
to trivalent arsenic by the reduction and complexing of
phenylarsonic acid (1b) with (L)-glutathione.3 However,
when the ligand is chiral and racemic there is the potential
for the formation of both (R,S)-, (R,R)- and (S,S)-forms
of the phenylarsonic acid–ligand2 complex. Initially, we
investigated these possibilities by examining the complexes
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2006; 44: 151–162

160
J. S. Edmonds et al.
Figure 5. Stereochemistry of the phenylarsonic acid – ꢁ1-mercapto-2-propanolꢂ2 complexes. a and b show the enantiomeric pair
with (R,R)- (a) and (S,S)-ligands (b). These enantiomers will, of course, have identical NMR properties, but the homomorphic ligands
will be diastereotopic in each case. Configuration at arsenic will be of no consequence. c and d show the diastereoisomers resulting
from the presence of ligands of different configuration with both configurations of the pseudoasymmetric arsenic atom. The arsenic
atom is shown having (r) and (s) configurations. The stereochemistry of the (phenylarsonic acid – 3-mercapto-1,2-propanediol)2
complexes will be directly analogous to this.
41
40
143
142
21.6
21.1
69
68
67
ppm
150
100
50
Figure 6. 13C NMR spectrum of the phenylarsonic acid – ꢁ1-mercapto-2-propanolꢂ2 complexes showing quadrupled signals for all
carbons except those assigned to aromatic residues. The latter show three sets of signals in ratios of 2 : 1 : 1. This is most clearly
seen for the C-As carbons as shown in the inset.
each case would be magnetically nonequivalent and would
show duplicated signals. There would be only one set of
signals, though, for the phenyl ring. This is as we previously
reported for the complex of phenylarsonic acid (1b) with
(L)-glutathione.3
Two further diastereoisomers are possible: those result-
ing from both configurations of the tetrahedral arsenic atom
with (R)- and (S)-ligands (Fig. 5(c) and (d)), and these would
be expected to have different NMR properties. These two
compounds (diastereoisomers) differ from one another in
the configuration of the achirotopic arsenic atom, which is
thus a pseudoasymmetric centre6 in the definition of Prelog.5
The configurational situation is directly analogous to that of
the 2,3,4-trihydroxyglutaric acids13 with a tetrahedral arsenic
atom and not a carbon as the pseudoasymmetric centre.
Thus, the NMR spectra of the adducts arising from reduc-
tion and complexing of phenylarsonic acid (1b) with racemic
chiral thiols would be expected to show four signals of
equal intensity for the carbons and non-exchangeable pro-
tons in the ligands (two from the magnetically nonequivalent
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2006; 44: 151–162

Phenylarsenic acid–thiol complexes studied by NMR 161
142.5
142.0
65.2
64.8
73
72
36
34
ppm
150
100
50
Figure 7. 13C NMR spectrum of the phenylarsonic acid – ꢁ3-mercapto-1,2-propanediolꢂ2 complexes showing quadrupled signals for
all carbons except those assigned to aromatic residues. The latter show three sets of signals in ratios of 2 : 1 : 1. Again this is most
clearly seen for the C-As carbons as shown in the inset.
ppm
ppm
20.0
2.0
40.0
3.0
4.0
60.0
80.0
ppm
4.0
3.0
2.0
ppm
4.0
3.0
2.0
Figure 9. HMQC spectrum of the phenylarsonic
acid – ꢁ1-mercapto-2-propanolꢂ2 complexes.
Figure 8. 1H–1H COSY spectrum of the phenylarsonic
acid – ꢁ1-mercapto-2-propanolꢂ2 complexes. Connectivities
consistent with the ligand in 4 different environment were
evident (diastereotopic homomorphic ligands in the (R,R)- and
(S,S)-enantiomers, and (R,S)-diastereoisomers with both
configurations, (r) and (s), of the pseudoasymmetric arsenic
atom.
Results for both 13C and 1H were entirely consistent with
these predictions. The 13C NMR spectra of the phenylarsonic
acid – 1-mercapto-2-propanol (1i) and the phenylarsonic
acid – 3-mercapto-1,2-propanediol (1j) complexes are shown
1
in Figs 6 and 7 repectively. The H–1H COSY spectrum of
the phenylarsonic acid – 1-mercapto-2-propanol complexes
(1i) is shown in Fig. 8. Connectivities consistent with the
presence of the ligand in four different environments
were evident. Insets to Figs 6 and 7 show the resonances
attributed to the carbons bonded to arsenic in each case.
It is apparent that, as expected, three species are present
in intensity ratios of approximately 2 : 1 : 1. These represent
the combined (R,R)- and (S,S)-enantiomers, and the two
(R,S) diastereoisomers that differ in configuration at the
arsenic atom as discussed above. The HMQC spectrum of
homomorphic ligands in the equivalent (R,R)- and (S,S)-
compounds, and two from the diastereoisomers with
(R)- and (S)-ligands with both configurations of the
pseudoasymmetric tetrahedral arsenic atom). Only three
sets of signals for the carbons and protons in the phenyl
ring are possible, though, and these should show inten-
sity ratios of 2 : 1:1 representing the enantiomeric (R,R)-
and (S,S)- (together) and the two (R,S)-diastereoisomers,
respectively.
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2006; 44: 151–162

162
J. S. Edmonds et al.
Figure 10. Partial 1H NMR spectrum of the phenylarsonic acid – ꢁ1-mercapto-2-propanolꢂ2 complexes with increasing quantities (a
to b) of the asymmetric lanthanide shift reagent 1f. That part of the spectrum depicting the –CH2 –S- protons is shown. Increasing
quantities of shift reagent caused an increase in multiplicity of signals resulting from two sets of ligands (marked by asterisks), and
these are therefore tentatively assigned to ꢁR, Rꢂ/ꢁS, Sꢂ-enantiomers.
the 1-mercapto-2-propanol complexes is shown in Fig. 9.
We made no attempt to separate diastereoisomers of these
complexes and can only say that no pyramidal inversion
at the arsenic atom was apparent on the NMR timescale.
Tentative assignment of resonances in the 1H NMR spectrum
of the phenylarsonic acid – 1-mercapto-2-propanol complex
to the ꢁR, Rꢂ/ꢁS, Sꢂ-enantiomers was achieved by further use
of an asymmetric lanthanide shift reagent (Fig. 10). Peaks
assigned to the methylene groups in the ligands proved
most amenable for this purpose. Resonances in the spectrum
were rather insensitive to the shift reagent, but, at a molar
ratio of 4 : 1 of shift reagent to complex, two sets of peaks
started to exhibit further multiplicity (indicated by asterisks
in Fig. 10(b)). These resonances were tentatively assigned
to the diastereotopic ligands of the enantiomeric pair, as
these might be expected to show the greatest differences in
chemical shifts in the asymmetric environment created by
Acknowledgement
We thank Ms M. Katsu for assistance.
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1
the shift reagent. The connectivities exhibited in the H–1H
COSY spectrum (Fig. 8) and the HMQC spectrum (Fig. 9)
then allowed the provisional assignment of all protons and
carbons for the ligands in these enantiomers.
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2006; 44: 151–162