Enzyme-catalysed oxygenation and deoxygenation routes to chiral thiosulfinates

Article abstract of DOI:10.1039/b203139f

Enantioenriched thiosulfinates have been obtained by dioxygenase- and chloroperoxidase-catalysed oxidation of 1,2-disulfides and dimethyl sulfoxide reductase- catalysed deoxygenation.

Full text of DOI:10.1039/b203139f

Enzyme-catalysed oxygenation and deoxygenation routes to chiral
thiosulfinates
Derek R. Boyd,*a Narain D. Sharma,a Martina A. Kennedy,a Steven D. Shepherd,a John F. Malone,a
André Alves-Areias,a Robert Holt,b Stig G. Allenmark,c Malin A. Lemurell (née Andersson),c Howard
Daltond and Heather Luckariftd
a School of Chemistry, The QueenAs University of Belfast, Belfast, UK BT9 5AG
b Avecia Pharmaceuticals, Billingham, Cleveland, UK TS23 1YN
c
Department of Chemistry, University of Göteborg, SE-412 96 Göteborg, Sweden
d Department of Biological Sciences, The University of Warwick, Coventry, UK CV4 7AL
Received (in Cambridge, UK) 28th March 2002, Accepted 15th May 2002
First published as an Advance Article on the web 6th June 2002
Enantioenriched thiosulfinates have been obtained by dioxy-
genase- and chloroperoxidase-catalysed oxidation of 1,2-dis-
ulfides and dimethyl sulfoxide reductase- catalysed deox-
ygenation.
(S) methyl phenyl sulfoxide 7 (11% yield, 94% ee) were
isolated. Furthermore, when 1,2-sulfides 2, 3 and 4 were each
added to P. putida UV4 or P. putida NCIMB 8859, diphenyl
disulfide 4 (2–34% yield) and sulfoxide 7 (1–4% yield) of (R)
and (S) configurations, respectively, were obtained. These
results are consistent with an intracellular reductive cleavage of
the S–S bond to yield the corresponding thiols. The less volatile
benzene thiol was then converted to disulfide 4 or gave methyl
phenyl sulfide 6 (after enzyme-catalysed methylation) and
sulfoxide 7 (after dioxygenase-catalysed sulfoxidation of 6).
Addition of benzene thiol as a substrate to P. putida UV4 also
yielded disulfide 4 and (R)-sulfoxide 7. The dioxygenase-
catalysed oxidation of methyl phenyl sulfide 1 had been found
to give either the (R)- ( > 98% ee, TDO) or (S)- isomer (91% ee,
NDO) enantiomer of sulfoxide 7.5,7 Chemically synthesised
thiosulfinate 5 (R = Me) was unstable and thus unlikely to
survive the biotransformation conditions.
The reduction of a 1,2-disulfide bond to the corresponding thiol
group (A ? B), and the reverse oxidation process (B ? A) are
common reactions in protein chemistry. By comparison,
enzyme-catalysed monooxygenation of 1,2-disulfides (A) to
yield isolable thiosulfinates (C) is uncommon while enzyme-
catalysed deoxygenation of thiosulfinates (C ? A) is unprece-
dented (Scheme 1).
Scheme 1 Oxidation and reduction reactions of 1,2-disulfides.
1,4-Dihydrobenzo-2,3-dithian 8, and the corresponding thio-
sulfinate 9 were adopted as model compounds due to their
greater stabilities. As NDO had earlier been found to catalyse
sulfoxidation of dialkyl sulfides,9 it was selected for the
biotransformation of the cyclic 1,2-disulfide 8; thiosulfinate 9
was the only metabolite isolated (11–23% yield, 10% excess of
the S enantiomer by chiral stationary phase [CSP] HPLC
analysis Whelk-O1 column, a 2.8, t-butyl methyl ether). Since
thiosulfinate 9 had not been resolved or stereochemically
assigned earlier, its enantiomers were separated by semi-
preparative CSP HPLC and (+)-enantiomer 9 ([a]D +250
CHCl3) was found to have an (S) configuration by an X-ray
crystal structure analysis10 (Fig. 1).†
1,2-Disulfide 8, as substrate to P. putida UV4, produced two
unexpected metabolites; the less polar was identified as
2-thiophthalide 10 (17% yield) and the more polar as cis-
dihydrodiol 11 ([a]D +169, CHCl3, 3% yield). The structure and
(4R,5S) absolute configuration of diol 11 was determined by
NMR spectral methods and by X-ray crystallographic analysis
of the hydrogenated derivative 12 ([a]D +20, CHCl3; CCDC
185045).† Possible metabolic pathways involving TDO-cata-
lysed benzylic hydroxylation to yield hemithioacetal 13,
followed by reductive cleavage of the S–S bond and loss of H2S
to give aldehyde 14 (in equilibrium with the cyclic hemiacetal
15) are shown in Scheme 3. Dehydrogenation of the hemiacetal
The eucaryotic oxidation of naturally occurring 1,2-dis-
ulfides A (e.g. R = CH2CNCH2, and n-Pr)1 to yield the
corresponding thiosulfinates C using cytochrome P-450 and
flavin-containing monooxygenases from human liver micro-
somes has recently been reported.2 Prior to this study, only one
report of a procaryotic enzyme-catalysed oxidation of 1,2-dis-
ulfides A to the corresponding thiosulfinates C has appeared.3
Thus, stereoselective sulfoxidation of symmetrical 1,2-dis-
ulfides A (R = n-Bu, i-Pr, t-Bu, 22–97% enantiomeric excess,
ee), utilising cyclohexanone monooxygenase from the bacte-
rium Acinetobacter calcoaceticus was observed.3
As toluene dioxygenase (TDO), naphthalene dioxygenase
(NDO) and chloroperoxidase (CPO) enzymes are known to
catalyse the stereoselective sulfoxidation of aryl alkyl sul-
fides,4–9 the focus of this study was to examine the potential of
these enzymes to catalyse the asymmetric sulfoxidation of
1,2-disulfides (A) to yield the corresponding thiosulfinates (C).
When methyl phenyl disulfide 1 was added to whole cell
cultures of Pseudomonas putida UV4 (TDO), diphenyl dis-
ulfide 4 (28% yield) and (R)-methyl phenyl sulfoxide 7 (6–9%
yield, 96% ee), rather than the anticipated thiosulfinate 5 (R =
Me), were formed (Scheme 2). With whole cells of P. putida
NCIMB 8859 (NDO) and substrate 1, disulfide 4 (5% yield) and
Scheme 2 TDO- or NDO-catalysed oxidation products from 1,2-dis-
ulfides.
Fig. 1 X-Ray structure of the (S)-enantiomer of thiosulfinate 9.
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cells of C. braakii DMSO-11, the residual samples of substrate
9, isolated after biotransformations, were consistently found to
have an excess of the (S) enantiomer (17–77% ee). A time-
course experiment with partially purified DMSOR enzyme
isolated from C. braakii DMSO-11, and racemic thiosulfinate 9
allowed both the 1,2-disulfide bioproduct 8, and the residual
thiosulfinate 9 (95% ee, (S)), to be isolated (Scheme 3).
Addition of racemic thiosulfinate 23 to whole cell cultures of C.
braakii DMSO-11 showed some evidence of kinetic resolution
(4% excess of the R configuration). Enantiomers of thiosulfi-
nate 23 were separated by CSP HPLC using identical conditions
to those used for thiosulfinate 19. The (+) enantiomer of
thiosulfinate 23 ([a]D +150, CHCl3) was found to have an (R)
configuration by comparison with the (2)-(S) enantiomer
whose configuration was assigned by X-ray crystal structure
analysis (CCDC 185046).†
In conclusion, the preliminary results contained herein
indicate that stereoselective oxygenation of 1,2-disulfides
(using NDO and CPO) and deoxygenation of racemic thiosulfi-
nates (using DMSOR) can yield enantiomerically enriched
thiosulfinates. In addition novel metabolic pathways for
1,2-disulfides and thiosulfinates have been found.
15 could account for the formation of 2-thiophthalide 10. cis-
Dihydrodiol 11 could be formed by a cis-dihydroxylation/
dehydration sequence via the triol intermediate 16 or a
dehydration/cis-dihydroxylation sequence via benzo[c]thio-
phene 17; addition of benzo[c]thiophene 17 as a substrate to P.
putida UV4 gave cis-dihydrodiol 11 of identical (4R,5S)
configuration. Since dioxygenase-catalysed benzylic monol ?
triol ? diol metabolic pathways are precedented using P. putida
UV4,11 the biosynthetic sequence 15 ? 16 ? 11 is preferred.
CPO enzyme from Caldariomyces fumago, a biocatalyst for
the synthesis of enantiopure sulfoxides from sulfide pre-
cursors,12–14 was applied to the asymmetric oxidation of
1,2-disulfides. The cyclic 1,2-disulfides 8 and 18 were selected
as substrates for reaction with a suspension of CPO (Sigma)
under previously reported conditions;13,14 1,2-disulfide 8 was
biotransformed into thiosulfinate 9 (ca: 60% yield) with an
excess of the (S) configuration (32–47% ee). The more water-
soluble monocyclic 1,2-disulfide 18 was a better substrate for
CPO; it gave the corresponding (S) thiosulfinate 19 in
essentially quantitative yield and in almost enantiopure form
(96% ee by CSP HPLC). The enantiomers of thiosulfinate 19
were separated by semi-preparative CSP HPLC (Whelk-01
column, a 1.4, t-butylmethyl ether+hexane; 1+1). The absolute
configuration of (2) enantiomer 19 ([a]D 2338, CHCl3) was
assigned as (R) by circular dichroism spectral correlation.
A new range of wild-type anaerobic bacterial strains was
found to catalyse the stereoselective deoxygenation of sulf-
oxides. Using one of these strains (DMSO-11) of the bacterium
Citrobacter braakii containing dimethyl sulfoxide reductase
(DMSOR), enantiomeric enrichment via kinetic resolution of
the racemic sulfoxide substrates was achieved. Thus, enriched
samples of alkylaryl sulfoxides (e.g. sulfoxide 20 [ > 98% ee]),
dialkyl sulfoxides (e.g. sulfoxide 21 [44% ee]) and cyclic
sulfoxides (e.g. sulfoxide 22 [97% ee]) were obtained. When
racemic thiosulfinate 9 was used as a substrate for the intact
We thank the BBSRC and DTI for postdoctoral support under
the Applied Biocatalysis LINK Programme (NDS and HL), the
ESF (MAK) and DENI (SS) for postgraduate studentships, Dr
O. Ishihara (Evotec OAI) and Dr G. Robinson (Astra-Zeneca)
for helpful discussion, Dr J. Hamilton for GC/MS analysis data,
and Dr R. Hamilton and Dr A. King for assistance with the
synthesis of 1,2-disulfide and thiosulfinate substrates.
Notes and references
† See http://www.rsc.org/suppdata/cc/b2/b203139f/ for crystallographic
files in .cif or other electronic format.
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10 Crystal data for (+)-(S)-thiosulfinate 9: C8H8OS2, M
= 184.3,
monoclinic, space group P21, a 7.712(1), b 4.705(1), c =
=
=
11.636(2) Å, b = 102.59(1)°, V = 412.1(1) Å3, Z = 2, Dc = 1.485 g
cm23, T = 300(2) K, F(000) = 192, P4 diffractometer, 1067 observed
reflections ( > 2s(I)), 101 parameters, R1 = 0.048, wR2 = 0.123 (all
data), GoF = 1.08, Flack x = 20.06(5). CCDC 183077.
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Scheme 3 NDO- and TDO-catalysed oxidation products from 1,2-disulfide
8
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