Enzyme-catalysed oxidation of 1,2-disulfides to yield chiral thiosulfinate, sulfoxide and cis-dihydrodiol metabolites

Article abstract of DOI:10.1039/c4ra02923b

Enantioenriched and enantiopure thiosulfinates were obtained by asymmetric sulfoxidation of cyclic 1,2-disulfides, using chemical and enzymatic (peroxidase, monooxygenase, dioxygenase) oxidation methods and chiral stationary phase HPLC resolution of racemic thiosulfinates. Enantiomeric excess values, absolute configurations and configurational stabilities of chiral thiosulfinates were determined. Methyl phenyl sulfoxide, benzo[c]thiophene cis-4,5-dihydrodiol and 1,3-dihydrobenzo[c]thiophene derivatives were among unexpected types of metabolites isolated, when acyclic and cyclic 1,2-disulfide were used as substrates for Pseudomonas putida strains. Possible biosynthetic pathways are presented for the production of metabolites from 1,4-dihydrobenzo-2,3-dithiane, including a novel cis-dihydrodiol metabolite that was also derived from benzo[c]thiophene and 1,3-dihydrobenzo[c]thiophene.

Full text of DOI:10.1039/c4ra02923b

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Enzyme-catalysed oxidation of 1,2-disulfides to
yield chiral thiosulfinate, sulfoxide and
cis-dihydrodiol metabolites†
a
Cite this: RSC Adv., 2014, 4, 27607
Derek R. Boyd, Narain D. Sharma,a Steven D. Shepherd,a Stig G. Allenmarkb
*
and Christopher C. R. Allenc
Enantioenriched and enantiopure thiosulfinates were obtained by asymmetric sulfoxidation of cyclic 1,2-
disulfides, using chemical and enzymatic (peroxidase, monooxygenase, dioxygenase) oxidation methods
and chiral stationary phase HPLC resolution of racemic thiosulfinates. Enantiomeric excess values,
absolute configurations and configurational stabilities of chiral thiosulfinates were determined. Methyl
phenyl sulfoxide, benzo[c]thiophene cis-4,5-dihydrodiol and 1,3-dihydrobenzo[c]thiophene derivatives
were among unexpected types of metabolites isolated, when acyclic and cyclic 1,2-disulfide were used
as substrates for Pseudomonas putida strains. Possible biosynthetic pathways are presented for the
Received 3rd April 2014
Accepted 2nd June 2014
DOI: 10.1039/c4ra02923b
production of metabolites from 1,4-dihydrobenzo-2,3-dithiane, including
a novel cis-dihydrodiol
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metabolite that was also derived from benzo[c]thiophene and 1,3-dihydrobenzo[c]thiophene.
hydroxylation and arene cis-dihydroxylation may occur, partic-
ularly when using benzyl-substituted dialkyl suldes.5a
Introduction
In Scheme 1a is shown alternative sulfoxide enantiomers (2R
and 2S) being produced by enantioselective toluene dioxygenase
(TDO)- and naphthalene dioxygenase (NDO)-catalysed oxida-
tions of methyl phenyl sulde 1, without further oxidation to
sulfone.4d Dioxygenase-catalysed sulfoxidation of alkylaryl
suldes occurs much faster than dialkyl suldes, while the
sulfoxidation of dialkyl suldes using NDO is faster than TDO.
The differences in regio- and stereo-selectivity, observed
during TDO- and NDO-catalysed sulfoxidation of an alkylaryl
sulde, in comparison with a dialkyl sulde, was exemplied,
when the acyclic 1,3-disulde 3 was used as substrate (Scheme
1b).5a The enantiopure alkylaryl sulfoxide enantiomer 4S was
produced from methylsulfanyl methyl phenyl sulde 3, by TDO
and NDO biocatalysts. The dialkyl sulfoxide 5S, with a lower ee
value, was obtained only when using NDO as biocatalyst.
The monosulfoxidation of bicyclic alkylaryl 1,4-disuldes,
e.g. compounds 6a and 6b (Scheme 1c), was also found to occur
in an enantiocomplementary manner, when using either TDO
or NDO to yield the corresponding sulfoxide enantiomers, 7aR
or 7aS and 7bR or 7bS.5b No evidence was obtained of further or
alternative arene dioxygenase-catalysed oxidations of alkylaryl
1,3-disuldes 3, or 1,4-disuldes 6a or 6b, to give the corre-
sponding disulfoxides, sulfones or cis-dihydrodiols.5b
The enzyme-catalysed oxidation of achiral suldes, to yield
chiral sulfoxides, has been of interest for many years, as sul-
foxidation occurs readily during drug metabolism and as it can
provide an alternative route to enantiopure chiral building
blocks of value in chemical synthesis and the pharmaceutical
industry. Enzymes involved in chiral sulfoxidations include
peroxidases, peroxygenases, monooxygenases, and dioxygena-
ses.1a–i The asymmetric sulfoxidation of monosulde substrates
has been extensively studied, as most of the resulting sulfoxide
metabolites are both thermally and congurationally stable
compounds, i.e. having a relatively high racemization barrier.
Sulfoxides derived from thiophenes,2a–c and 1,2-suldes3a–c are,
however, generally less stable and have much lower barriers to
racemization.
The value of arene dioxygenases, as biocatalysts for the
synthesis of chiral sulfoxides, has been reported.2c,4a–g,5a–c The
advantages of these enzymes include their abilities to: (i)
catalyse sulfoxidation of a wide range of alkylaryl suldes, (ii)
produce either enantiomer in a stereoselective manner and (iii)
decrease the possibility of further oxidation to sulfones. While
dioxygenase-catalysed sulfoxidation is the preferred metabolic
step for alkylaryl suldes, competition from benzylic
The earlier successful dioxygenase-catalysed sulfoxidation of
monosuldes,4a–g 1,3-disuldes,4a,5a,b 1,4-disuldes,5b and 1,5-
disuldes,5b prompted our preliminary investigation of the
enzymatic sulfoxidation of 1,2-disuldes, using similar con-
ditions.5c This more comprehensive study now reports chemical
resolution and asymmetric synthesis routes to several
aSchool of Chemistry and Chemical Engineering, Queen's University of Belfast, Belfast
BT9 5AG, UK. E-mail: dr.boyd@qub.ac.uk; Tel: +44 (0)2890974421
b
¨
¨
Department of Chemistry, University of Goteborg, SE-412 96, Goteborg, Sweden
cSchool of Biological Sciences, Queen's University of Belfast, Belfast, BT9 5AG, UK
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra02923b
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Scheme 1 TDO- and NDO-catalysed sulfoxidations of (a) monosulfide 1, (b) 1,3-disulfide 3 and (c) 1,4-disulfides 6a and 6b.
previously unavailable enantiopure thiosulnates; their abso-
The instability of 1,2-disuldes, in vivo, oen results from
lute congurations and chemical and congurational stabilities cleavage of the S–S bond via a reversible reduction–oxidation
were determined prior to their attempted enzymatic synthesis process with the corresponding thiols (Scheme 2). It is evident
via peroxidase-, monooxygenase- and dioxygenase-catalysed that thiol-disulde oxido-reductase activity (ORED) could
oxidations. Results obtained from enzyme-catalysed oxidation present a problem during biotransformations, using bacterial
reactions of acyclic and cyclic 1,2-disuldes and related whole cell systems, e.g. Pseudomonas putida. Further instability
substrates, including potential thiol, monosulde and mono- problems might also be encountered with thiosulnates,
sulfoxide intermediates, have led to possible new metabolic formed via dioxygenase (DO)-catalysed sulfoxidation of 1,2-
pathways for 1,2-disuldes being proposed.
disuldes, that could: (i) spontaneously disproportionate to
yield 1,2-disulde and thiosulfonate derivatives,3c (ii) undergo
Results and discussion
Enzyme-catalysed oxidation of monosuldes can provide a
route to many enantiopure sulfoxides,1a–i of interest to the
pharmaceutical industry, e.g. omeprazole (Fig. 1). However, few
reports have appeared on the enzymatic oxidations of 1,2-
disuldes, to yield enantiopure S-monoxides of 1,2-disulde
(thiosulnates) despite (i) the important role that the S–S bond
plays in many protein structures and natural products, e.g.
gliotoxin and (ii) the established potential of some naturally
occurring chiral thiosulnates in medicinal chemistry, e.g.
racemic allicin and leinamycin having an (S) thiosulnate
conguration.
Scheme 2 Enzyme-catalysed metabolism of 1,2-disulfides.
Fig. 1 Structures of omeprazole, gliotoxin, allicin and leinamycin.
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further enzymatic oxidation to yield unstable vic-disulfoxides, 1,2-disuldes 8a–c and alkylaryl 1,2-disulde 8e resulted in
which rearrange spontaneously to stable thiosulfonates the formation of the corresponding thiosulnates 9a (97% ee),
(Scheme 2),6a–d (iii) undergo sulfoxide reductase (SORED)-cata- 9b (70% ee), 9c (22% ee) and 9e (44% ee). This study10 estab-
lysed deoxygenation, when using P. putida cells and (iv) act as lished the feasibility of enantioselective synthesis of thio-
reactive oxygen species, under conditions of oxidative stress sulnates, via enzyme-catalysed oxidation of 1,2-disuldes, with
and interact with biological thiols, e.g. glutathione (GSH, absolute congurations being assigned to metabolites 9a and 9c
Scheme 2).7a–c
(Scheme 3a).
In addition to their chemical instability, some thiosulnates
have also been found to racemize, at ambient temperature, and
their congurational stabilities have been reported to vary with
their structure and the solution pH or solvent used.3c,8a–c
Despite the potential problems of both decomposition and
racemization occurring, several reports of the production of
enantiomerically enriched thiosulnates, by chemical asym-
metric oxidation of the corresponding 1,2-disuldes, have
appeared. Low enantiomeric excess (ee) values for thiosulnate
products were reported, when using chiral peroxyacids (<10%
ee)3a,8a or chiral oxaziridines (2–14% ee)8b as oxidants. Higher
enantiopurity values were observed when hydroperoxide or
other peroxide oxidizing agents were used in the presence of
chiral catalysts, including t-BuO2H–Ti(O-iPr)4-DET (13–52% ee)8c
or H2O2-VO(acac)2-imine ligands (98% ee).8d,e With the avail-
ability of more stable acyclic thiosulnates as single enantio-
mers, from chemical asymmetric synthesis, e.g. R ¼ t-Bu
(Scheme 2), their synthetic applications have been reported.8e,f
Although the structures of thiosulnate metabolites,
obtained from early enzymatic sulfoxidation studies of naturally
occurring 1,2-disuldes, e.g. diallyl and dipropyl disuldes,9a–d
and of anthropogenic 1,2-disuldes, e.g. 1,2-dithianes,9e were
determined, their stereochemistries were not assigned.9a–e The
rst evidence that the enzyme-catalysed oxidation of unnatural
1,2-disuldes can yield enantiomerically enriched thio-
sulnates, as isolable metabolites, was reported in a seminal
study by Colonna et al. (Scheme 3a).10 Thus, cyclohexanone
monooxygenase (CYMO)-catalysed sulfoxidation of dialkyl
(i) Biotransformations of acyclic alkylaryl 1,2-disuldes 8f–i
and diaryl disulde 8d using P. putida strains expressing TDO
or NDO
The successful arene dioxygenase-catalysed sulfoxidations of
alkylaryl 1,3-, 1,4-, 1,5-disuldes,4a,5a,b and dialkyl 1,3- disul-
des,5a to yield enantiopure monosulfoxides prompted our
preliminary communication,5c using whole cell systems
expressing the same TDO and NDO enzymes but with the
alkylaryl 1,2-disuldes 8f–h as potential substrates. These
acyclic 1,2-disuldes were initially selected, based on: (i) their
similarity in size to alkylphenyl suldes previously bio-
transformed to the corresponding enantiopure monosulf-
oxides, without evidence of further oxidation to sulfones,4d (ii)
the availability of these, and other 1,2-disuldes and the cor-
responding thiosulnates, either from commercial sources or
from literature synthesis routes, (iii) the strong preference
shown by TDO or NDO enzymes for alkylaryl sulde substrates
rather than dialkyl5a or diaryl suldes.4d
The racemic thiosulnates 9d, 9f–i (Scheme 3a) were
obtained by reported methods involving treatment of benze-
nesulnyl chloride with the appropriate thiols. The biotrans-
formations of disuldes 8d, 8f–h were studied, using whole cell
cultures of P. putida, as either a mutant strain (UV4, expressing
TDO) or a wild-type strain (NCIMB 8859, expressing NDO).
Following the extraction and separation of metabolites by PLC,
diphenyl disulde 8d was consistently isolated as the major
identied bioproduct (from substrates 8f–h) or recovered (from
Table 1 Yield, ee and absolute configuration (AC) of chiral metabolite
2 and yield of bioproduct 8d obtained from biotransformation of
substrates 8d, 8f–h and 10 using P. putida UV4 (TDO) and P. putida
NCIMB 8859 (NDO)
Metabolites
MeSOPh 2
Substrates
PhSSPh 8d
8d, 8f–h,10
Enzyme type
% Yield
% ee
AC
% yield
8d
8d
8f
TDO
NDO
TDO
NDO
TDO
NDO
TDO
NDO
TDO
NDO
2
1
6
11
3
1
4
1
97
97
92
94
91
95
85
97
97
—
R
S
R
S
R
S
R
S
10
2
2
10
28
5
34
4
8f
8g
8g
8h
8h
10
10
1
R
—
6
3
a
Scheme 3 Enzyme-catalysed reactions of 1,2-disulfides 8 to yield
thiosulfinates 9, 1,2-disulfide 8d and 8f–h to yield 1,2-disulfide 8d and
monosulfoxides 2R or 2S.
a
Sulfoxide 2 not observed.
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substrate 8d), using TDO (2–34% yield) or NDO (2–10% yield, P. putida UV4 and P. putida NCIMB 8859 cultures to yield thiol
Table 1). Methyl phenyl sulfoxide, metabolite 2, was similarly 10 as a metabolite (Scheme 3b).
isolated from disuldes 8d, 8f–h with high ee values but in low
While the possibility of a non-enzymatic cleavage of the S–S
yield (2–11%). Chiral stationary phase HPLC (CSP-HPLC) anal- bond in 1,2-disuldes occurring cannot be excluded, it was
ysis (Chiralcel-OD, IPA/hexane) was employed, to determine the considered more likely that the much weaker thiosulnate S–SO
enantiopurity of the methyl phenyl sulfoxide obtained using bond (ca. 47 kcal molꢁ1) would cleave during the biotransfor-
either TDO (2R, 85–97% ee) or NDO (2S, 94–97% ee). Despite mation or work-up. Thiosulnates are known to readily
employing either wild type or mutant P. putida strains, under disproportionate into 1,2-disuldes and thiosulfonates and
the previously reported sulfoxidation conditions,4a,5a,b no react with other thiols (Scheme 2). To investigate this possi-
evidence of thiosulnate metabolites was found. Possible bility, the stability of 1,2-thiosulnates 9d, 9f–i was examined by
metabolic pathways, to explain the absence of the expected NMR analysis at ambient temperature. While they showed no
thiosulnates 9d, 9f–h and presence of metabolites 2 and 8d, sign of decomposition in D2O solution over 24 h, the stability of
are shown in Scheme 3b.
some thiosulnates was found to be solvent dependent. Thus,
Several types of ORED enzymes have been reported to thiosulnate 9f, possibly the least stable member of the thio-
catalyse the reductive cleavage of 1,2-disuldes to yield thiols sulnates 9d, 9f–i, remained unchanged in D2O aer 72 h.
and the reverse oxidative S–S bond formation reaction. It is However, in CDCl3 solution, over the same period, 85% of it was
probable that a similar type of enzyme, and also a thiol found to have disproportionated, spontaneously, to yield a
S-methyl transferase (TSMT),11a were present in the P. putida mixture (1 : 1) of 1,2-disulde 8f and the corresponding thio-
whole cells. The ORED enzymes thus catalysed the formation sulfonate. The total disappearance of substrates 8f–h, and
of thiophenol 10 and diphenyl disulde 8d, while TSMT cat- absence of the corresponding more stable thiosulfonates
alysed the formation of methyl phenyl sulde 11 from during the biotransformation, furnished further evidence that
S-methylation of thiol 10. The reduction of 1,2-disuldes to thiosulnates 9f–h were not formed.
thiols, and thiol S-methylation, had been observed earlier
It was anticipated that six-membered ring 1,2-disuldes
during eukaryotic (animal) metabolism.11b As shown in would be more stable than the corresponding acyclic
Scheme 1a, sulde 1 had previously been biotransformed to compounds 8a–i, as reversible S–S bond ring cleavage and
either sulfoxide enantiomer using TDO (2R, >98% ee) or NDO closure could occur. Furthermore, earlier results obtained,
(2S, 91% ee).4d Although thiophenol 10 was not detected as a using 1,2-dithiane 12 as substrate with rabbit liver microsomes,
metabolite from substrates 8d, 8f–h, its addition as a substrate revealed that cytochrome P-450 monooxygenase-catalysed sul-
to P. putida UV4 and P. putida NCIMB 8859 and the resulting foxidation had occurred, to yield the isolable thiosulnate
formation of diphenyl disulde 8d, albeit in very low yield (3– metabolite 13 (Scheme 4a, [O] ¼ P-450, O2).9e Since thiosulnate
6%, Table 1), provided support for a mechanism involving a metabolites 9f–h could not be isolated from dioxygenase-cata-
thiol:disulde oxido-reductase enzyme (Scheme 3b). Methyl lysed oxidation of the corresponding acyclic precursors 8f–h,
phenyl sulfoxide 2R (97% ee) was also detected as a metabolite the cyclic 1,2-disuldes 12 and 14 were synthesised and utilized
of thiol 10, as a result of sequential TSMT-catalysed S-meth- as potential alternative substrates. They proved to be stable in
ylation and TDO-catalysed sulfoxidation. The generally very aqueous solution over a 24 h period; chemical oxidation using
low yields of extracted metabolites 2S, 2R and 8d, and our sodium periodate in aqueous MeOH (Schemes 4a and b, [O] ¼
inability to isolate the thiol intermediate 10, was assumed to NaIO4) yielded the corresponding racemic cyclic thiosulnates
be due to its formation and further metabolism including 13 and 15. The earlier liver microsomal metabolism study of 1,2-
oxidation to water-soluble metabolites.
dithiane 129e did not address the questions of enantiopurity,
absolute conguration or congurational stability of the
resulting thiosulnate metabolite 13, therefore particular
emphasis was placed on these aspects during this programme.
Thiosulnates were reported to undergo racemization more
readily than normal sulfoxides and several mechanisms were
(ii) Thermal and congurational stability studies of chiral
thiosulnate enantiomers obtained by chemical asymmetric
synthesis and CSP-HPLC resolution methods
In our preliminary study,5c it was suspected that the absence of proposed.3a–c The pyramidal sulfur atom in chiral alkylaryl or
the expected thiosulnate products 9d, 9f–h, among metabo-
lites from dioxygenase-catalysed sulfoxidation of the corre-
sponding 1,2-disuldes 8d, 8f–h (Scheme 3b), was a result of the
instability of substrates and/or bioproducts under the
biotransformation or work-up conditions. Further studies
showed that these acyclic 1,2-disuldes were thermally stable,
ꢀ
at the higher temperatures (50–130 C) used during their puri-
cation by distillation, and were also chemically stable for
extended periods, under aqueous conditions at ambient
temperature. It was, however, postulated that enzyme-catalysed
reductive cleavage of the S–S bond had occurred, during the
rst biotransformation step of 1,2-disuldes 8d, 8f–h, using 13S or 13R and (b) 1,2-disulfide 14 to yield thiosulfinates 15S or 15R.
Scheme 4 Sulfoxidation of (a) 1,2-disulfide 12 to yield thiosulfinates
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diaryl sulfoxides is generally assumed to racemize via a thermal methyl ether (t-BME) or t-BME/hexane as eluant. It was then
inversion process (DGs ¼ ca. 40 kcal molꢁ1). An alternative possible to process larger quantities (20 mg per injection) of the
mechanism was initially proposed for racemization of chiral corresponding racemates (13S/13R and 15S/15R), using a semi-
diaryl thiosulnates (DGs ¼ ca. 23 kcal molꢁ1) involving an preparative version of the Whelk-O1 CSP-HPLC column, which
internal displacement at the sulfenyl sulfur atom.8a,12a The gave a base-line separation of the enantiomers. This approach
possibility of thiosulnate racemization being catalysed by also proved successful for the isolation of single enantiomers of
traces of a sulfenic acid,12b and other acids or nucleophiles,3b acyclic thiosulnate 9iS/9iR (a ¼ 1.7) whose stability was
was later suggested. Congurational stability of chiral thio- improved by the presence of a bulky tert-butyl substituent. No
sulnates was dependent on both substituent size and solvent. evidence of decomposition of the thiosulnate enantiomers was
The presence of bulky substituents, e.g. tert-butyl, was found to observed when using any of the CSP-HPLC solvent systems.
increase the barrier to racemization. In this context, S-tert-butyl-
Furthermore, each of these enantiomers (13S, 13R, 15S, 15R,
tert-butanethiosulnate 9a was found to be congurationally 9iS, 9iR) was found to be congurationally stable, over a period
stable (DGs ¼ >23 kcal molꢁ1), aer heating in reuxing of 24 h at ambient temperature, in t-BME and mixtures of
benzene for 8 h.3a,b
t-BME/hexane or IPA/hexane. However, in more polar solvents,
To establish congurational stability of the cyclic thio- under the same conditions, varying degrees of racemization
sulnates 13 and 15, enantiomerically enriched samples were were found. Thus, CSP-HPLC analysis 24 h aer dissolution in a
rst produced by chemical asymmetric synthesis. Application of mixture of H2O/MeOH (1 : 1), showed a decrease in the ee values
the Kagan asymmetric oxidation method (t-BuO2H-Ti(O-iPr)4- for each of the thiosulnate enantiomers 13S or 13R (100 /
DET), to acyclic 1,2-disuldes, had earlier been found to yield 17%), 15S or 15R (100 / 86%) and 9iS or 9iR (100 / 83%).
thiosulnates with a maximum ee value of ca. 50%.8c When the Efforts were made to minimize the degree of racemization
Kagan oxidation method was applied to cyclic 1,2-disuldes 12 occurring, during biotransformation, isolation and stereo-
and 14 (Schemes 4a and 4b), the corresponding thiosulnates chemical analysis of thiosulnate metabolites.
were produced in moderate yield but of relatively low enantio-
Absolute congurations of the enantiomers of cyclic thio-
purity, based on CSP-HPLC analysis (13, 35% yield, 18% ee; 15, sulnates 13 and 15 were assigned using X-ray crystallography
69% yield, 13% ee, Table 2). Use of a chiral oxaziridine oxidant, and electronic circular dichroism (ECD) spectroscopy. The axial
[O] ¼ (ꢁ)-(1R)-(10-camphorsulfonyl)oxaziridine, resulted in an conformation of the oxygen atom, and (1S) absolute congu-
improvement in both yields and enantiopurity values of the ration, present in an enantiopure crystalline sample of (+)-1,4-
thiosulnates (13, 89% yield, 52% ee; 15, 95% yield, 20% ee).
dihydrobenzo-2,3-dithian-2-oxide 15S, [a]D +250 (c 0.4, CHCl3),
An alternative chemical approach, to obtaining enantiopure was established unequivocally by the X-ray crystal structure,
thiosulnates 13 and 15, involved a semi-preparative CSP-HPLC reported but not shown, in our preliminary communication.5c
separation of enantiomers from their racemates. The analytical The crystalline thiosulnate 9iS, [a]D ꢁ144 (c 0.4, CHCl3),
CSP-HPLC system used earlier for ee determination of sulfoxide obtained by CSP-HPLC separation of enantiomers, was also
2 (Chiralcel OD, IPA/hexane) was examined as a possible assigned an (S) absolute conguration by X-ray crystallogra-
method for separation of the individual thiosulnate enantio- phy.5c The absolute congurations of cyclic thiosulnate enan-
mers 13S/13R (a ¼ 1.1) and 15S/15R (a ¼ 1.6). It was, subse- tiomers 13S and 13R, were assigned by a comparison of their
quently, found that an alternative CSP-HPLC analytical column, ECD spectra with the corresponding spectra of cyclic thio-
(R,R)-Whelk-O1, provided a much better separation of enantio- sulnate enantiomers 15S and 15R of known congurations.
mers 13S/13R (a ¼ 1.4), and 15S/15R (a ¼ 2.8), using tert-butyl Based on the similarity between the ECD spectra of the early
eluted enantiomer 15S, [a]D +250 (c 0.40, CHCl3), and the early
eluted enantiomer 13S, [a]D +341 (c 0.43, CHCl3), an identical (S)
absolute conguration was assigned in each case.
Table 2 Yields, ee values and absolute configurations (AC) of thio-
sulfinates 13 and 15 obtained by chemical and enzyme-catalysed
asymmetric sulfoxidation of 1,2-disulfides 12 and 14
(iii) Asymmetric sulfoxidation of cyclic 1,2-disuldes 12 and
14 to yield thiosunates 13 and 15 using cyclohexanone
monooxygenase (CYMO) and chloroperoxidase (CPO)
Substrates
Products
Chemical and
%
Following our unsuccessful efforts to obtain the acyclic thio-
sulnates 9d, 9f–h from the 1,2-disuldes 8d, 8f–h, using whole
cells from P. putida strains expressing arene dioxygenases and
other enzymes, the biotransformation of 1,2-disulde
substrates 12, 14 and 8i with pure enzymes was investigated
(Table 2). It was anticipated that the more stable thiosulnate
bioproducts 13, 15 and 9i, if formed, would survive longer under
the shorter aqueous biotransformation and work-up conditions
and less racemisation would occur. A preparation of cyclohex-
anone monooxygenase, isolated from Acinetobacter calcoaceticus
NCIB 9871, had been used earlier by Colonna et al., to catalyse
Disulde
enzymatic oxidants Thiosulnate Yield % ee AC
12
12
12
12
14
14
14
14
Kagana
13
13
13
13
15
15
15
15
35
89
8
100
69
95
11
59
18
52
22
96
13
20
9
R
S
R
S
R
S
S
S
Oxaziridineb
CYMO/O2
CPO/H2O2
Kagana
Oxaziridineb
NDO/O2
CPO/H2O2
47
t-BuO2H-Ti(O-iPr)4-DET
Sulfoxide.
(1R)-(10-camphorsulfonyl)
a
b
oxaziridine.
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the sulfoxidation of 1,2-disuldes 8a–c. In each case, the cor-
responding enantioenriched thiosulnates 9a (90% yield), 9b
(4% yield) and 9c (4% yield) were obtained as metabolites.10
When 1,2-disuldes 12, 14 and 8i were evaluated as potential
substrates, under identical conditions (CYMO, NADPH, tris
buffer), only thiosulnate 13R was isolated, but in relatively low
yield (8%) and enantiopurity (22% ee) (Table 2, Scheme 4a, [O] ¼
CYMO, O2). The less water-soluble substrates 14 and 8i were
recovered without being metabolized by CYMO.
(iv) Dioxygenase-catalysed oxidation of 1,4-dihydrobenzo-
2,3-dithiane 14 using P. putida NCIMB 8859
Addition of the bicyclic dialkyl 1,2-disulde 14 as substrate, to
whole cell cultures of P. putida NCIMB 8859, resulted in NDO-
catalysed sulfoxidation to give the corresponding (+)-(S)-thio-
sulnate 15S (Scheme 4b, [O] ¼ NDO, O2) in lower yield (11%)
and enantiopurity (9% ee), compared with the earlier results
obtained using CPO (Scheme 4b, [O] ¼ CPO, H2O2, 58% yield
and 47% ee). The monocyclic dialkyl 1,2-disulde 12 was not
biotransformed into thiosulnate 13, when the arene hydroxyl-
ating dioxygenases, NDO (P. putida NCIMB 8859) or TDO
(P. putida UV4), were used.
Based on this limited enantioselectivity study of enzymatic
asymmetric sulfoxidation of 1,2-disuldes 12 and 14, to yield
the corresponding thiosulnates 13 and 15, the puried
peroxidase enzyme (CPO) gave better results than the mono-
oxygenase enzyme (CYMO) or whole cells expressing arene
dioxygenases (NDO and TDO).
Although it was not possible to obtain thiosulnate 15, as a
single enantiomer, by the enzyme-catalysed sulfoxidation
methods described herein, a complementary kinetic resolution
approach was developed in our laboratory. This alternative
method involved a sulfoxide reductase-catalyzed deoxygenation
process;15a,b enantioselective deoxygenation of racemic thio-
sulnate 15, yielded mainly the residual (S) enantiomer (15S,
>95% ee), when using puried dimethyl sulfoxide reductase
from Citrobacter braaki DMSO 11.
The earlier success of puried chloroperoxidase, in catalys-
ing the asymmetric sulfoxidation of monosuldes to sulfox-
ides,13a–g encouraged us to study its potential in the oxidation of
cyclic 1,2-disuldes 12 and 14, under similar conditions to
those used earlier by Allenmark et al. (CPO, H2O2, citrate buf-
fer).13b,d The biotransformation of 1,2-dithiane 12 yielded the
corresponding (+)-(S)-1,2-dithiane-1-oxide 13S in almost quan-
titative yield and excellent enantiopurity (Table 2, 96% ee,
Scheme 4a, [O] ¼ CPO, H2O2). The possibility of racemic thio-
sulnate being produced via hydrogen peroxide oxidation, and
a kinetic resolution process, by further oxidation to a disulf-
oxide or thiosulfonate, being partly responsible for the stereo-
selectivity, was excluded by: (a) the isolated yield of the product
and (b) the results obtained from a time course study. It
showed, unequivocally, that the enantiopurity of thiosulnate
metabolite 13S remained consistently high (96% ee), throughout
the period required for completion of the biotransformation
(40 minutes), and conrmed that a highly stereoselective CPO-
catalysed asymmetric synthesis process was responsible.
Following our preliminary report,5c Klibanov and Dzyuba also
used cyclic 1,2-disulde 12 as a substrate for horseradish
peroxidase (HRP).14 Thiosulnate 13S was again formed pref-
erentially (Scheme 4a, [O] ¼ HRP, H2O2) with stereoselectivity
being solvent-dependent, e.g. E ¼ 7.20 in aqueous buffer and
E ¼ 1.8 in neat methanol.14
(v) Biotransformation of 1,4-dihydrobenzo-2,3-dithiane 14,
benzo[c]thiophene 19, 1,3-dihydrobenzo[c]thiophene 20 and
1,3-dihydrobenzo[c]thiophene sulfoxide 21 using P. putida
UV4
The biotransformation of bicyclic 1,2-disulde substrate 14,
CPO-catalysed sulfoxidation of the less water-soluble cyclic with P. putida UV4, yielded three metabolites but the expected
1,2-disulde 14, under the conditions used for compound 12, thiosulnate 15 was not found. PLC separation of the metabo-
was also successful but the resulting (+)-(S)-thiosulnate 15 was lites, followed by analysis (MS and NMR spectroscopy) and
obtained in lower yield (59%) and enantiopurity (32% ee) along comparison with literature data, led to the identication of
with recovered substrate (Table 2, Scheme 4b, [O] ¼ CPO, H2O2). major bioproduct, 2-thiophthalide 16 (17% yield), and a mon-
This experiment, when repeated using 20% tert-butyl alcohol as ohydroxylated derivative, 6-hydroxy-2-thiophthalide 17, as a very
co-solvent, to improve the solubility and uptake of the 1,2- minor product (1% yield) (Scheme 5). A more polar minor (2%
disulde substrate 14, resulted in an increased ee value (47%). yield) chiral metabolite, cis-dihydrodiol 18, was also isolated
When acyclic 1,2-disulde 8i was used as substrate for CPO, in and identied, based upon NMR and CD spectroscopic analysis.
common with results obtained using the acyclic 1,2-disuldes
Bioproduct 18, contained a benzene cis-tetrahydrodiol ring
8d and 8f–h with TDO or NDO, it did not yield a thiosulnate. of similar structure to (1R,2S)-diol metabolite 24, obtained
The (S) absolute conguration of cyclic thiosulnates 13S using 1,3-cyclohexadiene as substrate with P. putida UV4.16a cis-
and 15S, obtained by CPO-catalysed asymmetric sulfoxidation of Diol 18 was a particularly novel metabolite, as virtually all
the corresponding 1,2-disuldes 12 and 14, was predominant previous TDO-catalysed cis-dihydroxylation products, from a
(Schemes 4a and b, [O] ¼ CPO, H2O2). This appeared to be at carbocyclic arene ring, were cis-dihydrodiols, e.g. metabolite 23
variance with the almost exclusive formation of the (R)- cyclic obtained from biotransformation of benzo[b]thiophene 22
sulfoxides enantiomers found earlier when using cyclic mono- using P. putida UV4.16b Metabolite 18 had similar structural
sulde substrates, e.g. 2,3-dihydrobenzo[b]thiophene (99% ee), features to cis-tetrahydrodiol 24, which was much more stable
thiochroman (96% ee) and 1-thiochroman-4-one (95% ee) under than the corresponding benzene cis-dihydrodiol. However, cis-
identical biotransformation conditions.13b This apparent diol 18 was unstable and readily aromatized, as found with all
reversal in preferred absolute congurations was accounted for arene cis-dihydrodiol metabolites including diol 23 (Fig. 2).16b
by a change in Sequence Rule priorities between the latter
monosulfoxides and thiosulnate metabolites 13S and 15S.
To prevent aromatization of metabolite 18, and to determine
its enantiopurity and absolute conguration, partial
a
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Scheme 5 Bioproducts 16–18 obtained from 1,2-disulfide 14 as substrate with P. putida UV4 and derivatives 25 and 26R/26S.
stable, has not been detected in the environment, and to our
knowledge, had not been used, previously, as a substrate for
biotransformation studies or identied as a metabolite. It was
thus surprising to nd that all three bioproducts 16–18,
obtained from 1,2-disulde 14 appeared to be possible metab-
olites of benzo[c]thiophene 19 (Scheme 5), or precursors
including 1,3-dihydrobenzo[c]thiophene 20 and 1,3-dihy-
drobenzo[c]thiophene-2-oxide 21 (Scheme 6); this unexpected
observation led to further studies of their biosynthetic origins.
When a pure sample of thiosulnate 15 was analysed by GC-
MS, three decomposition compounds were detected. Using
authentic samples, these were identied as 1,2-disulde 14, 1,3-
dihydrobenzo[c]thiophene 20 and benzo[c]thiophene 19
(Scheme 6). Benzo[c]thiophene 19 was obtained by dehydration
of 1,3-dihydrobenzo[c]thiophene-2-oxide 21, using the literature
method.18 GC-MS analysis of a pure sample of 1,2-disulde 14,
under identical conditions, again yielded the decomposition
products 19 and 20.
The previously reported thermal deoxygenation and dispro-
portionation reactions of thiosulnates, to yield 1,2-disuldes
(cf. Scheme 2) lent support for the initial step in the proposed
reaction sequence followed during GC-MS analysis of thio-
sulnate 15 (Scheme 6). Thermal disproportionation of thio-
sulnate 15 to yield 1,2-disulde 14 followed by thermal
extrusion of a sulfur atom at the elevated temperature of the GC
injection port (200ꢀ C), could generate 1,3-dihydrobenzo[c]
thiophene 20. Catalytic thermal dehydrogenation of interme-
diate 20 could then yield benzo[c]thiophene 19. A shorter
alternative sequence, involving thermal extrusion of a sulfur
atom from thiosulnate 15 and dehydration of the resulting
sulfoxide 21, could also account for the formation of benzo[c]-
Fig. 2 Structures of sulfoxide 21, benzo[b]thiophene 22, and cis-diol
metabolites 23, 24 and 30.
hydrogenation (Pd–C, H2 in EtOAc) was carried out, to give the
more stable cyclohexane cis-diol derivative 25. Formation of the
corresponding diastereoisomeric diMTPA ester derivatives, 26R
(using [R]-MTPA) and 26S (using [S]-MTPA) respectively, fol-
lowed by NMR analysis, indicated that both cis-diol 25 and its
cis-diol precursor 18 were enantiopure (Scheme 5, >98% ee).
Earlier studies had shown that di-MTPA esters of cis-tetrahy-
drodiols, obtained from partial hydrogenation of the corre-
sponding cis-dihydrodiol metabolites of polycyclic arenes and
heteroarenes, could be used to determine both their enantio-
purity values and absolute congurations.17 Using this method,
based on the smaller difference in chemical shi values,
observed between H-4 and H-5 (26R, Dd 0.76 ppm) when using
(+)-(R)-MTPA, compared with (ꢁ)-(S)-MTPA (26S, Dd 0.88 ppm),
cis-diol 25 was provisionally assigned a (4R,5S) conguration.
The structure, enantiopurity (>98% ee) and (4R,5S) absolute
conguration of the crystalline cis-diol 25, and of cis-dihy-
drodiol precursor 18 ([a]D +169 (CHCl3)), was conrmed
unequivocally by X-ray crystallography.5c
Benzo[b]thiophene 22 is a very stable compound, formed
during partial combustion of fossil fuels, prior to its release into
the atmosphere and ultimately its biodegradation in the envi-
ronment.16b Conversely, benzo[c]thiophene 19 is much less
Scheme 6 Thermal decomposition products 14, 20 and 19 identified during GC-MS analysis of thiosulfinate 15.
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thiophene 19. Further evidence for the formation of compounds
The formation of metabolites 16, 17 and 18 from 1,2-disul-
14, 19 and 20 as thermal decomposition products of thio- de 14 suggested that, in addition to TDO, other enzymes were
sulnate 15, was found as the latter compounds were not also involved. P. putida UV4 was already known to have a
detected by GC-MS analysis of compound 15 when the GC capacity for sulfoxide deoxygenation, catalysed by a sulfoxide
reductase enzyme (SORED).15a This could account for the
ꢀ
injection port temperature was reduced to 150 C.
Metabolites 16, 17 and 18 were isolated in a relatively low formation of sulde 20 from sulfoxide 21. The TDO enzyme in P.
combined yield (ca. 20%), aer the complete biotransformation putida UV4 was also shown to catalyse benzylic hydroxyl-
of 1,2-disulde 14 in P. putida UV4. It is probable that reductive ation,19a–d particularly in preference to sulfoxidation of dialkyl
cleavage of the disulde bond, found earlier with 1,2-disuldes suldes.5a Thus, TDO-catalysed benzylic hydroxylation of
8f–h, followed by oxidation of the resulting thiols to yield water- methyl benzyl sulde was previously found to occur as an initial
soluble bioproducts, was partly responsible for the low yield of step but ultimately resulting in an S dealkylation reaction and
extracted metabolites. Although benzo[c]thiophene 19 was a formation of an aldehyde intermediate.19e
possible metabolite of disulde 14, and a potential precursor
These earlier observations provide a precedent for the
of cis-dihydrodiol 18 as shown in Scheme 5, it was neither possible formation of thioacetal intermediate 27 from mono-
detected during, nor isolated following, the biotransformation sulde 20. A reversible ring opening process of cyclic thioacetal
(Scheme 7).
27, could then yield aldehyde intermediate 28 (Scheme 7). A
A metabolic pathway involving the extrusion of a sulfur atom similar reversible ring opening/closure reaction was also
from 1,2-disulde 14 to yield monosulde 20, as proposed involved in the spontaneous cis–trans isomerization of the cis-
during GC-MS analysis, although considered less likely to occur dihydrodiol metabolite 30 (Fig. 2) formed from TDO-catalysed
during the biotransformation process, was initially considered dihydroxylation of the heterocyclic ring of benzo[b]thiophene 22
(Scheme 7). In this context, potential intermediates 19–21 under similar conditions.16
involved in the biosynthesis of the unusual cis-dihydrodiol 18,
TDO-catalysed oxidation of aldehyde 28 and spontaneous
were then added separately as substrates to P. putida UV4 whole thiolactonization of the resulting carboxylic acid 29 could then
cells. Addition of 1,3-dihydrobenzo[c]thiophene 20 yielded yield the isolated thiolactone 16. A similar benzylic hydroxyl-
cis-diol 18 as the main isolated metabolite (6% yield), along with ation could also occur on 1,2-disulde 14, when followed by
2-thiophthalide 16 (1% yield) and 6-hydroxy-2-thiophthalide 17 ORED-catalysed cleavage of the S–S bond and decomposition of
(1% yield). The sulfoxide substrate 21 was deoxygenated to yield the resulting acyclic thioacetal intermediate to give aldehyde 28
sulde 20 (6% yield) and thiophthalide 16 (3% yield). When and thioacetal 27; this provides an alternative three step route
benzo[c]thiophene 19 was used as a substrate, the only identi- to intermediate 28 (Scheme 7).
ed metabolite was the corresponding cis-dihydrodiol 18 (8%
The metabolism of 1,2-disulde 14, to yield cis-dihydrodiol 18
yield). Although the isolated yields of compounds 18–21 were could also proceed via thioacetal 27 and the trihydroxylated
very low, based on these observations, the formation of cis-diol intermediate 31 (Scheme 7). A precedent was provided by a TDO-
18 from the 1,2-disulde 14 was in principle consistent with the catalysed monol/ triol / diol metabolic pathway discovered,
metabolic sequence: 14 / 20 / 21 / 19 / 18 (Scheme 7).
during earlier biotransformations of 1,2-dihydronaphthalene19d
Scheme 7 Possible biosynthetic pathways for metabolites 16–18 during the biotransformation of 1,2-disulfide 14 in P. putida UV4.
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Table 3 Chiral stationary phase high performance liquid chromatography (CSP-HPLC) separation of racemic thiosulfinates 9i, 13 and 15 into
enantiomers using a Whelk-O1(R,R) column
ECD spectra (MeCN)
Optical rotations
Thiosulnate
[a]D, (CHCl3)
Separation factor (a)
Elution order (min.)
10.3
l (nm)
D3
9iR
+150 (c 0.88)
1.5a
289
226
200
290
224
200
250
218
208
188
250
218
208
198
258
221
197
258
221
197
ꢁ2.30
ꢁ5.40
+16.15
+2.02
+5.74
ꢁ16.06
ꢁ2.77
ꢁ2.75
+3.99
ꢁ3.32
+1.75
+1.77
+3.32
+2.34
ꢁ5.39
ꢁ6.32
+16.95
+14.5
+17.6
ꢁ46.8
9iS
ꢁ144 (c 0.44)
ꢁ338 (c 0.27)
1.5a
1.4a
13.2
36.6
13R
13S
+341 (c 0.43)
1.4a
31.6
15R
15S
ꢁ246 (c 0.40)
+250 (c 0.40)
2.8b
2.8b
41.3
17.3
a
50% tert-butyl methyl ether/hexane. b 100% tert-butyl methyl ether.
and 2,3-dihydrobenzo[b]furan,5b using P. putida UV4. In these of thiosulnates were obtained, by chemical and enzymatic
examples, monohydroxylation yielded the corresponding asymmetric synthesis and enantiopure samples by semi-
isolable benzylic alcohols, which were further metabolized, via preparative CSP-HPLC. Their chemical and congurational
transient triol intermediates, that in turn spontaneously dehy- stabilities, and absolute congurations, were investigated by
drated to yield the corresponding cis-dihydrodiol derivatives of NMR, ECD spectroscopy and X-ray crystallography. Enzymatic
naphthalene19d and benzo[b]furan.5b
Benzylic hydroxylation of dialkyl sulde 20 and cis-dihy- yielded thiosulnates 13 (22–96% ee) and 15 (9–47% ee).
droxylation catalysed by TDO could thus yield the trihydroxy- The presence of TDO in whole cells of P. putida UV4, shown
asymmetric sulfoxidation of cyclic 1,2-disuldes 12 and 14
lated intermediate 31 via the thioacetal 27. Dehydration of earlier to catalyse sulfoxidation, benzylic hydroxylation, deal-
compound 31 could yield either the cis-diol metabolite 18 or the kylation, aldehyde oxidation and arene ring cis-dihydroxylation,
thioacetal intermediate 32. A similar three-step metabolic was responsible for the majority of metabolites derived from
sequence to that proposed for the conversion of thioacetal 27 1,2-disuldes. Other redox enzymes, expressed in P. putida UV4
into thiolactone 16 (27 / 28 / 29 / 16) could also apply to whole cells, were assumed to catalyse: (a) the reversible reduc-
the formation of thiolactone 17 from thioacetal 32 (32 / 33 / tive cleavage of S–S bonds, (b) thiol S-methylation and (c) and
34 / 17).
sulfoxide deoxygenation. A combination of these enzymes was
Scheme 7 indicates that two metabolic pathways could be required to rationalize the unexpectedly wide range of metab-
involved in the formation of compound 18 from 1,2-disulde 14 olites formed from 1,2-disulde substrates. Possible biosyn-
i.e. (i) 14 / 20 / 21 / 19 / 18 and (ii) 14 / 28 / 27 / 31 thetic mechanisms were proposed, for the formation of the
/ 18. However, based on earlier studies with P. putida UV4 novel benzo[c]thiophene cis-dihydrodiol 18 and 1,3-dihy-
which indicated (a) a reluctance of TDO to catalyse sulfox- drobenzo[c]thiophene derivatives 16 and 17, isolated from 1,4-
idation of a dialkyl sulde, e.g. 20 / 21, and (b) a lack of dihydrobenzo-2,3-dithiane 14.
precedent for the extrusion of a sulfur atom from a 1,2-disulde
cis-Dihydrodiol 18 was formed as a biotransformation
to yield a monosulde e.g. 14 / 20, sequence (ii) is currently product, when 1,2-disulde 14, cyclic monosulde 20 and
the preferred option.
benzo[c]thiophene 19 were each used as substrates with
P. putida UV4 whole cells.
Conclusion
Experimental
The oxidative metabolism of cyclic and acyclic 1,2-disulde
substrates, to yield thiosulnates and other bioproducts, was Melting Points (m.p.) were reported in degrees Celsius using a
studied, using monooxygenase (CYMO), dioxygenase (TDO, Reichert block and are uncorrected. Infrared Spectra (IR) were
NDO) and peroxidase (CPO) enzymes. Enantioenriched samples recorded either on a Bio-Rad 185 FT-IR spectrometer coupled to a
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PC running the Bio-Rad Win-IR soware package or a Perkin- procedures used earlier for other monosulde and 1,3-disulde
Elmer Spectrum RX1 FT-IR spectrometer. 1H Nuclear Magnetic sulfoxidations.4a,d In each case, methyl phenyl sulfoxide 2 and
Resonance (NMR) spectra were recorded at 300 MHz on either 1,2-disulde 8d were obtained as the only identied metabolites
General Electric QE 300 or Bruker Avance DPX-300 equipment, and (Table 1). The enantiopurity and absolute conguration of the
at 500 MHz on either General Electric GN 500 or Bruker Avance methyl phenyl sulfoxide product 2 was determined by CSP-
DPX-500 instruments. Chemical shi values are reported in d HPLC (Chiralcel OD Column, 25 cm ꢂ 4.6 mm, 0.8 cm3 minꢁ1
,
(ppm) downeld from TMS and coupling constants (J) are reported 10% iso-propyl alcohol (IPA)/hexane), a ¼ 1.25, 16.4 min, early
in Hz. 13C NMR spectra were recorded at 75 MHz on either a major R peak, 20.5 min, late minor S peak.
General Electric QE 300 or a Bruker Avance DPX-300 instrument,
and at 125 MHz on either a General Electric GN 500 or a Bruker
Avance DPX-500 instrument. Mass spectra were recorded at 70 eV,
on a VG Autospec Mass Spectrometer, using a heated inlet system.
Accurate molecular weights were determined by the peak match-
Chemical and enzyme-catalysed sulfoxidation of 1,2-
disuldes 12 and 14 to yield the corresponding thiosulnates
13 and 15
ing method, with peruorokerosene as the standard.
Flash column chromatography and preparative layer chro- using tert-BuO2H-Ti(iso-PrO)4-DET
(a) Asymmetric sulfoxidation of 1,2-disuldes 12 and 14
matography (PLC) were performed on Merck Kieselgel type 60
General procedure. Titanium(IV) iso-propoxide (0.44 cm3, 1.47
(250–400 mesh) and PF254/366 plates respectively. Merck Kie- mmol) and (+)-diethyl tartrate (0.50 cm3, 2.94 mmol) were dis-
selgel type 60F254 analytical plates were employed for TLC. solved in dry CH2Cl2 (75 cm3) under nitrogen atmosphere at
Chiral Stationary Phase-High Performance Liquid Chromatog- room temperature. Water (0.026 cm3, 1.47 mmol) was added
raphy (CSP-HPLC) analyses were run using a Beckman System through a septum and the mixture stirred until the solution was
Gold 128 solvent module attached to a 168 UV detector coupled homogeneous (ca. 0.5 h). 1,2-Disulde (1.47 mmol) was added
ꢀ
to a PC running “Gold Nouveau Chromatography Data System” and the solution cooled to ꢁ20 C followed by the addition of
version 1.72 integration soware. Columns and solvent systems tert-butyl hydroperoxide (0.32 cm3, 1.76 mmol). The reaction
used were as individually specied. Optical rotations ([a]D) were mixture was monitored by TLC for the disappearance of the
determined on a Perkin-Elmer automatic precision polarimeter starting material. On completion of the reaction, water (0.26
Model 241. Concentrations were measured in g per 100 cm3 cm3, 14.7 mmol) was added, the solution stirred for 1 h at
using the specied solvent at a wavelength of 589 nm (sodium D ꢁ20 ꢀC and for another 1 h at room temperature. The reaction
line) and ambient temperature. Electronic circular dichroism mixture was ltrated aer treatment with a small amount of
spectra were recorded using a Jasco J-720 instrument and alumina. The ltrate was stirred for 1 h with an equivalent
acetonitrile solvent as specied (concentration approximately volume of 5% sodium hydroxide solution and then for another
0.1 mg cmꢁ3), D3 in molꢁ1 dm.
The acyclic 1,2-disuldes 8d (Sigma-Aldrich) and 8f (Acros), (Na2SO4), ltered and evaporated to give the crude product.
Oxidation of 1,2-dithiane 12 (1.0 g, 8.33 mmol) and puri-
1 h with brine solution. The organic phase was separated, dried
racemic methyl phenyl sulfoxide 2, (1R)-(ꢁ)-(10-cam-
phorsulfonyl) oxaziridine and chloroperoxidase Caldariomyces cation of the crude product by ash chromatography (CH2Cl2)
fumago (Sigma-Aldrich) were obtained commercially. The other gave an excess of (ꢁ)-(R)-1,2-dithiane-2-oxide 13R (0.40 g, 35%);
acyclic 1,2-disuldes 8g–i,20a–d and cyclic 1,2-disuldes 1220e,f ee 18% by CSP-HPLC (Chiralcel OD Column, 25 cm ꢂ 4.6 mm,
and 14,20g were synthesised using literature procedures. Other 0.5 cm3 minꢁ1, 25% IPA/hexane, a ¼ 1.1, 27.4 min, early R major
substrates including benzo[c]thiophene 19,21a 1,3-dihydrobenzo peak, 28.6 min, late S minor peak).
[c]thiophene 20,21a,b 1,3-dihydrobenzo[c]thiophene sulfoxide
Oxidation of 1,4-dihydrobenzo-2,3-dithiane 14 (0.20 g 1.19
21,21c and metabolite 2-thiophthalide 1621d were also obtained mmol) and purication of the crude product by ash chroma-
by following the literature methods and were found to have tography (30% EtOAc/hexane) furnished an excess of (ꢁ)-(R)-1,4-
identical characteristics (ESI†).
dihydro-2,3-benzodithiane-2-oxide 15R (0.151 g, 69%); ee 13%
The CSP-HPLC enantiomeric separations of thiosulnates 9i, by CSP-HPLC (Chiralcel OD Column, 25 cm ꢂ 4.6 mm, 0.5 cm3
13 and 15 were carried out using a semi-preparative Whelk-O1 minꢁ1, 2.5% IPA/hexane, a ¼ 1.6, 29.7 min, early S minor peak,
(R,R) column (25 cm ꢂ 4.6 mm) with a mobile phase ow rate of 45.1 min, late R major peak).
1.0 cm3 minꢁ1 and a UV detector set at 254 nm. The chemically
(b) Asymmetric oxidation of 1,2-disuldes 12 and 14 by
synthesised racemic thiosulnate samples were dissolved in the (1R)-(ꢁ)-(10-camphorsulfonyl)oxaziridine to yield the corre-
mobile phase and injected as 1 cm3 samples. The optical rota- sponding thiosulnates 13 and 15
tions, separation factors, elution sequence and ECD spectra of
the thiosulnate enantiomers 9iR, 9iS, 13R, 13S, 15R and 15S are (0.2–0.4 mmol) was added to a stirring solution of 1,2-disulde
General procedure. (1R)-(ꢁ)-(10-Camphorsulfonyl)oxaziridine
shown in Table 3.
(0.05 g, 0.2–0.4 mmol) in CHCl3 (25 cm3) at ambient tempera-
ture. The reaction mixture was monitored by TLC for the
disappearance of the starting material. On completion of the
reaction, removal of the solvent under reduced pressure yielded
the crude product.
Biotransformation of 1,2-disuldes 8d, 8f–h by Pseudomonas
putida UV4 and Pseudomonas putida NCIMB 8859
Biotransformations with Pseudomonas putida UV4 and Pseudo-
Oxidation of 1,2-dithiane 12 (0.050 g, 0.42 mmol) yielded
monas putida NCIMB 8859 were conducted following the (+)-(S)-1,2-dithiane-1-oxide 13S, which was puried by PLC
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(CH2Cl2); (0.050 g, 89%); ee 52% by CSP-HPLC (Chiralcel OD
Column 25 cm ꢂ 4.6 mm, 0.5 cm3 minꢁ1, 2.5% IPA/hexane, a ¼
1.1, 27.4 min, early R minor peak, 28.6 min, late S major peak).
Oxidation of 1,4-dihydrobenzo-2,3-dithiane 14 (0.050 g, 0.20
mmol) yielded (+)-(S)-1,4-dihydro-2,3-benzodithiane-2-oxide
15S, which was puried by PLC (3% MeOH/CHCl3); (0.052 g,
95%); ee 20% by CSP-HPLC (Chiralcel OD Column, 25 cm ꢂ 4.6
mm, 0.5 cm3 minꢁ1, 25% IPA/hexane, a ¼ 1.6, 29.7 min, early S
major peak, 45.1 min, late R minor peak).
(c) Enzymatic oxidation of 1,2-disuldes to yield the cor-
responding thiosulnates and other metabolites
(i) Cyclohexanone monooxygenase (CYMO)-catalysed sulfox-
idation of 1,2-dithiane 12
(iv) Biotransformation of 1,4-dihydrobenzo-2,3-dithiane 14 by
Pseudomonas putida UV4. Biotransformation of 1,4-dihy-
drobenzo-2,3-dithiane 14 (0.20 g, 1.19 mmol) by P. putida UV4
followed by the usual work up yielded 2-thiophthalide 16, 6-
hydroxy-2-thiophthalide 17 and (+)-(4R,5S)-4,5-dihydroxy-4,5-
dihydrobenzo[c]thiophene 18, aer PLC separation (3% MeOH/
CHCl3).
2-Thiophthalide 16. White solid (31 mg, 17%); m.p. 55–56 ꢀC
(CHCl3/hexane) (lit.,21d,e m.p. 56–57 ꢀC); Rf 0.69 (3% MeOH/
CHCl3); (Found: C, 63.9; H, 4.0. C8H6OS requires C, 64.0; H,
4.0%); (Found: M+, 150.0141. C8H6OS requires 150.0139); dH
(500 MHz, CDCl3) 4.48 (2H, s, CH2), 7.46–7.49 (1H, td, J5,4 ¼ J5,6
7.5, J5,7 0.4, 5-H), 7.54 (1H, d, J7,6 7.7, 7-H), 7.61–7.64 (1H, td, J6,5
¼ J6,7 7.5, J6,4 1.2, 6-H), 7.83 (1H, d, J4,5 7.8, 4-H); dC (125 MHz,
CDCl3) 34.6, 123.9, 126.4, 128.0, 133.2, 135.9, 147.0, 197.8; m/z
(EI) 150 (M+, 100%), 122 (81), 121 (96), 105 (18), 89 (24), 78 (40),
63 (30), 51 (20); nmax (KBr)/cmꢁ1 3082, 2922, 2850, 1686, 772.
6-Hydroxy-2-thiophthalide 17. An oil (1.9 mg, 1%); Rf 0.20 (3%
MeOH/CHCl3); (Found: M+, 166.0092. C8H6O2S requires
166.0089); dH (500 MHz, CDCl3) 4.39 (2H, s, CH2), 5.80 (1H, br s,
OH), 7.14–7.17 (1H, dd, J5,4 8.4, J5,7 2.6 5-H), 7.24–7.25 (1H, d,
J7,5 2.5, 7-H), 7.38–7.40 (1H, d, J4,5 8.4, 4-H), saturation at d 4.39
gave a 0.8% nOe at d 7.38–7.40; dC (125 MHz, CDCl3) 34.1, 109.1,
121.5, 127.1, 137.4, 139.1, 156.1, 197.7; m/z (EI) 166 (M+, 100%),
150 (20), 138 (35), 137 (63), 121 (13), 121 (15), 91 (27), 85 (28), 71
(43), 57 (54), 43 (43), 28 (26); nmax (KBr)/cmꢁ1 3421, 3033, 2920,
2852, 1652, 1482, 1109, 720.
(ꢁ)-(R)-1,2-dithiane-1-oxide 13R. Biotransformation of 1,2-
dithiane 12 (0.025 g, 0.21 mmol, 18 h) by CYMO (70 units) with
NADPH (0.175 g) and tris buffer (0.05 M, pH 8.6) followed by
ethyl acetate extraction of the aqueous biotransformed material
yielded (ꢁ)-(R)-1,2-dithiane-1-oxide 13R (2.2 mg, 8%); ee 22% by
CSP-HPLC, Table 3, [a]D ꢁ69.5 (c 0.18, CHCl3). The 1,2-disulde
14 was not biotransformed under the same conditions.
(ii) Chloroperoxidase (CPO)-catalysed sulfoxidation of 1,2-
dithianes 12 and 14
General procedure. 1,2-Disulde (25 mmol) and chloroperox-
idase (Caldariomyces fumago 30 units) were stirred magnetically
in 2.9 cm3 of a 0.1 M citrate ion buffer (pH 5.0) and maintained
at 25 ꢀC, using a thermostatically controlled water bath.
Hydrogen peroxide (0.113 cm3, 50 mmol, 0.44 M) was injected,
using a continuous addition syringe pump over a period of 55
min. The reaction mixture was quenched aer a further 10 min,
by adding saturated sodium sulte solution (2 cm3). The
mixture was extracted with CH2Cl2 (3 ꢂ 2 cm3), the extract dried
(Na2SO4) and ltered. Removal of the solvent gave the crude
product, which was analysed by CSP-HPLC.
(+)-(S)-1,2-Dithiane-1-oxide 13S. Biotransformation of 1,2-
dithiane 12 (3 mg, 25 mmol, 65 min) by CPO followed by CH2Cl2
extraction of the biotransformed material yielded (+)-(S)-1,2-
dithiane-1-oxide 13S (3.4 mg, 100%); ee 96% by CSP-HPLC
(Table 3).
(+)-(S)-1,4-Dihydrobenzo-2,3-dithiane-2-oxide 15S. Biotransfor-
mation of 1,4-dihydrobenzo-2,3-dithiane 14 (4.2 mg, 25 mmol,
65 min) by CPO followed by CH2Cl2 extraction of the bio-
transformed material yielded (+)-(S)-1,4-dihydrobenzo-2,3-
dithian-2-oxide 15S (2.7 mg, 59%); ee 32% by CSP-HPLC (Table
3). When the above biotransformation was repeated using tert-
butyl alcohol as co-solvent (0.9 cm3) with citrate buffer (2.00
cm3), (+)-(S)-oxide 15S of 47% ee was obtained.
(+)-(4R,5S)-4,5-Dihydro-4,5-dihydroxybenzo[c]thiophene
18.
ꢀ
White crystalline solid (3.8 mg, 2%); m.p. 114–116 C (CHCl3/
hexane); Rf 0.06 (3% MeOH/CHCl3); [a]D +169 (c 0.23, CHCl3);
(Found: M+, 168.0253. C8H8O2S requires 168.0251); dH (500
MHz, CDCl3) 2.35 (2H, br s, 2 ꢂ OH), 4.35–4.39 (1H, td, J5,4 ¼ J5,6
4.3, J5,7 1.1, 5-H), 4.76–4.77 (1H, d, J4,5 4.5, 4-H), 5.95–5.98 (1H,
dd, J6,7 9.7, J6,5 4.0, 6-H), 6.59–6.61 (1H, d, J7,6 9.8, 7-H), 7.07–
7.08 (1H, d, J1,3 2.7, 1-H), 7.34–7.35 (1H, d, J3,1 2.7, 3-H); dC (125
MHz, CDCl3) 69.1, 69.2, 121.9, 124.1, 125.0, 128.7, 129.6, 138.9;
m/z (EI) 168 (M+, 75%), 150 (59), 139 (42), 122 (100), 111 (35), 96
(27), 77 (42), 55 (31), 45 (53), 39 (37), 29 (29); ECD (MeCN): 253
nm D3 +1.11, 239 nm D3 +2.17, 219 nm D3 +4.07, 192 nm D3
ꢁ4.25; nmax (KBr)/cmꢁ1 3421, 3032, 2924, 2853, 1651, 1099, 797.
(+)-(4R,5S)-4,5,6,7-Tetrahydro-4,5-dihydroxybenzo[c]thiophene
25. A solution of (+)-(4R,5S)-4,5-dihydro-4,5-dihydroxybenzo[c]
thiophene 18 (9 mg, 0.054 mmol) containing Pd/C (10%, 5 mg)
in ethyl acetate (5 cm3) was stirred overnight under a hydrogen
atmosphere. The catalyst was removed by ltration, the ltrate
dried (Na2SO4) and concentrated under reduced pressure. The
crude product obtained was puried by ash chromatography
(EtOAc) to yield (+)-(4R,5S)-4,5,6,7-tetrahydro-4,5-dihydrox-
ybenzo[c]thiophene 25 as a white crystalline solid (9 mg, 99%);
m.p. 128–129 ꢀC (CHCl3/hexane); [a]D +19.6 (c 0.43, CHCl3);
(Found: M+, 170.0409. C8H10O2S requires 170.0402); dH (500
MHz, CDCl3) 1.82–1.88 (1H, dtd, J6eq,6ax 13.3, J6eq,5 ¼ J6eq,7ax 6.6,
J6eq,7eq 2.7, 6-Heq), 2.07–2.14 (1H, dddd, J6ax,6eq 13.4, J6ax,7ax 8.1,
J6ax,5 7.8, J6ax,7eq 5.6, 6-Hax), 2.32 (2H, br s, 2 ꢂ OH), 2.66–2.72
(1H, dt, J7ax,7eq 16.6, J7ax,6ax ¼ J7ax,6eq 6.9, 7-Hax), 2.89–2.95 (1H,
(iii) Naphthalene dioxygenase-catalysed sulfoxidation of 1,4-
dihydrobenzo-2,3-dithiane 14 by Pseudomonas putida NCIMB
8859
(+)-(S)-1,4-Dihydrobenzo-2,3-dithiane-2-oxide 15S. Biotransfor-
mation of 1,4-dihydrobenzo-2,3-dithiane 14 (0.10 g, 0.60 mmol)
by P. putida NCIMB 8859 followed by ethyl acetate extraction of
the centrifuged culture medium yielded (+)-(S)-1,4-dihy-
drobenzo-2,3-dithiane-2-oxide 15S as the only identied metab-
olite (12 mg, 11%) aer purication by PLC (30% EtOAc/hexane);
ee 9% by CSP-HPLC (Table 3). The monocyclic 1,2-disulde 12
was not biotransformed under the same conditions.
dt, J7eq,7ax 16.6, J7eq,6ax ¼ J7eq,6eq 6.4, 7-Heq), 4.06 (1H, dt, J5,6ax
¼
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J5,6eq 8.8, J5,4 3.3, 5-H), 4.76 (1H, d, J4,5 3.5, 4-H), 6.91–6.92 (1H,
d, J1,3 3.0, 1-H), 7.36–7.37 (1H, d, J3,1 3.0, 3-H), saturation at d
4.76 gave a 1.1% nOe at d 4.06 and a 0.4% nOe at d 7.36–7.37; dC
(125 MHz, CDCl3) 21.4, 26.9, 68.2, 69.9, 119.7, 124.0, 136.6,
139.5; m/z (EI) 170 (M+, 43%), 152 (65), 126 (100), 125 (96), 97
(38), 45 (32); ECD (MeCN): 235 nm D3 +0.47, 221 nm D3 +0.85,
209 nm D3 +2.37; nmax (KBr)/cmꢁ1 3447, 3105, 2924, 2847, 1651,
1120, 669.
DiMTPA esters 26 of cis-diol 25. (+)-(R)-MTPA gave ester 26R;
typical signals: dH (500 MHz, CDCl3) 5.50 (5-H), 6.26 (4-H);
(ꢁ)-(S)-MTPA furnished ester 26S; typical signals: dH (500 MHz,
CDCl3) 5.54 (5-H), 6.42 (4-H).
(v) Biotransformation of benzo[c]thiophene 19 by Pseudo-
monas putida UV4. Biotransformation of benzo[c]thiophene 19
(0.134 g, 1.00 mmol) by P. putida UV4 followed by ethyl acetate
extraction of the centrifuged medium yielded (+)-(4R,5S)-4,5-
dihydro-4,5-dihydroxybenzo[c]thiophene 18 (14 mg, 8%) as the
only metabolite aer purication by PLC (3% MeOH/CHCl3).
The sample of compound 18 was indistinguishable from the
sample obtained from the 1,2-disulde 14.
(vi) Biotransformation of 1,3-dihydrobenzo[c]thiophene 20 by
Pseudomonas putida UV4. Biotransformation of 1,3-dihy-
drobenzo[c]thiophene 20 (0.20 g, 1.47 mmol) by P. putida UV4
followed by the usual work up and PLC (2% MeOH/CHCl3)
separation of the crude bioproduct yielded 2-thiophthalide 16
(3 mg, 1.4%), 6-hydroxy-2-thiophthalide 17 (2.4 mg, 1%) and
(+)-(4R,5S)-4,5-dihydro-4,5-dihydroxybenzo[c]thiophene 18 (15
mg, 6%). cis-Dihydrodiol 18 was found to have an identical ee
value (>98%) and absolute conguration to the sample isolated
from 1,2-disulde 14.
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drobenzo[c]thiophene 20 (11 mg, 6%) and 2-thiophthalide 16
(7 mg, 3.6%) aer separation by PLC (2% MeOH/CHCl3).
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Acknowledgements
We wish to thank Dr Malin A Lemurell (nee Andersson)
(Department of Chemistry, University of Goteborg) for helpful
advice and invaluable assistance with the CPO-catalysed sul-
foxidations of 1,2-disuldes 12 and 14 to thiosulnates 13 and
15 and Professor Giacomo Carrea (Institute of Chemistry of
Molecular Recognition CNR, Milan) for the CYMO-catalysed
sulfoxidation of 1,2-disulde 14. We also wish to acknowledge
the contribution of Dr Martina Kennedy during preliminary
biotransformation studies of the 1,2-disuldes and the
Department of Education for Northern Ireland for a post-
graduate studentship (to SDS).
7 (a) G. I. Giles, K. M. Tasker and C. Jacob, Gen. Physiol.
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