Toluene dioxygenase-catalyzed cis-dihydroxylation of benzo[b]thiophenes and benzo[b]furans: Synthesis of benzo[b]thiophene 2,3-oxide

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PAPER

Toluene dioxygenase-catalyzed cis-dihydroxylation of benzo[b]thiophenes and

benzo[b]furans: synthesis of benzo[b]thiophene 2,3-oxide†

Derek R. Boyd,*^aNarain D. Sharma,^aIan N. Brannigan,^aTimothy A. Evans,^aSimon A. Haughey,^a

Brian T. McMurray,^aJohn F. Malone,^aPeter B. A. McIntyre,^aPaul J. Stevenson^aand

Christopher C. R. Allen^b

Received 11th June 2012, Accepted 23rd July 2012

DOI: 10.1039/c2ob26120k

Enzymatic cis-dihydroxylation of benzo[b]thiophene, benzo[b]furan and several methyl substituted

derivatives was found to occur in both the carbocyclic and heterocyclic rings. Relative and absolute

conﬁgurations and enantiopurities of the resulting dihydrodiols were determined. Hydrogenation of the

alkene bond in carbocyclic cis-dihydrodiols and ring-opening epimerization/reduction reactions of

heterocyclic cis/trans-dihydrodiols were also studied. The relatively stable heterocyclic dihydrodiols of

benzo[b]thiophene and benzo[b]furan showed a strong preference for the trans conﬁguration in aqueous

solutions. The 2,3-dihydrodiol metabolite of benzo[b]thiophene was utilized as a precursor in the

chemoenzymatic synthesis of the unstable arene oxide, benzo[b]thiophene 2,3-oxide.

Although benzo[b]furans, B[b]Fs, have only been detected as

Introduction

minor products, during combustion processes (e.g. coal gasiﬁca-

tion, coke production and tobacco smoke),¹ⁱmany members of

Benzo[b]thiophene, B[b]T, 1a, and methylated derivatives (MB-

[b]Ts) including 1b and 1c are present in fossil fuels (e.g. crude

oil, coal and shale). As they are liberated into the atmosphere

through combustion, this has prompted a number of studies into

the fate of these thiaarenes in the environment (Scheme 1).^1a–i

Earlier reports, from these laboratories,^2a–dhave shown that the

corresponding sulfoxides 2a–c and their derivatives were formed

via mono- and di-oxygenase-catalyzed heteroatom oxidations of

the corresponding B[b]T substrates 1a–c.

The concomitant formation of cis-diols 3a–c, 5a_cisand 5b_cis,

along with sulfoxides 2a–c, from biocatalytic oxidation of the

carbocyclic and heterocyclic rings of B[b]T substrates 1a–c using

several dioxygenase enzymes, was brieﬂy mentioned in our recent

paper.^2aThe results from this comprehensive study of the sulfox-

ide metabolites 2a–c (and their decomposition products), obtained

using B[b]T 1a and the MB[b]T substrates 1b and 1c with bac-

terial monooxygenase or dioxygenase enzymes, were presented

initially.^2aHowever, in the interest of brevity neither the charac-

terization nor reactivity of the corresponding dihydrodiols 3a–c,

5a and 5b were discussed. Full structural and stereochemical

this family occur in the environment as secondary plant meta-

bolites³(e.g. furanocoumarins and furanoquinolines) and undergo

further biodegradation. In comparison with B[b]Ts, the biotrans-

formation of B[b]F 1e, and methyl substituted benzo[b]furans,

MB[b]Fs, 1f and 1g has, to date, received relatively little atten-

tion (Scheme 1). Our earlier studies of arene dioxygenase-cata-

lyzed cis-dihydroxylation of bicyclic aromatic heterocycles^2b,c,4a,b

included only one member, compound 1e, of the B[b]F family

as a substrate. This study allows a comparison of the results

obtained using three B[b]F substrates 1e–g with the correspond-

ing B[b]T substrates 1a–d.

Most of the earlier reports, on the bacterial biotransformations

of B[b]T substrates from other laboratories, were based largely

on GC-MS analyses of bioproducts.^1b–iWhile the approach was

successful in detecting the more stable products, it did not

permit a full structural or stereochemical analysis. This was par-

ticularly relevant to the thermally unstable metabolites, including

those with a propensity to racemize or epimerize spontaneously.

In this study, we report (i) full structural characterizations of

the vic-dihydrodiol metabolites from B[b]T substrates (withheld

from our recent report^2a) which were not included in earlier com-

munications,^2b,c,4a(ii) new dihydrodiols from methyl substituted

B[b]T 1d and B[b]F substrates 1f and 1g, (iii) results of solvent-

dependent cis/trans equilibration studies of heterocyclic 2,3-

dihydrodiol metabolites and (iv) examples of the reactivity of

cis-dihydrodiols, including regioselective hydrogenation, aroma-

tization and other reactions of value, e.g. in the chemoenzymatic

synthesis of B[b]T 2,3-oxide.

data of the vic-dihydroxylation bioproducts 3a–c, 5a_cis/5a_trans

,

5b_cis, and 5d_cis/5d_trans(from 5-methylbenzo[b]thiophene, 5MB[b]-

T 1d), and the derived products 7–12 are presented in this paper.

^aSchool of Chemistry and Chemical Engineering, Queens University,

Belfast, N Ireland. E-mail: dr.boyd@qub.ac.uk; Tel: +02890974421

^bSchool of Biological Sciences, Queens University, Belfast, N Ireland

†Electronic supplementary information (ESI) available. CCDC 871978,

871979 and 871980. For ESI and crystallographic data in CIF or other

electronic format see DOI: 10.1039/c2ob26120k

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Results and discussion

strain containing toluene dioxygenase (TDO). Under previously

reported conditions,^2awhere heteroatom oxidation (sulfoxida-

tion) was encountered, cis-dihydroxylation was also found to

occur in both the carbocyclic and heterocyclic rings. The regio-

selectivity was dependent on the nature of the heteroatom and

position of the methyl group (Table 1). Characterization of the

corresponding sulfoxide bioproducts 2a–d and their derivatives,

(a) Structural and stereochemical assignments of cis-

dihydrodiol metabolites and derivatives from TDO-catalyzed

oxidation of carbocyclic rings in substrates 1a–c, 1e and 1g

The biotransformation of substrates 1a–g was carried out using

whole cells of Pseudomonas putida UV4, a constitutive mutant

Scheme 1 Metabolites from TDO-catalyzed cis-dihydroxylation (3–5) and heteroatom oxidation (2) of B[b]T or B[b]F substrates (1) and derived pro-

ducts (7–12).

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Table 1 Relative ratios (%) of cis-dihydrodiols 3a–c, 4e, 4g and cis or

trans-diols 5a, 5b, 5d–f from TDO-catalyzed cis-dihydroxylation of

substrates 1a–g

Table 2 Relative (J_cis) and absolute conﬁgurations (Ab. conﬁg.) of cis-

dihydrodiols (3a, 3b, 3c, 4e and 4g) and cis-tetrahydrodiols (3a′, 3b′,

3c′, 4e′ and 4g′)

Carbocyclic

Heterocyclic cis or

trans-diol^a(%)

Phenol

cis-Dihydro-

diol

cis-Tetra-

Substrate

cis-diol^a(%)

products^a,b

J_cis(Hz)^aAb. conﬁg. hydrodiol J_cis^a(Hz) Ab. conﬁg.

1a

3a (0, 12)^c

5a (94, 71)^c

5b (11)

7a (3, 8)^c, 8a

3a

3b

3c

4e

4g

J

_4,55.6 (+)-(4R,5S) 3a′

J_4,53.6 (−)-(4R,5S)

J_4,53.7 (−)-(4R,5S)

J_6,73.9^c(−)-(6S,7S)

J_6,74.2 (−)-(6S,7S)

(3, 9)^c

J_4,55.7 (+)-(4R,5S) 3b′

b

1b

1c

1d

1e

3b (89)

3c (100)

(+)-(4R,5S) 3c′

(−)-(6S,7S) 4e′

(−)-(6S,7S) 4g′

b

5d (100)

4e (44, 35)^c

5e (0, 39)^c

9e (15, 7)^c, 10e

(34, 19)^c

9f (25), 10f (8)

9g (25)

^aVicinal coupling constants. ^bSuperimposed ¹H-NMR signals. ^cRef.

4b.

1f

1g

5f (67)

4g (75)

^aRelative ratio excludes sulfoxidation bioproducts. ^bBioproducts

resulting from further reactions of cis- and cis/trans-dihydrodiols.

^cResults from two biotransformations carried out under slightly different

conditions.

obtained after chromatographic separation from the correspond-

ing cis-dihydrodiol metabolites 3a–c and 5a–d, was reported

earlier.^2a

(i) Structural assignments for cis-diols 3a–c, 4e and 4g. Car-

bocyclic ring dihydroxylation of the parent substrate B[b]T 1a

occurred, exclusively, at the 4,5-bond to yield cis-dihydrodiol

3a, along with several phenol derivatives, e.g. 7a and 8a, which

were separable by PLC (Table 1). A similar pattern of 4,5 bond

regioselectivity was found for MB[b]T substrates 1b and 1c to

give the corresponding cis-diols 3b and 3c. However, the methyl

group of 5MB[b]T 1d appeared to block dihydroxylation at the

4,5 bond.

Fig. 1 X-ray crystal structure of 3a′_cisMTPA(R)

.

g were assumed to result from decomposition of the correspond-

ing cis-dihydrodiol metabolites 3a, 4e–g.

Biotransformation of the parent B[b]F substrate 1e also

resulted in carbocyclic cis-dihydroxylation, exclusively at the 6,7

bond to give cis-dihydrodiol 4e and the derived phenol 9e. Sub-

strate 3MB[b]F 1g yielded diol 4g and phenol 9g products. The

corresponding 6,7-cis-dihydrodiol metabolite of 2MB[b]F 1f

was not isolated. Isolation of phenol 9f suggested that this pre-

sumed relatively unstable carbocyclic cis-dihydrodiol precursor

had been formed but decomposed during the course of biotrans-

formation or workup procedure. This change of regioselectivity

indicates that, during cis-dihydroxylation of B[b]T (4,5 bond

oxidation) and B[b]F substrates (6,7 bond oxidation), proximity

to an aromatic heteroatom is less important than the type of

heteroatom present, when bound within the TDO active site. A

different trend was observed during TDO-catalyzed cis-dihy-

droxylation of quinoline and isoquinoline, where carbocyclic

ring oxidation at the 5,6 and 7,8 bonds occurred.⁵

The chiral cis-dihydrodiol metabolites 3a–c, 4e and 4g were

found to be enantioenriched and showed the characteristic NMR

spectral patterns and coupling constants reported for carbocyclic

cis-diol metabolites from other bicyclic arene substrates, e.g.

naphthalene, quinoline, and isoquinoline.⁵To date, the only

member of the carbocyclic cis-dihydrodiol family from either

B[b]T or B[b]F substrates to have been structurally and stereo-

chemically characterized was cis-dihydrodiol 4e.^4bThis biopro-

duct, along with carbocyclic cis-diols 3a–c and 4g, was

relatively unstable and readily aromatized upon heating or treat-

ment with acid. Thus, the isolated phenolic products 7a, 8a, 9e–

(ii) Absolute conﬁgurations/ee values of diols 3a–c, 4e and

4g. In view of the instability of carbocyclic cis-dihydrodiols

3a–c, 4e and 4g, the relative and absolute conﬁgurations and

enantiopurity values (% ee) were determined after their catalytic

partial hydrogenation (H₂, Pd–C). Hydrogenation of the 6,7

alkene bonds of cis-diols 3a–c and the 4,5 bonds of cis-diols 4e,

4g yielded the corresponding stable cis-tetrahydrodiol derivatives

3a′–c′, 4e′ and 4g′ with typical vicinal coupling constants (J_4,5

or J_6,7) within the range 3.6–4.2 Hz (Table 2). These values were

consistent with those previously found for cis-tetrahydrodiols

derived from other polycyclic arenes.⁵

In view of the instability of carbocyclic cis-dihydrodiols 3a–c,

4e and 4g, the relative and absolute conﬁgurations and enantio-

purity values (% ee) were determined after their catalytic partial

hydrogenation (H₂, Pd–C). Hydrogenation of the 6,7 alkene

bonds of cis-diols 3a–c and the 4,5 bonds of cis-diols 4e, 4g

yielded the corresponding stable cis-tetrahydrodiol derivatives

3a′–c′, 4e′ and 4g′ with typical vicinal coupling constants (J_4,5

or J_6,7) within the range 3.6–4.2 Hz (Table 2). These values were

consistent with those previously found for cis-tetrahydrodiols

derived from other polycyclic arenes.⁵

The absolute conﬁguration of carbocyclic cis-dihydrodiol

metabolite 3a was established as (4R,5S) by X-ray crystallo-

graphic analysis of diMTPA ester derivative 3a′_cisMTPA(R)

,

formed using cis-tetrahydrodiol 3a′ and the acid chloride from

(R)-MTPA (Fig. 1). The cyclohexene ring adopted a half-chair

conformation with the OMTPA groups equatorial on C-5 and

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pseudoaxial on C-4. The dihedral angle between the cis-OMTPA

substituents was −51°.

Spontaneous epimerization with the corresponding trans isomers

(5_cis⇌ 6 ⇌ 5_trans, Scheme 1) involved ring opening/closure via

undetected isomeric aldehyde or ketone intermediates 6 (muta-

rotation). Initial epimerization studies were conducted on the

2,3-diol 5a metabolite from the parent B[b]T 1a. Similar results

were observed for 2,3-diols 5d and 5e derived from 5MB[b]T 1d

and B[b]F 1e respectively.

The very similar ECD spectra, i.e. strong negative Cotton

effects in the region 205–209 nm and weak negative Cotton

effects about 250 nm, observed for cis-tetrahydrodiols 3a′ and

3b′ indicated that the corresponding cis-dihydrodiol metabolites

3a and 3b had identical (4R,5S) absolute conﬁgurations (see

ESI†). A similar absolute conﬁguration for cis-diol 3c was

assigned, after comparison of the ECD spectrum of its cis-tetra-

hydrodiol 3c′ with that of cis-tetrahydrodiol 3a′. An alternative

approach, involving acetylation (Ac₂O/pyridine), oxidative clea-

vage (RuO₂/NaIO₄) of the corresponding diacetate, and dimethyl

ester formation 13 (CH₂N₂) of the resulting dicarboxylic acid,

was adopted for the absolute conﬁguration assignment of cis-

tetrahydrodiols 3c′ and 4g′. This stereochemical correlation

sequence relating these compounds to (2S,3S)-dimethyl(2,3-di-

acetoxy)adipate 13, similar to that used earlier for assigning the

absolute conﬁguration of compound 4e′,^4bconﬁrmed that cis-

dihydrodiols 3c and 4g had (4R,5S) and (6S,7S) absolute

conﬁgurations respectively.

(i) Structure and relative stereochemistry of diols 5a, 5b,

5d–f. ¹H-NMR (CDCl₃) analysis showed that the initially

formed cis-dihydrodiols 5a_cis, 5d_cis, and 5e_cishad larger vicinal

coupling constants (J_2,3cis4.1–5.1 Hz) compared with the trans-

isomers, 5a_trans, 5d_trans, and 5e_trans(J_2,3trans< 1.0–1.7 Hz,

Table 3). Application of the GOESY method to cis-dihydrodiols

gave larger NOE values (2.3–3.5%) between H-2 and H-3 atoms

and smaller NOE values (0.8–1.2%) for the corresponding trans-

diols.

It was found that the equilibrium position attained by meta-

bolites 5a, 5d and 5e favoured the trans-epimer (90–100%) in

polar solvents, e.g. D₂O or CD₃OD (Table 3). A similar trans

preference (80–92%), in polar solvents and spontaneous equili-

bration process for cis- (J_2,3cis4.1–5.1 Hz) and trans-diol meta-

bolites (J_2,3trans1.0 Hz), had been observed earlier with 2,3-diol

metabolites derived from monocyclic thiophenes.⁶Indeed, the

TDO-catalyzed dihydroxylation of substrates 1a, 1d and 1e

resulted in trans-diols 5a_trans, 5d_transand 5e_transbeing the predo-

minant (or only) epimers present in the aqueous culture medium.

As expected from earlier biotransformation studies of bicyclic

azaarene substrates, e.g. quinoline, isoquinoline, quinoxaline and

quinazoline,⁵the TDO-catalyzed cis-dihydroxylation of the car-

bocyclic rings in bicyclic thiaarenes 1a–c and oxaarenes 1e and

1g was, consistently, found to yield the corresponding cis-dihy-

drodiols 3a–c, 4e and 4g having identical absolute conﬁgurations

(non-benzylic S) and ee values (>98%).

(b) Structural and stereochemical assignments of

cis-dihydroxylation products and derivatives from the

heterocyclic rings of substrates 1a, 1b, 1d–f

This “trans-dihydroxylation” of arenes was evidently the com-

bined result of an initial cis-dihydroxylation followed by a rapid

epimerization process. In a less polar solvent, e.g. CDCl₃, the cis

conﬁguration was found to be dominant for diols 5a, 5d and 5e

(60–80%) both in this study and in our earlier analysis of mono-

cyclic thiophene 2,3-diols.⁶A steric preference for the cis

conﬁguration is expected in non-hydroxylic solvents, where

favourable intramolecular H-bonding can occur.

Due to their conﬁgurational instability, characterization of TDO-

catalysed dihydroxylation products 5, formed by oxidation of the

heterocyclic rings of the corresponding substrates 1, was more

challenging, compared with the carbocyclic ring derived cis-

dihydrodiols 3 and 4. The labile cyclic thioacetal, acetal or ketal

groups were present in the initially formed cis-dihydrodiols 5_cis.

Recrystallization of dihydrodiol 5a, from MeOH, yielded a

pure sample of trans-isomer 5a_trans. The NMR spectrum of this

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Table 3 Relative conﬁgurations (J_cisor J_trans), absolute conﬁgurations (Ab. conﬁg.), enantiomeric excess values (% ee), equilibrium ratios of

cis-dihydrodiols 5a_cis, 5b_cis, 5d_cis–f_cisand trans-dihydrodiols 5a_trans, 5d_transand 5e_trans(cis/trans ratio)

cis-Dihydrodiol

J_2,3cis^a(Hz)

(% ee)

Ab. conﬁg.

trans-Dihydrodiol

J_2,3trans^a(Hz)

Ab. conﬁg.

(2R,3R)

cis/trans ratio

5a_cis

5b_cis

5d_cis

5e_cis

4.1

60^b, 63^c>98^b,c

(2S,3R)

5a_trans

1.6

80 : 20^a, 0 : 100^d

100 : 0^a

9^b,c

e

f

4.3

5.1

>98^c

55^b,c

(2S,3R)

5d_trans

5e_trans

<1.0

(2R,3R)

78 : 22^a, 0 : 100^d

60 : 40^a, 55 : 45^g

10 : 90^h, 5 : 95ⁱ

100 : 0^a

e

5f_cis

80^c

(2R,3S)

^aCDCl₃. ^{b 1}H-NMR analysis of diMTPA esters. ^{c 1}H-NMR analysis of MEBBA derivatives. ^dCD₃OD. ^eAbsent. ^fNot determined. ^gd₆-benzene.

^hD₂O. ⁱd₅-pyridine.

sample conﬁrmed that only the trans-epimer (J_2,31.6 Hz; [α]_D

−97) was present at equilibrium in D₂O. Conversely, recrystalli-

zation of dihydrodiol 5a from a mixture of CH₂Cl₂–hexane

yielded a pure sample of cis-isomer 5a_cis(J_2,34.1 Hz; [α]_D+117

in CHCl₃), which equilibrated rapidly in CDCl₃solution. Pure

formation of enol 11a (δ_H6.5 for H-2), as the minor component

(5%) at equilibrium, in CDCl₃solution. The equilibrium ratio of

tautomers 11a : 12a was found to favour the enol tautomer (2 : 1)

in CD₃OD solution as reported earlier.^7bSimilar TFA treatment

of dihydrodiol 5e, at room temperature for an extended period

(24 h), showed no evidence of decomposition, but dehydration

was found to occur at an elevated temperature (60 °C, 4 h),

giving keto tautomer 12e (δ_H4.7 for H-2) exclusively. It is note-

worthy that the dehydration products (phenols 7–9) derived from

the less stable carbocyclic dihydrodiols 3a, 4e, and 4g exhibited

increased aromatic character compared with the dehydration pro-

ducts (keto/enols 11 and 12), formed from the more stable

heterocyclic diols, e.g. 5a and 5e.

Early biotransformation studies of B[b]F 1e, using P. putida

UV4,^2bprovided no direct evidence of the heterocyclic dihydro-

diol 5e (Table 1). Isolation of the phenolic acyclic diol 10e,

however, suggested that it could have resulted from ring opening

and reduction of aldehyde 6e. This premise was conﬁrmed by a

subsequent time-course study which showed that while dihydro-

diol 5e was the major metabolite (45%), after a biotransform-

ation period of 12 h, its proportion decreased to zero over a 24 h

biotransformation period with the concomitant formation of phe-

nolic diol 10e (35% after 24 h). Dihydrodiol 5e was successfully

intercepted and isolated, when the biotransformation was termi-

nated after 12 h (19% isolated yield, Table 1).

Addition of metabolite 5e as a substrate, to P. putida UV4, or

treatment with NaBH₄, both resulted in its total conversion to

phenolic diol 10e (55% ee, determined by the cyclic boronate

method^5,6). The absolute conﬁguration of (−)-phenolic diol 10e

was established as (1R), in our earlier study^2bof B[b]F 1e meta-

bolites. A similar phenolic diol 10f, isolated as a very minor

metabolite of 2MB[b]F 1f, was found to be a single enantiomer

(>98% ee). It was assigned as a syn diastereoisomer, by compari-

son of its ¹H NMR spectrum with that reported in the literature.⁸

This result clearly shows that a carbonyl reductase (CRED)

enzyme, capable of catalyzing stereoselective aldehyde and

ketone reductions, is present in P. putida UV4 cultures. A similar

ring opening and aldehyde reduction sequence of B[b]T dihydro-

diol 5a did not appear to have occurred, since the corresponding

exocyclic thiophenolic diol was not detected within the mixture

of bioproducts.

samples of the individual epimeric metabolites 5e_cisand 5e_trans

,

derived from B[b]F 1e, could not be detected or isolated using

similar recrystallization methods.

TDO-catalyzed dihydroxylation of the 2,3 bond in 2-methyl

substituted substrates 1b and 1f, each, yielded a single diol

product whose NMR spectrum and MS data were consistent

with the corresponding cis-diol structures, 5b_cisand 5f_cis. The

TDO-catalyzed cis-dihydroxylation of alkyl substituted bonds in

the carbocyclic arene rings is unusual but a precedent was found

earlier using benzocyclobutene as a substrate.^9bIt was not poss-

ible to use the relative magnitude of vicinal coupling constants

(J_2,3), or relative NOE values for H-2 and H-3, as an indicator of

cis or trans conﬁgurations. Both ¹H- and ¹³C-NMR spectra

showed only the presence of single epimers, i.e. diols 5b_cisand

5f_ciswhich lack proton H-3.

Recrystallization of diol 5b_cishad earlier yielded a racemic

sample suitable for X-ray crystal structure analysis. This

conﬁrmed the relative stereochemistry of diol 5b as cis.^2cWhile,

in principle, the monothioacetal diol 5b_ciscould epimerize to its

trans-isomer 5b_trans, it was expected that the process would be

slower and that the cis-epimer would be dominant at equilibrium,

due to steric interactions between the vicinal Me and OH groups.

For similar reasons, it is assumed that the heterocyclic ring diol

5f would also have a cis conﬁguration.

A marked difference in stabilities was found between carbo-

cyclic cis-dihydrodiols 3a–c, 4e and 4g and the heterocyclic cis/

trans diols 5a, 5b and 5d–f. This resulted in the isolation of a

range of phenols, e.g. 7a, 8a, 9e–g, from the mixture of biopro-

ducts of the corresponding substrates 1a, 1e–g. No evidence of

spontaneous aromatization of heterocyclic dihydrodiols 5a, 5b,

5d–f to yield the corresponding phenols, e.g. 11a and 11e (or

their keto tautomers 12a and 12e), was found during the bio-

transformation. These observations were supported by NMR

analysis of products formed by acid-catalyzed (TFA) dehydration

of the representative heterocyclic diol 5a, in CDCl₃solution over

several hours, at room temperature. The major product (95%)

found was ketone 12a whose structure was conﬁrmed by chemi-

cal synthesis using the literature method.^7aA crystalline sample

of ketone 12a showed the characteristic NMR signal for H-2 (δ_H

3.8) in CDCl₃solution. Rapid tautomerization resulted in the

(ii) Absolute conﬁgurations and ee values of diols 5a, 5b,

5d–f. As the instability of carbocyclic cis-dihydrodiols 3a–c, 4e

and 4g led to their decomposition, during the attempted direct

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Fig. 2 X-ray crystal structure of 5a_cisMTPA(R)

.

Fig. 3 X-ray crystal structure of 5e_transMTPA(S)

.

formation of their diMTPA esters, the diMTPA esters from the

stable cis-tetrahydrodiols 3a′–c′, 4e′ and 4g′ were utilized for

the determination of enantiopurity. The heterocyclic 2,3-diols

5a, 5b, 5d–f, as indicated, were found to be much more resistant

to aromatization and could be converted, directly, to the corres-

ponding diMTPA esters. Treatment of dihydrodiols 5a_cis/5a_trans

with the acid chloride of (+)-(R)-MTPA, in pyridine solution,

gave a mixture (40 : 60) of the corresponding diMTPA esters

4g, as single enantiomers, an alternative explanation based on

the partial racemization of the single chiral centre, present in the

elusive ring-opened intermediate 6, should be considered

(Scheme 1). Tautomerization of the aldehyde or ketone group in

compound 6, to form a conjugated achiral enol, could explain

this partial racemization of the thiophene and furan ring dihydro-

diols 5a, 5b, 5e and 5f.

5a_cisMTPA(R)and 5a_transMTPA(R).

¹H-NMR spectral analysis of

Reaction of the acid chloride from (−)-(S)-MTPA with the

metabolite 5e_trans/5e_cisyielded four diastereoisomers from which

the major (85%) trans isomers were separated by PLC. Frac-

tional crystallization of the trans diastereoisomers yielded a

sample of a single diastereoisomer. X-ray crystal structure analy-

sis showed it to be the (2S,3R)-diMTPA ester 5e_transMTPA(S)

(Fig. 3). This conﬁrmed the trans conﬁguration and provided

unequivocal evidence of a (2R,3R) absolute conﬁguration for the

precursor diol 5e_trans(Table 3). This result is in accord with the

established (1R) conﬁguration of the derived phenolic diol 10e

the diMTPA esters showed that the dihydrodiol metabolites 5a_cis/

5a_transhad been formed as single enantiomers (>98% ee,

Table 3). PLC separation of the diMTPA esters, followed by

recrystallization, provided a suitable sample of ester 5a_cisMTPA(R)

for X-ray crystallographic analysis (Fig. 2). The analysis

conﬁrmed the relative conﬁguration as cis and the absolute

conﬁguration as (2S,3R). In the crystal, the heterocyclic ring

adopts an envelope conformation with C-2 0.38 Å above the

plane of the ring (as viewed in Fig. 2). The OMTPA group on

C-2 is axial and on C-3 pseudoequatorial. The dihedral angle

between the cis-related oxygen atoms at C-2 and C-3, shown in

Fig. 2, is +28°. When the biotransformation of B[b]T 1a was

repeated at a later date, formation of the diMTPA esters, surpris-

ingly, revealed that dihydrodiols 5a_cis/5a_transwere of lower enan-

tiopurity (60% ee).

which was found to be a further metabolite of diols 5e_cis/5e_trans

.

In the crystal of 5e_transMTPA(S), the heterocyclic ring adopts an

envelope conformation with C-2 0.27 Å below the plane of the

ring. This fold is smaller, and in the opposite sense, to that

observed in 5a_cisMTPA(R). It thus retains the axial conformation

for the oxygen atom on C-2, as 5e_transMTPA(S)and 5a_cisMTPA(R)

have opposite absolute conﬁgurations at C-2. It follows that the

OMTPA group on C-3 in 5e_transMTPA(S)is pseudoaxial. The

dihedral angle between the trans-related oxygen atoms at C-2

and C-3, shown in Fig. 3, is −153°.

An alternative approach, to the determination of ee values of

dihydrodiols, involved forming cyclic boronate derivatives,

using

(−)-(S)-2-(1-methoxyethyl)-benzene

boronic

acid

(MEBBA). This method had earlier been successfully used for

cis/trans-dihydrodiol metabolites of monocyclic thiophenes,⁶

and was again applied to dihydrodiols 5a_cis/5a_trans. The MEBBA

derivative 5a_cisMEBBAwas formed, exclusively, from the equili-

brating epimeric mixture.

The preference for axial substituents at C-2 in solution, even

when the bulky OMTPA group is absent, is supported by the

2c

crystal structure of cis-diol 5b_ciswhich also has an envelope

Analysis of the ¹H-NMR spectrum of boronate 5a_cisMEBBA

gave an ee value of 63% which was in good agreement with the

value obtained, using the diMTPA method, for the later sample

of dihydrodiols 5a_cis/5a_trans(60%, Table 3). Formation of the

corresponding cyclic MEBBA derivatives of dihydrodiols 5b

and 5f provided further evidence that both existed exclusively as

cis-diol epimers 5b_cisand 5f_cisat equilibrium.

The ee values for heterocyclic dihydrodiols 5b and 5d–f,

determined using the MEBBA derivatives, covered a remarkably

wide range (<10 to >98% ee, Table 3). The different ee values,

found among the heterocyclic dihydrodiols in Table 3, could

have resulted from variable enantioselectivity occurring during

the TDO-catalyzed dihydroxylation. However, in view of the

consistent formation of the carbocyclic cis-diols 3a–c, 4e and

conformation for the heterocyclic ring. In the (2S,3R) enantio-

mer, within the diol racemate 5b_cis, C-2 lies above the plane of

the ring by 0.59 Å and 0.61 Å in the two crystallographically

independent molecules. As in 5a_cisMTPA(R), the oxygen atom on

C-2 in diol 5b_cisis axial and on C-3 pseudoequatorial. The di-

hedral angles between the cis-related oxygen atoms at C-2 and

C-3, in the two independent molecules of diol 5b_cis, are +50°

and +53°. The preference for axial substituents at C-2 appears to

be analogous to the anomeric effect found in sugars and substi-

tuted tetrahydropyrans or thianes.

Electronic circular dichroism (ECD) spectra of dihydrodiols

5a_cis/5a_transand 5d_cis/5d_trans, in acetonitrile solvent, proved to be

very similar and provided conﬁrmation that they had identical

absolute conﬁgurations (Table 3 and also ESI†). The relative

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Scheme 2 Absolute conﬁguration assignment of metabolites 5f_cisand 10f.

Scheme 3 Monooxygenase-catalyzed sulfoxidation and epoxidation of 2-phenylthiophene (16) to yield sulfoxide (17) and arene oxide (18) meta-

bolites and their glutathione adducts (19 and 20).¹³

Scheme 4 Synthesis of B[b]T-2,3-oxide 21a and derived products, 11a and 12a.

conﬁguration of the 2-methyl substituted diol metabolites, from

substrates 1b and 1f, was established as cis by X-ray crystallo-

graphy (5b_cis)^2cand MEBBA derivative formation (5f_cis). The

crude sample of heterocyclic ring metabolite 5b_ciswas found to

have a very low ee value (9%, Table 3), which on recrystalliza-

tion yielded a racemic sample.^2cX-ray crystallography of the

sample^2ctherefore proved only the relative conﬁguration.

This resulted in a (2R,3S) conﬁguration being assigned to cis-

diol metabolite 5f_cis. As the conﬁguration at C-3 is opposite to

that found for the other heterocyclic cis diols, e.g. 5a_cis, 5d_cis

and 5e_cis, this assignment is tentative; it is based only on the

assumption that the Sharpless mnemonic for AD-mix-α dihy-

droxylations¹⁰is applicable to the ortho substituted phenyl

alkene 14.

The assignment of absolute conﬁguration to cis-diol 5f_ciswas

carried out indirectly through stereochemical correlation to the

phenolic diol metabolite 10f (Scheme 2). It was assumed to be

formed via ring opening of the diol 5f_cis, to yield the correspond-

ing undetected acyclic ketone 5f′. Treatment of phenolic diol

metabolite 10f with diazomethane yielded the corresponding

methyl ether 15 ([α]_D+22, EtOH, Scheme 2). A sample of

methyl ether 15, with an [α]_D+26 (EtOH), was independently

synthesized by cis-dihydroxylation of trans-1-(2′-methoxyphe-

nyl)prop-1-ene 14 using the chiral dihydroxylating reagent, AD-

mix-α. Based on the reported cis-dihydroxylation of trans-

β-methylstyrene by AD-mix-α, which produced mainly the (S,S)

enantiomer (97% ee) of the corresponding diol,¹⁰it was

assumed that methyl ether 15 would have a similar conﬁguration.

(c) Synthesis of B[b]T 2,3-oxide 21a from 2,3-dihydroxy-2,3-

dihydrobenzo[b]thiophene 5a_cis/5a_trans

Dansette et al.¹³have proposed that monooxygenases, e.g. the

cytochrome P450, CYP1A1, can catalyze the concomitant sul-

foxidation and epoxidation of thiophene rings. Thus, 2-phe-

nylthiophene 16 was biotransformed into the unstable thiophene

oxide 17 and arene oxide 18 (Scheme 3). Evidence for the pres-

ence of these transient intermediates was obtained by trapping

them as stable glutathione adducts 19 and 20. This report,¹³

allied to our earlier isolation of the unstable B[b]T sulfoxide

2a,^2aprompted the current synthesis of the previously unknown

B[b]T 2,3-arene oxide 21a.

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Dioxygenase-catalyzed oxidation of indene 1h,^11a–eB[b]T 1a

and B[b]F 1e, using TDO, has been shown to produce the corres-

ponding cis-diols 5h_cis,^11a–e5a_cis, 5e_cisand sulfoxide 2a^2aas

the initial metabolites (Scheme 1). Using styrene monooxygen-

ase (SMO) as a biocatalyst, the oxidation of indene 1h was

found to form, exclusively, indene 1,2-epoxide 21h

(Scheme 4).^12a–cThe formation of indigo as a metabolite of

indole 1i (R = R′ = R′′ = H, X = NH, Scheme 1), when using

either monooxygenase or dioxygenase biocatalysts, is also con-

sistent with an epoxidation (SMO-catalyzed) to yield arene

oxide 21i, or a dihydroxylation (TDO-catalyzed) to give cis-

dihydrodiol 5i_cis(R = R′ = R′′ = H, X = NH, Scheme 1) as the

initial metabolite. Both oxidation products 21i and 5i are

assumed to be transient metabolites and would spontaneously

decompose to yield indoxyl, which in turn undergoes autoxida-

tion to yield indigo.^12a–cIn this context, the question of whether

the SMO enzyme could similarly catalyze the epoxidation of

B[b]T 1a to yield metabolite 21a, albeit as a very minor meta-

bolic pathway compared to the formation of unstable sulfoxide

2a,^2awas also addressed in this study.

Adam et al. using dimethyldioxirane (DMD) as an oxidant.^15a–d

Our efforts to synthesise B[b]T 2,3-oxide using DMD oxidation

yielded only the corresponding sulfoxide and derivatives.^2aNo

attempt was made to synthesize the parent 2,3-oxide from B[b]F

1e via the ortho-ester method, as the corresponding cis/trans

dihydrodiols 5e_cis/5e_transwere only available in lower yields and

were less stable than diols 5a_cis/5a_trans. To date, the correspond-

ing 2,3-arene oxides of indole (21i) and B[b]F (21e) have not

been reported; they are expected to be much less stable than the

isolated 2,3-dimethyl substituted 2,3-oxides of B[b]F^15a–cand

N-acylindoles.^15d

Conclusions

The TDO-catalyzed dihydroxylation of a series of B[b]T 1a–d

and B[b]F 1e–g substrates yielded (i) ﬁve carbocyclic cis-diols

3a–c, 4e, 4g, (ii) eight heterocyclic cis-diols 5a_cis, 5b_cis, 5d_cis–

f_cis, 5a_trans, 5d_transand 5e_transand (iii) two acyclic diols 10e and

10f. The structures, ee values and absolute conﬁgurations of

these chiral metabolites were determined by a combination of X-

ray crystallography, ECD spectroscopy and stereochemical corre-

lation methods. The heterocyclic ring cis-diols 5a_cis, 5d_cis, and

5e_ciswere assumed to be the initial metabolites and were found

to equilibrate spontaneously (mutarotation), preferring the trans

conﬁguration 5a_trans, 5d_trans, and 5e_transin aqueous solution.

The chemoenzymatic synthesis of the unstable heterocyclic

arene oxide B[b]T 2,3-oxide 21a has been achieved in three

steps from the corresponding cis/trans-diol bioproduct 5a_cis/

5a_trans. No evidence for this transient metabolite was found,

during the biotransformation of B[b]T 1a, using SMO as a bio-

catalyst. It is probable that the mutarotation process of the

initially formed 2,3-cis-diol metabolites of B[b]T 1a and B[b]F

1e (via TDO biocatalysis), and formation of a heterocyclic 2,3-

oxide (via SMO biocatalysis), in the ﬁve membered heterocyclic

rings of B[b]T and B[b]F also occurs during TDO- and SMO-

catalyzed biotransformations of other ﬁve-membered aromatic

heterocyclic ring systems, e.g. indole 1i.

Earlier publications^14a–chad shown that the relatively stable

carbocyclic K-region cis-dihydrodiols of polycyclic aromatic

hydrocarbons, and their ortho-ester derivatives, can be used as

precursors in the synthesis of the corresponding K-region arene

oxides (e.g. from phenanthrene, pyrene, chrysene, benz[a]anthra-

cene, benzo[c]phenanthrene, benzo[a]pyrene and benzo[e]-

pyrene). Adapting this ortho-ester approach to the relatively

stable heterocyclic ring dihydrodiol 5a_cis/5a_trans, a chemoenzy-

matic synthesis of B[b]T 2,3-oxide 21a was developed

(Scheme 4).

Diol epimers 5a_cis/5a_transwere converted into an inseparable

mixture of dioxolane stereoisomers 22 (77 : 27). The mixture

was reacted, without further puriﬁcation, with trimethylsilyl

chloride to yield trans-chloroacetate 23. In common with the

ortho-ester procedure, used earlier for the synthesis of K-region

14a–c

arene oxides of PAHs,

dioxolane mixture 22 and the rela-

tively unstable trans-chloroacetate intermediates 23 were charac-

terized mainly by ¹H-NMR spectroscopy and mass spectrometry.

A sample of chloroacetate 23, obtained by rapid puriﬁcation

using PLC, was treated with NaOMe in THF-d₈solvent to yield

the desired unpuriﬁed arene oxide 21a in a yellow solution. The

NMR spectra and MS data of the crude arene oxide 21a were

fully consistent with its structure. As expected, arene oxide 21a

was found to be very unstable and during attempted PLC puriﬁ-

cation, prior to [α]_Dmeasurement, it rapidly isomerized to give

the keto tautomer 12a which was found to equilibrate, spon-

taneously, to phenol 11a in MeOH solution.

Acid-catalyzed dehydration of diols 5a_cis/5a_transand isomeri-

zation of the arene oxide 21a both yielded tautomers 11a and

12a. Neither LC-TOFMS nor NMR spectroscopic analyses

showed any evidence for the presence of arene oxide 21a or the

expected decomposition products 11a and 12a, among the

metabolites of B[b]T 1a, following biotransformation using

E. coli BL21 (DE3) as the SMO source. While SMO-catalyzed

oxidation of indene 1h followed an epoxidation pathway, exclu-

sively, in this study sulfoxidation was observed as the only

SMO-catalyzed oxidation pathway during B[b]T 1a metabolism.

A direct single step synthesis of methyl substituted 2,3-oxide

derivatives of B[b]Fs and N-acylindoles has been reported by

Experimental

¹H and ¹³C NMR spectra were recorded on Bruker Avance 400,

DPX-300 and DRX-500 instruments. Chemical shifts (δ) are

reported in ppm relative to SiMe₄and coupling constants (J) are

given in Hz. Mass spectra were run at 70 eV, on a VG Autospec

mass spectrometer, using a heated inlet system. IR spectra were

recorded on a Perkin-Elmer Model 983G instrument, coupled to

a Perkin-Elmer 3700 data station, in potassium bromide (KBr)

disks unless otherwise stated. High resolution mass spectra

(HRMS) were determined by the peak matching method, with

perﬂuorokerosene as the standard. A PerkinElmer 341 polari-

meter was used for optical rotation ([α]_D) measurements. ECD

spectra were recorded in spectroscopic grade acetonitrile using a

JASCO J-720 instrument. Flash column chromatography and

preparative layer chromatography (PLC) were performed on

Merck Kieselgel type 60 (250–400 mesh) and PF_254/366plates

respectively. Merck Kieselgel type 60F₂₅₄analytical plates were

employed for TLC. Enantiomeric excess values (% ee) for diols

were determined by formation of MTPA (Method A) and

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MEBBA (Method B) derivatives using previously described

methods.⁴X-ray crystal structure analysis of diMTPA esters was

carried out using a Siemens P3 diffractometer. All B[b]T and

B[b]F substrates were available from commercial sources.

Biotransformations, with Pseudomonas putida UV4 whole

cells, as a source of TDO, were conducted employing the

reported conditions.^2a,6The relative yields of diols and derived

products are given in Table 1. Separation of bioproducts was

achieved by ﬂash column chromatography and PLC on silica

gel. R_fvalues were recorded using either Et₂O : hexane (1 : 1,

eluent A) or EtOAc : hexane (55 : 45, eluent B).

(Found: C, 58.9; H, 5.5; S, 17.6. C₉H₁₀O₂S requires C, 59.3; H,

5.5; S, 17.6%); H-NMR (500 MHz, CDCl₃) δ 1.72 (1 H, br s,

1

OH), 2.29 (3 H, s, Me), 2.79 (1 H, br s, OH), 4.56–4.60 (2 H,

m, 4-H, 5-H), 5.80 (1 H, d, J_7,69.8, 7-H), 6.42 (1 H, dd, J_6,79.8,

J_6,52.5, 6-H), 6.81 (1 H, s, 2-H); ¹³C-NMR (125 MHz, CDCl₃)

δ 13.5, 65.2, 71.1, 120.2, 120.3, 129.2, 134.8, 135.8, 137.0;

LRMS (EI): m/z 182 (M⁺, 50%), 164 (M⁺− H₂O, 100); ν_max

(KBr) 3255 cm⁻¹(OH); >98% ee (Method A).

(iv) Metabolites from benzo[b]furan 1e

(−)-(6S,7S)-cis-6,7-Dihydroxy-6,7-dihydrobenzo[b]furan 4e. Yield

(1.9 g, 15%); mp (dec.) 60–61 °C (from CHCl₃–hexane) (lit.,^4b

61–63 °C); R_f0.39 (eluent B); [α]_D−33.0 (c 0.77, MeOH)

1

(lit.,^4b−34.7, MeOH); H-NMR (500 MHz, CDCl₃) δ 3.99 (2

Biotransformations of benzo[b]thiophene 1a, 2-methyl benzo[b]-

thiophene 1b, 3-methyl benzo[b]thiophene 1c, benzo[b]furan 1e

and 3-methyl benzo[b]furan 1g using P. putida UV4 to yield the

corresponding carbocyclic cis-diol and phenol bioproducts

H, br s, OH), 4.57–4.65 (2 H, m, 6-H, 7-H), 6.57 (1 H, dd, J_5,4

9.6, J_5,62.1, 5-H), 6.24 (1 H, dd, J_4,59.6, J_4,62.5, 4-H), 6.29

(1 H, d, J_3,21.8, 3-H), 7.35 (1 H, d, J_2,31.8, 2-H); >98% ee

(Method A).

6-Hydroxybenzofuran 9e. Yield (0.034 g, 3%); R_f0.83 (eluent

B). It was found to be spectrally identical to an authentic

sample.^4b

(i) Metabolites from benzo[b]thiophene 1a

(+)-(4R,5S)-cis-4,5-Dihydroxy-4,5-dihydrobenzo[b]

thiophene

3a. Yield (0.79 g, 6%); mp (dec.) 62–76 °C (from CHCl₃–

hexane); R_f0.20 (eluent A); [α]_D+98.0 (c 0.57, CHCl₃) (Found:

C, 57.5; H, 4.6; S, 18.9. C₈H₈O₂S requires C, 57.1; H, 4.8; S,

19.1%); ¹H-NMR (300 MHz, CDCl₃) δ 2.35 (2 H, br s, 2 ×

OH), 4.47 (1 H, m, 5-H), 4.71 (1 H, d, J_4,55.6, 4-H), 5.91 (1 H,

dd, J_6,79.8, J_6,53.3, 6-H), 6.51 (1 H, dd, J_7,69.8, J_7,51.6, 7-H),

7.13 (1 H, d, J_3,25.0, 3-H), 7.20 (1 H, d, J_2,34.9, 2-H); LRMS

(EI): m/z 168 (M⁺, 100%), 150 (M⁺− H₂O, 66.8); >98% ee

(Method A).

(v) Metabolite from 2-methyl benzo[b]furan 4g

6-Hydroxy-2-methylbenzo[b]furan 9f. Yield (0.168 g, 3%); mp

48–49 °C (Et₂O–hexane); R_f0.85 (eluent B) (Found: C, 72.8; H,

1

5.6. C₉H₈O₂requires C, 73.0; H, 5.4%); H-NMR (300 MHz,

CDCl₃) δ 2.41 (3 H, s, Me), 6.27 (1 H, s, 3-H), 6.72 (1 H, dd,

J_5,48.3, J_5,72.2, 5-H), 6.91 (1 H, d, J_7,51.9, 7-H), 7.28 (1 H, d,

J

_4,58.2, 4-H); LRMS (EI): m/z 148 (M⁺, 100%), 91(56), 77(83);

ν_max(KBr): 3498 cm⁻¹(OH).

4-Hydroxybenzo[b]thiophene 7a. Yield (0.52 g, 4%); mp

74–76 °C (from Et₂O–hexane) (lit.¹⁶76–78 °C); R_f0.88 (eluent

(vi) Metabolites from 3-methylbenzo[b]furan 1g

(−)-(6S,7S)-cis-6,7-Dihydroxy-6,7-dihydro-3-methyl

1

A); H-NMR (300 MHz, CDCl₃) δ 5.19 (1 H, br s, OH), 6.71

benzo[b]-

(1 H, d, J_5,67.3, 5-H), 7.19 (1 H, dd, J_6,77.6, J_6,57.3, 6-H),

7.35 (1 H, d, J_2,35.6, 2-H), 7.47 (2 H, dd, J_7,67.5, J_3,25.7, 7-H,

3-H); LRMS (EI): m/z 150 (M⁺, 100%), 121 (25).

furan 4g. Yield (0.60 g, 12%); mp (dec.) 82–84 °C (from Et₂O–

hexane); R_f0.41 (eluent B); [α]_D−48 (c 0.78, CHCl₃); HRMS

(Found: M⁺166.06384. C₉H₁₀O₃requires 166.06389); ¹H-NMR

(500 MHz, CDCl₃) δ 2.06 (3 H, s, Me), 4.58–4.62 (2 H, m, 6-H,

7-H), 5.75 (1 H, d, J_5,410.3, 5-H), 6.34 (1 H, dd, J_4,510.0, J_4,6

2.3, 4-H), 7.08 (1 H, s, 2-H); LRMS (EI): m/z 166 (M⁺, 44%),

148 (100), 123 (74); ν_max(KBr): 3468 (OH); >98% ee

(Method A).

6-Hydroxy-3-methyl benzofuran 9g (from cis-diol 4g). Yield

(0.175 g, 4%); mp 90–91 °C (CHCl₃–hexane); R_f0.88 (eluent

B) (Found: C, 72.5; H, 5.2. C₉H₈O₂requires C, 73.0; H, 5.4%);

¹H-NMR (500 MHz, CDCl₃) δ 2.16 (3 H, s, Me), 6.80 (1 H, dd,

J_5,48.7, J_5,72.6, 5-H), 6.92 (1 H, d, J_7,52.5, 7-H), 7.29 (1 H,

d, J_4,58.7, 4-H), 7.37 (1 H, s, 2-H); LRMS (EI): m/z 148

(M⁺, 100%), 147 (82); ν_max(KBr): 3550 cm⁻¹(OH).

5-Hydroxybenzo[b]thiophene 8a. Yield (0.71 g, 5%); mp

70–71 °C (from Et₂O–hexane) (lit.¹⁶72–77 °C); R_f0.85 (eluent

1

A); H-NMR (300 MHz, CDCl₃) δ 6.95 (1 H, dd, J_6,78.7, J_6,4

2.4, 6-H), 7.23 (1 H, d, J_3,25.6, 3-H), 7.27 (1 H, d, J_4,62.5, 4-

H), 7.47 (1 H, d, J_2,35.6, 2-H), 7.74 (1 H, d, J_7,68.6, 7-H);

LRMS (EI): m/z 150 (M⁺, 100%), 121 (14).

(ii) Metabolite from 2-methyl benzo[b]thiophene 1b

(+)-2-Methyl-(4R,5S)-cis-4,5-dihydroxy-4,5-dihydrobenzo[b]thio-

phene 3b. Yield (1.80 g, 25%); mp (dec.) 96–102 °C (from

CHCl₃–hexane); R_f0.23 (eluent A); [α]_D+125.0 (c 0.55,

MeOH) (Found: C, 54.3; H, 5.8; S, 16.4. C₉H₁₀O₂S·H₂O

requires C, 54.0; H, 6.0; S, 16.0%); ¹H-NMR (500 MHz,

CDCl₃) δ 2.38 (1 H, br d, J_OH,46.5, OH), 2.46 (1 H, s, Me),

2.59 (1 H, d, J_OH,54.9, OH), 4.39–4.44 (1 H, m, 5-H), 4.59 (1

H, dd, J_4,55.7, J_4,OH7.9, 4-H), 5.80 (1 H, dd, J_6,79.7, J_6,53.2,

6-H), 6.38 (1 H, dd, J_7,69.9, J_7,51.9, 7-H), 6.78 (1 H, s, 2-H);

¹³C-NMR (125 MHz, CDCl₃) δ 15.3, 67.8, 69.6, 121.1, 125.5,

126.9, 132.1, 136.5, 139.6; LRMS (EI): m/z 182 (M⁺, 55%), 164

(100); ν_max(KBr) 3250 cm⁻¹(OH).

Heterocyclic ring cis/trans diol bioproducts 5a, 5b, 5d–f and

derivatives 5a_cisMTPA(R), 5e_transMTPA(S), 10a, 10e, 10f, 11a from

substrates 1a, 1b, 1d–f using P. putida UV4

(i) Metabolites from benzo[b]thiophene 1a

2,3-Dihydroxy-2,3-dihydrobenzo[b]thiophene 5a_cis/5a_trans. The

isomeric mixture of diols 5a_cis/5a_transwas obtained by ﬂash

chromatography (Et₂O : hexane, 1 : 1); yield (4.86 g, 37%); R_f

0.5 (eluent A) (Found: C, 57.1; H, 4.8; S, 19.0. C₈H₈O₂S

requires C, 57.1; H, 4.8; S, 19.1%); LRMS (EI): m/z 168

(M⁺, 86%), 150 (12), 139 (100); 60–63% ee (Methods A and B).

(iii) Metabolite from 3-methyl benzo[b]thiophene 1c

(+)-3-Methyl-(4R,5S)-cis-4,5-dihydroxy-4,5-dihydrobenzo[b]thio-

phene 3c. Yield (4.1 g, 45%); mp (dec.) 83–89 °C (from

CHCl₃–hexane); R_f0.2 (eluent A); [α]_D+103.0 (c 0.46, MeOH)

7300 | Org. Biomol. Chem., 2012, 10, 7292–7304

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(+)-(2S,3R)-2,3-Dihydroxy-2,3-dihydrobenzo[b]thiophene 5a_cis

.

82.4, 94.5, 123.4, 125.9 (×2), 129.7, 137.9, 138.8; LRMS (EI):

m/z 182 (M⁺, 30%), 139 (100); ν_max(KBr): 3300 cm⁻¹(OH);

9% ee (Methods A and B).

Mp 99–100 °C (from CH₂Cl₂–hexane); [α]_D+116.8 (c 0.41,

CHCl₃); ¹H-NMR (300 MHz, CDCl₃) δ 5.13 (1 H, d, J_3,24.1, 3-

H), 5.58 (1 H, d, J_2,34.1, 2-H), 7.13–7.25 (3 H, m, 4-H, 5-H, 6-

H), 7.40 (1 H, d, J_7,67.3, 7-H); ¹³C-NMR (125 MHz, CDCl₃) δ

78.8, 82.0, 123.6, 125.5, 125.7, 129.8, 136.7, 138.4; ν_max(KBr):

3412 cm⁻¹(OH).

(iii) Metabolites from 5-methyl benzo[b]thiophene 1d

5-Methyl 2,3-dihydroxy-2,3-dihydrobenzo[b]thiophene 5d_cis

/

5d_trans. The isomeric mixture of diols 5d_cis/5d_transwas obtained

by ﬂash chromatography (Et₂O : hexane, 3 : 1); yield (2.9 g,

79%); R_f0.5 (eluent B) (Found: C, 59.0; H, 5.6; S, 17.3.

C₉H₁₀O₂S requires C, 59.3; H, 5.5; S, 17.6%); LRMS (EI): m/z

182 (M⁺, 51%), 166 (100); ν_max(KBr): 3408 cm⁻¹(OH); >98%

ee (Method B).

(+)-(2S,3R)-bis-[(R)-2′-Methoxy-2′-phenyl-2′-triﬂuoromethylace-

toxy]-2,3-dihydrobenzo[b]thiophene 5a_cisMTPA(R). To a mixture

(40 : 60) of cis : trans-diols 5a_cis/5a_trans(0.050 g, 0.3 mmol) in

anhydrous pyridine (0.5 ml) was added (+)-MTPA chloride

(0.154 g, 0.66 mmol). The reaction mixture was left overnight at

room temperature. Excess of pyridine was removed under high

vacuum by addition of an equivalent volume of toluene and the

mixture concentrated under high vacuum. Puriﬁcation of the

crude product by PLC (two elutions using 10% Et₂O : hexane)

afforded diMTPA ester (+)-5a_cisMTPA(R)as a white crystalline

compound (0.029 g, 16%); mp 123–125 °C (from Et₂O–

hexane); R_f0.59; [α]_D+243 (0.78, CHCl₃) (Found: C, 56.3; H,

3.8; S, 5.8. C₂₈H₂₂O₆F₆S requires C, 56.0; H, 3.7; S, 5.4%);

¹H-NMR (300 MHz, CDCl₃) δ 3.07 (3 H, s, OMe), 3.35 (3 H, s,

OMe), 6.32 (1 H, d, J_3,24.8, 3-H), 6.72 (1 H, d, J_2,34.9, 2-H),

6.82–7.49 (14 H, m, Ar-H); LRMS (EI): m/z 600 (M⁺, 8%), 189

(74), 57 (100); ν_max(KBr): 1758 cm⁻¹(CvO).

(+)-(2S,3R)-5-Methyl-2,3-dihydroxy-2,3-dihydrobenzo[b]thio-

phene 5d_cis. Mp 126–128 °C (from CHCl₃); [α]_D+135.6

1

(c 0.54, CHCl₃); H-NMR (500 MHz, CDCl₃) δ 2.34 (3 H, s,

Me), 2.69 (1 H, br s, OH), 2.87 (1 H, br s, OH), 5.11 (1 H, d,

J_3,24.3, 3-H), 5.60 (1 H, d, J_2,34.7, 2-H), 7.06–7.27 (3 H, m,

Ar-H).

(−)-(2R,3R)-5-Methyl-2,3-dihydroxy-2,3-dihydrobenzo[b]thio-

phene 5d_trans. Mp 126–128 °C (from MeOH); [α]_D−80.0

1

(c 0.45, MeOH); H-NMR (500 MHz, CD₃OD) δ 2.29 (3 H, s,

Me), 4.98 (1 H, bs, 3-H), 5.48 (1 H, bs, 2-H), 7.02–7.04 (2 H,

m, 6-H, 7-H), 7.18 (1H, s, 4-H).

(iv) Metabolites from benzo[b]furan 1e

Crystal data for 5a_cisMTPA(R). C₂₈H₂₂F₆O₆S, M = 600.5, mono-

clinic, a = 10.186(3), b = 12.692(4), c = 10.984(3) Å, β = 93.75

(2)°, U = 1417.0(7) Å³, T = 293(2) K, space group P2₁(no. 4),

Mo-Kα radiation, λ = 0.71073 Å, Z = 2, F(000) = 616, D_x=

1.407 g cm⁻³, μ = 0.194 mm⁻¹, Siemens P3 diffractometer,

ω scans, 3.7° < 2θ < 55.1°, measured/independent reﬂections:

3595/3423, direct methods solution, full-matrix least squares

2,3-Dihydroxy-2,3-dihydrobenzo[b]furan 5e_cis/5e_trans. The iso-

meric mixture of diols 5e_cis/5e_transwas obtained as a gum by

ﬂash chromatography (EtOAc : hexane, 2 : 3); yield (2.2 g,

17%); [α]_D−14.0 (c 0.76, CHCl₃), [α]_D−34.0 (c 0.84, H₂O);

HRMS (Found: M⁺152.0470. C₈H₈O₃requires 152.0473);

¹H-NMR (500 MHz, CDCl₃) δ (cis/trans 60 : 40), 4.99 (1H, s,

3-H_trans), 5.00 (1 H, d, J_3,25.1, 3-H_cis), 5.73 (1 H, s, 2-H_trans),

5.75 (1 H, d, J_2,35.1, 2-H_cis), 6.80–6.98 (2 H, m, 5-H, 7-H),

7.25 (1 H, ddd, J_6,7= J_6,58.1, J_6,42.1, 6-H), 7.37 (1 H, dd, J_4,5

7.8, J_4,62.0, 4-H); ν_max(neat): 3472 cm⁻¹(OH); ¹H-NMR

(500 MHz, C₆D₆) (cis/trans, 55 : 45) δ 4.35 (1 H, d, J_3,25.1, 3-

H_cis), 4.78 (1 H, s, 3-H_trans), 5.39 (1 H, d, J_2,35.2, 2-H_cis), 5.49

(1 H, s, 2-H_trans), 6.66–7.10 (4H, m, ArH); ¹H-NMR (500 MHz,

D₂O) (cis/trans, 10 : 90) δ 5.04 (1 H, s, 3-H_trans), 5.18 (1 H, d,

J_3,25.3, 3-H_cis), 5.77 (1 H, d, J_2,30.8, 2-H_trans), 5.89 (1 H, d,

J_2,35.2, 2-H_cis), 6.78–7.22 (2 H, m, ArH), 7.25–7.52 (2 H, m,

ArH); ¹³C-NMR (125 MHz, CDCl₃) (cis isomer) δ 70.5, 100.5,

110.5, 121.5, 125.6, 126.2, 130.9, 157.1; (trans isomer) 77.7,

106.8, 110.7, 121.7, 126.0, 126.3, 131.1, 158.6; LRMS (EI): m/z

152 (M⁺, 58%), 134 (44), 121 (100); 55% ee (Methods A

and B).

2

reﬁnement on F_o, anisotropic displacement parameters for non-

hydrogen atoms; all hydrogen atoms located in a difference

Fourier synthesis but included at positions calculated from the

geometry of the molecule using the riding model, with isotropic

vibration parameters. R₁= 0.063 for 1474 data with F_o> 4σ(F_o),

373 parameters, wR₂= 0.122 (all data), GoF = 1.03, Δρ_min,max

=

−0.17/0.21 e Å⁻³. CCDC 871979. The absolute conﬁguration is

established as (2S,3R) from the anomalous scattering arising

from the sulfur atom (Flack parameter, x = 0.05(17)) and inde-

pendently from the known absolute conﬁguration of the (R)-

MTPA group.

(−)-(2R,3R)-2,3-Dihydroxy-2,3-dihydrobenzo[b]thiophene 5a_trans

.

Mp 97–98 °C (from MeOH); [α]_D−97.0 (c 0.48, MeOH) and

1

[α]_D−98.3 (c 0.57, D₂O); H-NMR (300 MHz, D₂O) δ 5.19 (1

H, d, J_3,21.6, 3-H), 5.58 (1 H, d, J_2,31.6, 2-H), 7.23–7.26 (1 H,

m, 4-H), 7.34–7.36 (2 H, m, 5-H, 6-H), 7.46 (1 H, d, J_7,67.2, 7-

H); ¹³C-NMR (125 MHz, CD₃OD) δ 84.7, 90.0, 123.7, 125.6,

127.3, 130.7, 140.2, 141.3; ν_max(KBr): 3256 cm⁻¹(OH).

(+)-(2S,3R)-bis-[(S)-2′-Methoxy-2′-phenyl-2′-triﬂuoromethylace-

toxy]-2,3-dihydrobenzo[b]furan 5e_transMTPA(S). To a mixture of

cis/trans-diols 5e_cis/5e_trans(0.016 g, 0.1 mmol) in anhydrous

pyridine (0.2 ml) was added (−)-MTPA chloride (0.056 g,

0.22 mmol). The reaction mixture was shaken gently and then

left overnight at room temperature. Excess of pyridine was

removed from the mixture under high vacuum to yield an oily

(ii) Metabolite from 2-methyl benzo[b]thiophene 1b

2-Methyl-2,3-dihydroxy-2,3-dihydrobenzo[b]thiophene

5b_cis.

Yield (0.20 g, 3%); mp 87–89 °C (from CH₂Cl₂–hexane); R_f0.4

(eluent A); HRMS (Found: M⁺182.0394. C₉H₁₀O₂S requires

182.0402); [α]_D−2.9 (c 0.51, CHCl₃) and [α]_D+5.0 (c 0.51,

1

residue. H-NMR analysis of this crude product showed that (a)

it was mainly (95%) composed of a mixture of trans diMTPA

esters 5e_transMTPA(S)and (b) the starting mixture of diols 5e_cis/

5e_transwas estimated to be of 55% ee. From PLC (6% Et₂O in

hexane) of the oily residue, a mixture of (2S,3R)- and (2R,3S)-

diMTPA diastereoisomers of diol 5e_trans(the major component)

1

MeOH); H-NMR (500 MHz, CDCl₃) δ 1.84 (3 H, s, Me), 2.48

(1 H, d, J_OH,39.9, OH), 3.27 (1 H, s, OH), 4.78 (1 H, d, J_3,OH

9.9, 3-H), 7.12–7.16 (1 H, m, Ar-H), 7.22–7.28 (2 H, m, Ar-H),

7.38 (1 H, d, J 7.7, Ar-H); ¹³C-NMR (125 MHz, CDCl₃) δ 25.7,

Org. Biomol. Chem., 2012, 10, 7292–7304 | 7301

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was separated (0.012 g, 30%, R_f0.42); trans-isomer 5e_transMTPA(S)

,

(EI): m/z 168 (M⁺, 13%), 123 (100), 95 (31); ν_max(CH₂Cl₂):

the major (85%) compound in this separated mixture, was then

crystallized out as a white solid; mp 116–118 °C (from CH₂Cl₂–

hexane); [α]_D+68 (0.73, CHCl₃) (Found: C, 57.4; H, 3.4.

3480, 3468 cm⁻¹(OH); >98% ee (Method A).

Enantiomeric excess values and absolute conﬁgurations of

diol metabolites 3a–c, 4e, 4g and 10f. The enantiopurity of cis-

dihydrodiol 4e, obtained from P. putida UV4 biotransformation

of benzo[b]furan 1e, had been determined as >98% ee, ^4bfollow-

ing partial hydrogenation (10% Pd–C, EtOAc) to yield the corre-

sponding cis-tetrahydrodiol 4e′, formation of the diMTPA ester

derivative and analysis of its ¹H-NMR spectrum. A similar

approach was used for cis-dihydrodiols 3a–c and 4g. Formation

of diMTPA esters of the corresponding cis-tetrahydrodiols 3a′–c′

1

C₂₈H₂₂O₇F₆requires C, 57.5; H, 3.8%); H-NMR (500 MHz,

CDCl₃) δ 3.47 (3 H, s, OMe), 3.51 (3 H, s, OMe), 6.20 (1 H, s,

3-H), 6.73 (1 H, d, J_2,30.7, 2-H), 7.00 (1H, d, J 8.1, Ar-H), 7.05

(1 H, m, Ar-H), 7.37–7.50 (12 H, m, Ar-H); LRMS (EI): m/z

584 (M⁺, 14%), 351 (37), 189 (100); ν_max(KBr): 1733 cm⁻¹

(CvO).

Crystal data for 5e_transMTPA(S). C₂₈H₂₂F₆O₇, M = 584.5, ortho-

rhombic, a = 6.783(2), b = 16.637(6), c = 23.581(9) Å, U =

2661.1(16) Å³, T = 293(2) K, space group P2₁2₁2₁(no. 19),

Cu-Kα radiation, λ = 1.54178 Å, Z = 4, F(000) = 1200, D_x=

1.459 g cm⁻³, μ = 1.150 mm⁻¹, Siemens P3 diffractometer, ω

scans, 6.5° < 2θ < 110.2°, 1961 independent reﬂections, direct

1

and 4g′ and their H-NMR analyses showed that each of the cis-

dihydrodiol precursors was a single enantiomer (>98% ee).

(−)-(4R,5S)-4,5-Dihydroxy-4,5,6,7-tetrahydrobenzo[b]thiophene

3a′ (from diol 3a). Yield (0.067 g, 66%); mp 135–137 °C (from

CHCl₃–hexane); [α]_D−56.0 (c 0.77, CHCl₃); HRMS (Found:

M⁺170.0408. C₈H₁₀O₂S requires 170.0408); ¹H-NMR

(300 MHz, CDCl₃) δ 1.91–1.99 (1 H, m, 6-H), 2.01–2.13 (1 H,

m, 6′-H), 2.72–2.83 (1 H, m, 7-H), 2.93–3.02 (1 H, m, 7′-H),

3.97–4.02 (1 H, m, 5-H), 4.69 (1 H, d, J_4,53.6, 4-H), 7.01 (1 H,

d, J_3,25.1, 3-H), 7.13 (1 H, d, J_2,35.1, 2-H); LRMS (EI): m/z

170 (M⁺, 22%), 126 (100); ν_max(KBr): 3250 cm⁻¹(OH).

2

methods solution, full-matrix least squares reﬁnement on F_o,

anisotropic displacement parameters for non-hydrogen atoms; all

hydrogen atoms located in a difference Fourier synthesis but

included at positions calculated from the geometry of the mo-

lecule using the riding model, with isotropic vibration parameters.

R₁= 0.069 for 1364 data with F_o> 4σ(F_o), 373 parameters, wR₂

= 0.206 (all data), GoF = 1.02, Δρ_min,max= −0.27/0.28 e Å⁻³

.

(−)-(4R,5S)-bis-[(R)-2′-Methoxy-2′-phenyl-2′-triﬂuoromethylace-

toxy]-4,5,6,7-tetrahydrobenzo[b]thiophene 3a′_cisMTPA(R). A solu-

tion of cis-tetrahydrodiol 3a′ (0.06 g, 0.35 mmol) in anhydrous

pyridine (ca. 0.5 ml) was treated with (+)-MTPA chloride

(0.194 g, 0.77 mmol) and the mixture left at room temperature

overnight. Excess of pyridine was removed under high vacuum

and the residue puriﬁed by PLC (10% Et₂O in hexane) to afford

crystals of bis-(R)-MTPA ester 3a′_cisMTPA(R)(0.83 g, 39%); mp

111–112 °C (MeOH–CHCl₃); [α]_D−53.0 (c. 0.66, CHCl₃)

(Found: C, 55.9; H, 3.9; S, 5.0. C₂₈H₂₄O₆F₆S requires C, 55.8;

CCDC 871980. The absolute conﬁguration is established as

(2S,3R) relative to the known absolute conﬁguration of the

(S)-MTPA group.

(1R)-1,2-Dihydroxy-1-(2′-hydroxyphenyl)ethane 10e. The most

polar fractions obtained from puriﬁcation by ﬂash chromato-

graphy (EtOAc : hexane, 2 : 3) yielded phenolic diol 10e (1.05 g,

8%); mp 68–69 °C (from CH₂Cl₂–hexane) (lit.,^4bmp

69–71 °C); R_f0.3 (eluent B); [α]_D−23.4 (c 0.89, MeOH) (lit.,^4b

−24, MeOH); ¹H-NMR (300 MHz, CDCl₃) δ 3.83 (2 H, m, 2-H,

2-H), 4.95 (1 H, dd, J_1,2a5.0, J_1,2b7.5, 1-H), 6.82–6.89 (2 H, m,

3′-H, 5′-H), 7.01 (1 H, d, J 7.5, 4-H), 7.19 (1H, dd, J_6,57.8, J_6,4

1.4, 6′-H).

1

H, 4.0; S, 5.3%); H-NMR (500 MHz, CDCl₃) δ 2.24–2.29 (1

H, m, 6-H), 2.37–2.45 (1 H, m, 6′-H), 2.98–3.03 (2 H, m, 7-H,

7′-H), 3.28 (3 H, s, OMe), 3.50 (3 H, s, OMe), 5.50–5.53 (1H,

m, 5-H), 6.20 (1 H, d, J_4,52.6, 4-H), 6.95 (1 H, d, J_3,25.3, 3-H),

7.12 (1 H, d, J_2,35.3 2-H), 7.23–7.54 (10 H, m, Ar-H); LRMS

(EI): m/z 602 (M⁺, 3%), 189 (100); ν_max(KBr): 1756 cm⁻¹

(CvO).

(v) Metabolite from 2-methyl benzo[b]furan 1f

(+)-(2R,3S)-2,3-Dihydroxy-2,3-dihydro-2-methyl[b]benzofuran

5f_cis. Diol 5f_ciswas isolated as a gum, using ﬂash chromato-

graphy (5–25% EtOAc in hexane) followed by PLC (10%

EtOAc in hexane) (0.444 g, 8%); [α]_D+206 (c 1.1, CHCl₃);

HRMS (Found: 166.0632. C₉H₁₀O₃requires 166.0630);

¹H-NMR (500 MHz, CDCl₃) δ 2.12 (3 H, s, Me), 5.23 (1 H, s,

3-H), 6.85 (1 H, d, J 8.2, 4-H), 6.95 (1 H, m, 5-H), 7.23 (2 H,

m, 6-H, 7-H); ¹³C-NMR (125 MHz, CDCl₃) δ 23.5, 77.5, 116.7,

118.0, 120.2, 121.5, 129.5, 131.1, 155.0; LRMS (EI): m/z 166

(M⁺, 4%), 148 (100), 91 (58); ν_max(KBr): 3540 cm⁻¹(OH);

80% ee (Method B).

Crystal data for 3a′_cisMTPA(R). C₂₈H₂₄F₆O₆S, M = 602.5,

orthorhombic, a = 10.026(4), b = 12.876(4), c = 21.444(7) Å,

U = 2768.3(17) Å³, T = 293(2) K, space group P2₁2₁2₁(no. 19),

Mo-Kα radiation, λ = 0.71073 Å, Z = 4, F(000) = 1240, D_x=

1.446 g cm⁻³, μ = 0.198 mm⁻¹, Siemens P3 diffractometer, ω

scans, 3.7° < 2θ < 55.1°, 3593 independent reﬂections, direct

2

methods solution, full-matrix least squares reﬁnement on F_o,

anisotropic displacement parameters for non-hydrogen atoms; all

hydrogen atoms located in a difference Fourier synthesis but

included at positions calculated from the geometry of the mo-

lecule using the riding model, with isotropic vibration parameters.

R₁= 0.061 for 1853 data with F_o> 4σ(F_o), 373 parameters,

wR₂= 0.136 (all data), GoF = 1.02, Δρ_min,max= −0.24/0.25 e Å⁻³.

CCDC 871978. The absolute conﬁguration is established as

(4R,5S) from the anomalous scattering arising from the sulfur

atom (Flack parameter x = 0.2(2)) and independently from the

known absolute conﬁguration of the (R)-MTPA group.

(+)-(1S,2S)-1,2-Dihydroxy-1-(2′-hydroxyphenyl)propane

10f.

Phenolic diol 10f, the most polar bioproduct, was obtained as a

yellow gum, after puriﬁcation by PLC (EtOAc : hexane, 9 : 1)

(0.02 g, 1%); [α]_D+13.3 (c 0.8, EtOH); HRMS (Found: M⁺

168.0789. C₉H₁₂O₃requires 168.0787); ¹H-NMR (500 MHz,

CDCl₃) δ 1.11 (3 H, d, J_Me,26.4, Me), 4.05 (1 H, dq, J_2,17.9, J_2,Me

6.5, 2-H), 4.90 (1 H, d, J_1,27.9, 1-H), 6.85 (2 H, m, 3′-H, 5′-H),

7.01 (1 H, ddd, J_4′,3′= J_4′,47.6, J_4′,6′1.6, 4′-H), 7.20 (1 H, dd,

J_6′,5′7.3, J_6′,4′1.6, 6′-H); ¹³C-NMR (125 MHz, CDCl₃) 19.3,

70.3, 80.3, 117.4, 119.7, 124.3, 129.0, 129.5, 155.7; LRMS

(−)-2-Methyl-(4R,5S)-4,5-dihydroxy-4,5,6,7-tetrahydrobenzo[b]-

thiophene 3b′ (from diol 3b). Yield (0.06 g, 55%); mp

7302 | Org. Biomol. Chem., 2012, 10, 7292–7304

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155–157 °C (from CHCl₃–hexane) (Found: C, 58.2; H, 6.5; S,

17.7. C₉H₁₂O₂S requires C, 58.7; H, 6.5; S, 17.4%); [α]_D−57.6

acetate (1.36 ml, 10.7 mmol) was gently reﬂuxed under nitrogen

until the starting diol was consumed. The cooled reaction

mixture was dried (Na₂CO₃), ﬁltered and the ﬁltrate concentrated

under reduced pressure. The crude diastereoisomeric mixture of

dioxoles 22, obtained as a pale yellow oil (0.57 g, 95%), was

used without puriﬁcation in the next step due to its instability on

silica gel. HRMS (EI) (Found: M⁺224.0512. C₁₁H₁₂O₃S

requires 224.0507); LRMS (EI): m/z 224 (M⁺, 30%), 151 (38),

150 (76), 122 (100), 121 (83).

1

(c 0.83, MeOH); H-NMR (300 MHz, CDCl₃) δ 1.58 (2 H, br s,

OH), 1.92–2.10 (2 H, m, 6-H, 6′-H), 2.42 (3 H, s, Me),

2.72–2.77 (1 H, m, 7-H), 2.84–2.92 (1 H, m, 7′-H), 3.94–4.00 (1

H, m, 5-H), 4.62 (1 H, d, J_4,53.6, 4-H), 6.66 (1 H, s, 3-H);

LRMS (EI): m/z 184 (M⁺, 22%), 166 (13), 140 (100).

(−)-3-Methyl-(4R,5S)-4,5-dihydroxy-4,5,6,7-tetrahydrobenzo[b]-

thiophene 3c′ (from diol 3c). Yield (0.33 g, 65%); mp

158–160 °C (from CHCl₃–hexane); [α]_D−101.0 (c 0.45,

MeOH) (Found: C, 58.2; H, 6.8; S, 17.1. C₉H₁₂O₂S requires C,

58.7; H, 6.5; S, 17.4%); ¹H-NMR (300 MHz, CDCl₃) δ

1.91–1.99 (2 H, m, 6-H, 6′-H), 2.26 (3 H, s, Me), 2.78–2.84 (1

H, m, 7-H), 2.91–2.97 (1 H, m, 7′-H), 3.90–3.97 (1 H, m, 5-H),

4.62 (1 H, d, J_4,53.7, 4-H), 6.76 (1 H, s, 2-H); LRMS (EI): m/z

184 (M⁺, 19%), 166 (14), 140 (100).

1

Major diastereoisomer: H-NMR (400 MHz, CDCl₃) δ 1.62 (3,

s, Me), 2.93 (3 H, s, OMe), 5.88 (1 H, d, J_8b,3a6.5, 8b-H), 6.18

(1 H, d, J_3a,8b6.5, 3a-H), 7.09–7.18 (2 H, m, 4-H, 6-H),

7.21–7.28 (1 H, m, 5-H), 7.36 (1 H, d, J 7.4, 7-H); ¹³C-NMR

(100 MHz, CDCl₃) δ 19.9, 48.4, 84.4, 84.9, 120.2, 122.0, 122.9,

124.3, 128.1, 134.8, 137.3.

1

Minor diastereoisomer: H-NMR (400 MHz, CDCl₃) δ 1.46

(3H, s, Me), 3.33 (3H, s, OMe), 5.89 (1H, d, J_8b,3a6.0, 8b-H),

6.24 (1H, d, J_3a,8b6.0, 3a-H), 7.04–7.40 (4H, m, 4-H, 5-H, 6-H,

7-H); ¹³C-NMR (100 MHz, CDCl₃) δ 22.0, 47.5, 84.6, 85.3,

120.5, 122.0, 123.2, 124.6, 128.5, 134.2, 137.3.

(−)-3-Methyl-(6S,7S)-6,7-dihydroxy-4,5,6,7-tetrahydrobenzo[b]-

furan 4g′ (from diol 4g). Yield (0.164 g, 81%); an oil; [α]_D

−76.0 (c 0.74, MeOH) (Found: C, 64.1; H, 7.4. C₉H₁₂O₃

1

requires C, 64.3; H, 7.2%); H-NMR (300 MHz, CDCl₃) δ 1.92

2-Chloro-2,3-dihydrobenzo[b]thiophen-3-yl acetate 23. To

cooled (0 °C) and stirred solution of dioxole isomers 22 (0.5 g,

2.23 mmol) in dried CH₂Cl₂(20 ml) containing Et₃N (0.1 ml)

a

(2 H, m, 5-H), 2.13 (3 H, s, Me), 2.47 (1 H, m, 4-H), 2.73 (1 H,

m, 4-H′), 4.06 (1 H, m, 6-H), 4.68 (1 H, d, J_7,64.2, 7-H), 7.10

(1 H, s, 2-H); LRMS (EI): m/z 168 (M⁺, 23%), 124 (100); ν_max

(neat): 3546 cm⁻¹(OH).

was added, dropwise,

a solution of chlorotrimethylsilane

(0.56 ml, 4.46 mmol) in CH₂Cl₂(5 ml) over a period of 15 min.

After leaving the stirred reaction mixture for a further 10 min,

the solvent was removed under reduced pressure to give crude

chloroacetate 23 as an oil (yield ca. 70%). A small sample

(0.1 g) of the crude product was rapidly puriﬁed by PLC (10%

EtOAc in hexane) to furnish chloroacetate 23 as a light yellow

oil, R_f0.62; [α]_D−421.6 (c 0.45, CHCl₃); HRMS (Found: M⁺

228.0003. C₁₀H₉O₂SCl requires 228.0011); ¹H-NMR

(400 MHz, CDCl₃) δ 2.07 (3 H, s, Me), 5.67 (1 H, s, 2-H), 6.32

(1 H, s, 3-H), 7.21 (1 H, ddd, J_5,4= J_5,67.4, J_5,71.2, 5-H), 7.32

(1 H, dm, J_7,67.8, 7-H), 7.36–7.41(1 H, m, 6-H), 7.53 (1 H, dm,

J_4,57.4, 4-H); ¹³C-NMR (100 MHz, CDCl₃) δ 21.1, 70.1, 85.1,

123.2, 126.0, 128.0, 131.2, 134.0, 140.4, 170.2; LRMS (EI): m/z

228 (M⁺, 5%), 168 (47), 150 (100). ν_max(CH₂Cl₂): 1742 cm⁻¹

(CvO), 1225 (C–O), 753 (aromatic).

(+)-(1S,2S)-1,2-Dihydroxy-1-(2′-methoxyphenyl)propane 15. To

a stirred mixture of tert-butyl alcohol (5 ml), H₂O (5 ml) and

AD-mix-α (1.4 g) was added methanesulphonamide (0.095 g)

and the mixture cooled to 0 °C. trans-1-(2′-Methoxyphenyl)

prop-1-ene 14 (0.150 g, 1 mmol) was added and vigorous stir-

ring, at 0 °C, continued for 16 h. The reaction mixture was

treated with sodium sulphite (1.0 g), stirred for another 0.5 h at

ambient temperature, and diluted with EtOAc (20 ml). The

organic layer was separated and the remaining aqueous phase

extracted with EtOAc. The extract was washed with water, dried

(Na₂SO₄), and concentrated to yield the crude diol 15. Puriﬁ-

cation by PLC (EtOAc : hexane, 2 : 3) gave diol 15 as a white

solid (0.156 g, 86%); mp 69–70 °C (from CHCl₃–hexane); [α]_D

+26.0 (c 0.9, EtOH) (Found: C, 66.3; H, 7.7. C₁₀H₁₄O₃requires

1

C, 65.9; H, 7.7%); H-NMR (500 MHz, CDCl₃) δ 1.07 (3 H, d,

Benzo[b]thiophene-2,3-oxide 21a. Chloroacetate 23 (0.050 g,

0.22 mmol) was dissolved in THF-d₈(1 ml) under nitrogen and

sodium methoxide (0.024 g, 0.44 mmol) was added to the sol-

ution. The mixture was stirred for 2 h at 0 °C, ﬁltered and the

resulting yellow ﬁltrate submitted immediately for NMR and

mass spectrometry analyses. HRMS (EI) (Found: M⁺150.0134.

C₈H₆OS requires 150.0139); ¹H-NMR (400 MHz, THF-d₈) δ

4.80 (1 H, d, J 3.0, 3-H), 5.47 (1 H, d, J₂3.0, 2-H), 7.11 (1 H,

ddd, J 7.5, 6.3, 2.0, 6-H), 7.23–7.27 (2 H, m, 4-H, 5-H), 7.59 (1

H, ddd, J 7.5, 1.2, 0.7, 7-H); ¹³C-NMR (100 MHz, THF-d₈) δ

62.8, 67.1, 124.4, 125.2, 128.0, 130.3, 136.1, 143.8; LRMS

(EI): m/z 150 (M⁺, 74%), 121 (89), 43 (100).

J

_Me,26.3, Me), 3.86 (3 H, s, OMe), 3.99 (1 H, dq, J_2,17.1, J_2,Me

6.4, 2-H), 4.57 (1 H, d, J_1,27.4, 1-H), 6.86–7.00 (2 H, m, 3-H,

5-H), 7.25–7.31 (2 H, m, 4-H, 6-H); LRMS (EI): m/z 182

(M⁺, 34%), 137 (100); ν_max(CH₂Cl₂): 3502 cm⁻¹(OH).

(+)-(1S,2S)-1,2-Dihydroxy-1-(2′-methoxyphenyl)propane

15

(from diol 10f). A sample of the phenolic diol metabolite 10f

(0.01 g) was methylated by treating it at 0 °C with an excess of

freshly prepared solution of ethereal diazomethane. Puriﬁcation

by PLC (5% MeOH in CHCl₃) gave the methoxy derivative 15

(0.009 g, 83%), [α]_D+22.0 (c 0.4, EtOH). This sample was spec-

trally indistinguishable from the sample of diol 15 synthesized

from alkene 14.

2,3-Dihydrobenzo[b]thiophen-3-one 12a. Attempts to purify

arene oxide 21a led to its decomposition to yield ketone 12a,

which on puriﬁcation by PLC (10% EtOAc in hexane) gave a

light pink coloured solid. Crystallization from hexane furnished

white needles; mp 64–66 °C (hexane) (lit.^7amp 65–66 °C); R_f

0.55 (10% EtOAc in hexane); H-NMR (400 MHz, CDCl₃) δ

3.80 (2 H, s, CH₂), 7.22 (1 H, ddd, J_5,48.0, J_5,67.2, J_5,71.0, 5-

H), 7.44 (1 H, dd, J_7,68.0, J_7,51.0, 7-H), 7.56 (1 H, ddd, J_6,7

Synthesis of benzo[b]thiophene-2,3-oxide 21a. The synthesis

of compounds 22, 23 and 21a was carried out using an ortho-

ester procedure, similar to that reported earlier for other types of

arene oxides employing THF-d₈solvent in the ﬁnal step.^14a–c

2-Methoxy-2-methyl-3a,8b-dihydrobenzo[b]thiophene-1,3-dioxole

22. A mixture of diol 5a_cis/5a_trans(0.45 g, 2.68 mmol), dry

benzene (30 ml), benzoic acid (0.030 g) and trimethyl ortho-

1

Org. Biomol. Chem., 2012, 10, 7292–7304 | 7303

View Article Online

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Acknowledgements

We thank Dr Kevin E. O’Connor and Dr J. Nikodinovic-Runic

for the biotransformation work using SMO [E. coli BL21(DE3)-

pRSET-styAB] and the Department of Education of Northern

Ireland for Postgraduate Quota (INB, TAE, SH) and CAST

awards (BMcM).

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7304 | Org. Biomol. Chem., 2012, 10, 7292–7304

Article Doi

Toluene dioxygenase-catalyzed cis-dihydroxylation of benzo[b]thiophenes and benzo[b]furans: Synthesis of benzo[b]thiophene 2,3-oxide

DOI: 10.1039/c2ob26120k

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Article abstract of DOI:10.1039/c2ob26120k

Full text of DOI:10.1039/c2ob26120k

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