Structure, stereochemistry and synthesis of enantiopure cyclohexenone cis-diol bacterial metabolites derived from phenols

View Article Online / Journal Homepage / Table of Contents for this issue

Organic &

Biomolecular

Chemistry

This article is part of the

OBC 10^thanniversary

themed issue

All articles in this issue will be gathered together

online at

www.rsc.org/OBC10

Dynamic Ar t^Vic^iel^we^AL^ri^tn^ick^les^Online

Organic &

Biomolecular

Chemistry

Cite this: Org. Biomol. Chem., 2012, 10, 6217

www.rsc.org/obc

PAPER

Structure, stereochemistry and synthesis of enantiopure cyclohexenone

cis-diol bacterial metabolites derived from phenols†‡

Derek R. Boyd,*^aNarain D. Sharma,^aJohn F. Malone,^aPeter B. A. McIntyre,^aPaul J. Stevenson,^a

Christopher C. R. Allen,^bMarcin Kwit^cand Jacek Gawronski*^c

Received 11th January 2012, Accepted 22nd February 2012

DOI: 10.1039/c2ob25079a

Biotransformation of 3-substituted and 2,5-disubstituted phenols, using whole cells of P. putida UV4,

yielded cyclohexenone cis-diols as single enantiomers; their structures and absolute conﬁgurations have

been determined by NMR and ECD spectroscopy, X-ray crystallography, and stereochemical correlation

involving a four step chemoenzymatic synthesis from the corresponding cis-dihydrodiol metabolites. An

active site model has been proposed, to account for the formation of enantiopure cyclohexenone cis-diols

with opposite absolute conﬁgurations.

1. Introduction

Phenols are major aromatic metabolites formed via hydroxylase-

catalysed monohydroxylation, monooxygenase-catalysed epoxi-

dation (followed by spontaneous isomerisation), and dioxygen-

ase-catalysed cis-dihydroxylation of arenes followed by

spontaneous dehydration.^1aThe metabolic fate of phenols is thus

an important topic, particularly in the environment. The bacterial

monohydroxylation of phenols 1 to yield catechols 2,^1a–kand

hydroquinones 3,^1j,khas been widely recognized as a major min-

eralization pathway in the environment (Scheme 1). Recent

studies in these laboratories have focused on new families of

chiral metabolites from phenol substrates 1 using mutant and

recombinant bacterial strains.^2a–cIn this context, 3-substituted

phenols 1 (R′ = H) were found to adopt cis-dihydroxylation path-

ways involving toluene dioxygenase (TDO)-catalysed oxidation

to yield the corresponding, relatively stable, cyclohexenone cis-

diols 4_Sas single enantiomers.^2a–cThe latter type of cis-diols,

Scheme 1 Biotransformation products obtained from substituted

phenols using P. putida UV4.

often formed in modest yields, has received little attention com-

pared with the corresponding less stable benzene cis-dihydro-

diols 6^3a–nfrom non-phenolic substrates.

The cyclohexenone cis-diol bioproducts 4 (R′ = H), derived

from the TDO-catalysed biotransformation of 3-substituted

phenols 1 (R′ = H) in whole cells of P. putida UV4, were found

to be enantiopure, and of identical (5S) absolute conﬁgura-

^aSchool of Chemistry and Chemical Engineering, Queen’s University,

tion.^2a–cSurprisingly, the 2,5-disubstituted phenol, p-xylenol 1

Belfast, BT9 5AG, UK. E-mail: dr.boyd@qub.ac.uk; Fax: +44 (0) 28

(R = R′ = Me), also gave a cyclohexenone cis-diol 4 (R = R′ =

Me) as a single cyclohexenone cis-diol diastereoisomer (>98%

ee) but had the opposite (5R) absolute conﬁguration compared

with cyclohexenone cis-diol metabolites derived from 3-substi-

tuted phenols.^2aThe objectives of the current study were to: (i)

extend the range of substituted phenol substrates 1, found to

undergo dioxygenase-catalysed cis-dihydroxylation using whole

cell cultures of P. putida UV4, and provide evidence of the

general applicability of this new metabolic pathway to other

9097 4687; Tel: +44 (0) 28 9097 4421

^bSchool of Biological Sciences, Queen’s University, Belfast BT9 7BL,

UK

^cDepartment of Chemistry, A. Mickiewicz University, Grunwaldzka 6,

60–780 Poznan, Poland. E-mail: gawronsk@amu.edu.pl; Fax: (+48)-

61-8658-008

†This article is part of the Organic & Biomolecular Chemistry 10th

Anniversary issue.

‡Electronic supplementary information (ESI) available. CCDC 852568,

852569 and 852570. For ESI and crystallographic data in CIF or other

electronic format see DOI: 10.1039/c2ob25079a

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 6217–6229 | 6217

View Article Online

types of chiral cyclohexenone cis-diols (e.g. 4 and 5), (ii) carry

out full structural and stereochemical characterizations of new

cyclohexenone cis-diol diastereoisomers (4 and 5) and enantio-

mers having opposite absolute conﬁgurations (4_R/4_Sand 5_R/5_S)

based on ECD spectroscopy, X-ray crystallography and stereo-

chemical correlation methods, (iii) develop a chemoenzymatic

synthetic route to cyclohexenone cis-diols 4_S(R′ = H) from

enantiopure substituted benzene cis-dihydrodiol precursors 6 of

established absolute conﬁguration which were readily available

in multigram quantities (iv) rationalize the formation of enantio-

pure cyclohexenone cis-diol metabolites 4_Sand 4_Rof opposite

absolute conﬁgurations, following TDO-catalysed cis-dihydroxy-

lation of the corresponding aromatic substrates 1 and provide a

simple predictive active site model.

8c and picolinic acid 9c as minor metabolites.^2cThe biotrans-

formation of phenol 1c also gave a cis-dihydrodiol product 6 (R

= 3–HO·C₆H₄) from oxidation of the unsubstituted phenyl ring,

thus reducing the isolated yield of cyclohexenone cis-diol 4c_S.

The formation of metabolite 4c_Swas noteworthy, since

it conﬁrmed that a member of the cyclohexenone cis-diol

family could be formed using different types of arene dioxy-

genases, e.g. TDO, naphthalene dioxygenase and biphenyl

dioxygenase.^2c

Following on from the use of 3-iodophenol 1a as substrate for

TDO,^2a–cduring this study several other halogenated phenols

were examined as substrates with P. putida UV4. Surprisingly,

addition of 3-ﬂuorophenol 1 (R = F, R′ = H) as substrate pro-

duced no evidence of cyclohexenone cis-diol 4 (R = F, R′ = H)

being formed; the corresponding catechol 2 (R = F, R′ = H)

was the only identiﬁed metabolite. When 3-chlorophenol 1d and

3-bromophenol 1e were used as substrates, the corresponding

catechols 2d and 2e again proved to be the major bioproducts

with cyclohexenone cis-diols 4d and 4e as minor products in

relatively low isolated yields (≤5%). This prompted us to

develop a chemoenzymatic route to these minor metabolites

from the readily available cis-dihydrodiols 6d and 6e (see

Section 2.3).

2. Results and discussion

It was difﬁcult to obtain accurate estimates of the relative

yields of halogenated cyclohexenone cis-diols 4 and catechols 2

since: (i) further metabolism often occurred to different degrees

among repeat biotransformations and (ii) LC-TOFMS analysis

was unsuitable for the detection of catechols, and NMR spectro-

scopic analysis of the crude mixture of products, in some

cases, proved to be less reliable due to the presence of both

unreacted substrates and aromatic products. Despite these

limitations, a qualitative estimate based on NMR spectroscopic

analysis can be made of the relative proportions of cyclohexe-

none cis-diols 4a, 4d, 4e and the corresponding catechols

2a, 2d, 2e obtained by TDO-catalysed oxidation. Thus, it

was observed that higher yields of cyclohexenone cis-diols

were generally obtained when using phenol substrates with

larger substituents, including halogen atoms i.e. 4a > 4e > 4d >

4 (R = F, R′ = H). As already mentioned in the context of metab-

olite 4a,^2a,cand in view of conversion of substrate generally

being complete, the variable yields of cyclohexenone cis-diols

obtained herein are mainly assumed to result from further metab-

olism or alternative metabolic pathways to give water-soluble

products.

Methoxyphenols are commonly found in ecosystems as

natural and combustion products derived from plants, as well as

from anthropogenic sources. As the initial step of a wider pro-

gramme to investigate the dioxygenase-catalysed biotransform-

ation of methoxyphenols, including those found in the

environment, 3-methoxyphenol 1f was examined as a substrate

for P. putida UV4. It was gratifying to ﬁnd that the isolated

yields of cyclohexenone cis-diol 4f, using substrate 1f, were con-

sistently higher (>50%) than those obtained with halogenated

phenols 1d and 1e and most other 3-substituted phenols.

Although little evidence was found for the formation of catechol

2f, a minor unidentiﬁed bioproduct was also detected and separ-

ated during puriﬁcation of cis-diol 4f. The structures of cyclo-

hexenone cis-diols 4d, 4e and 4f were established by a

combination of spectroscopic and chiroptical methods that had

2.1 TDO-catalysed biotransformation of phenols 1d–1g, 1i and

1j to yield the corresponding cyclohexenone cis-diols 4d–4g, 4i,

4j, 5g and 5i

The phenol substrates 1 and catechol bioproducts 2 for this study

were available either commercially or from earlier studies.^4,53-

Iodophenol 1a, 3-triﬂuoromethyl phenol 1b, 3-phenylphenol 1c

and 2,5-dimethylphenol 1h had been used earlier as substrates

for P. putida UV4 and the corresponding cyclohexenone cis-

diols 4a_S–4c_Sand 4h_Rwere isolated and characterized.^2a–c

These cis-diols have been included in Scheme 1, to allow com-

parison of their calculated and experimentally recorded ECD

spectra (Fig. 2) with those of the new cis-diol bioproducts 4d–

4g, 4i, 4j, 5g and 5i from the corresponding phenol substrates

1d–1g, 1i and 1j.

The earlier study^2ahad shown that biotransformation of 3-

iodophenol 1a with P. putida UV4 yielded cyclohexenone cis-

diol 4a_Sas a major metabolite and only traces of catechol 2a.

The yields of cyclohexenone cis-diol metabolite 4a_S, formed

from phenol 1a, were found to vary greatly among repeated bio-

transformations. This may have been the result of several com-

peting reactions occurring simultaneously, e.g. (a) reduction of

the keto group in compound 4a_Sto form cis,cis-triol 7a, (b) for-

mation of dehalogenation products and their derivatives, (c) cate-

chol 2a formation which could have inhibited TDO activity.^2a,c

The reduction of the keto group was particularly favoured during

the biotransformation of 3-triﬂuoromethylphenol 1b, using P.

putida UV4, where cyclohexenone cis-diol 4b_Swas formed and

then rapidly reduced to give cis,cis-triol 7b as the major

bioproduct.^2c

While the phenol ring of substrate 1c was also cis-dihydroxy-

lated, using P. putida UV4 to yield the corresponding cyclohexe-

none cis-diol 4c_S, only traces of catechol bioproduct 2c were

detected. However, it was found that a ring opening reaction of

catechol 2c had occurred to yield the acyclic ketocarboxylic acid

6218 | Org. Biomol. Chem., 2012, 10, 6217–6229

This journal is © The Royal Society of Chemistry 2012

View Article Online

Scheme 2 TDO-catalysed formation of cis,cis-4g_R–4j_Rand cis,trans-cyclohexenone diols 5g_R–5j_Rfrom the initial cis-dihydrodiol intermediates

10g_R–10j_Rand phenol substrates 1g–1j.

been used earlier for the other members of this family of meta-

bolites.^2a–c

indicated a preference for the half-chair conformation and a cis,

cis relationship between substituents i.e. (pseudo)equatorial C4–

OH and C6–Me and a (pseudo)axial C5–OH. It is probable that

this is also the preferred conformation in a solution of the cis-

diol moiety in metabolites 4a–4j, based on NMR spectroscopy

analysis. The structures of all the cyclohexenone cis-diols shown

in Scheme 1 (4a_S–4f_S, 4g_R–4j_R, 5g_Rand 5i_R) were assigned

using a combination of NMR spectroscopy and mass spec-

trometry. The vicinal coupling constants J_4,5were consistently

located within the range found earlier for the cis-diol conﬁgur-

ation among other members of this family of metabolites

(2.5–3.6 Hz).^2a–cThe ¹H coupling constant values at C6 and C5,

i.e. J_5,6= 2.6, in cis-diol 4g and 2.3 in cis-diol 4i, were consist-

ent with a cis relationship. The larger trans C5–C6 coupling con-

stants found in cis-diols 5g and 5i (J_5,6= 10.3 and 8.4 Hz

respectively) were consistent with C5 and C6 substituents adopt-

ing (pseudo)equatorial conformations and C4 a (pseudo)axial

conformation.

The major cis,cis-diastereoisomers 4g–4j showed little evi-

dence of epimerization in CDCl₃or D₂O solution at ambient

temperature over an extended period (>10 days). This contrasted

with the behaviour of the minor halogenated cis,trans metab-

olites 5g and 5i which in CDCl₃solution, were observed to epi-

merize via an inversion of conﬁguration at the C6 chiral centre,

over a period of several days, to yield the corresponding cis,cis

diastereoisomers 4g and 4i (Scheme 2). This observation

suggested that both the cis,cis isomers 4g and 4i and the corre-

sponding cis,trans diastereoisomers 5g and 5i were formed sim-

ultaneously under kinetic control in the aqueous culture medium.

Equilibration could then occur via undetected enol intermediates

10g and 10i with the less stable cis,trans isomers epimerizing to

give the thermodynamically preferred cis,cis isomers 4g and 4i

in CDCl₃solution.

Due to the remarkable stability of cis-diol 4f, compared to the

methoxy substituted cis-dihydrodiol metabolite 6f derived from

methoxybenzene,⁵and the reproducible yields recorded during

shake ﬂask biotransformations, large-scale (>100 litre) biotrans-

formation studies are in progress to optimize the yield and obtain

multigram quantities for examination of its potential as a multi-

functional chiral precursor in chemoenzymatic synthesis.

The earlier communication on this study^2ashowed that TDO-

catalysed cis-dihydroxylation of 2,5-dimethylphenol 1h (a

metabolite of p-xylene) by P. putida UV4 yielded metabolite

4h_R, as the ﬁrst disubstituted member of the cyclohexenone cis-

diol family. With most of the other previously reported cyclohex-

enone cis-diol metabolites having been derived from monosub-

stituted phenols,^2a–ccyclohexenone cis-diol 4h_Rwas the ﬁrst

member of this family to possess three endocyclic chiral centres.

X-ray crystallography and NMR spectroscopic analysis of a

monocamphanate derivative showed that the two hydroxyl

groups (at C4 and C5) and the methyl group (at C6) had a cis,cis

relationship in metabolite 4h_R.^2a

Three more 2,5-disubstitututed phenols 1g, 1i and 1j have

now been found to be acceptable substrates for P. putida UV4.

The corresponding cyclohexenone cis-diols 4g_R, 4i_R, 4j_Rwere

identiﬁed as major metabolites, initially on the basis of NMR

spectroscopy (Scheme 2). The cis,cis metabolites 4h_Rand 4j_R,

appeared to be the only identiﬁed cyclohexenone cis-diols

present in the crude mixtures. However, a careful analysis of the

NMR spectra of the crude mixture, recorded immediately after

biotransformation of phenols 1h and 1j, also showed traces of

the minor diastereoisomers 5h_Rand 5j_R, which disappeared

during isolation of the major metabolites 4h_Rand 4j_R, presum-

ably via epimerization. This premise was supported when the

two cis,trans diastereoisomers 5g_Rand 5i_Rwere found as signiﬁ-

cant minor metabolites (30% and 10% relative yields respect-

ively) in the crude culture media and were readily separable from

the major cyclohexenone cis-diols 4g_Rand 4i_Rby PLC.

The cyclohexenone cis-diols chlorosphaeropsidone 5k and

epi-chlorosphaeropsidone 4k have been isolated as natural pro-

ducts from cultures of Sphaeropsis sapinea f. sp. Cupressi, a

fungus responsible for canker among cypress trees (Scheme 3).^6a

Compounds 4k and 5k, although possibly derived from epoxide

precursors,^6a,bwere found to have very similar structures to

The relative conﬁguration of cyclohexenone cis-diol 4h_Rhad

been established earlier by X-ray crystal structure analysis.^2aIt

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 6217–6229 | 6219

View Article Online

Scheme 3 Base-catalysed epimerization of chlorosphaeropsidone 5k

to epi-chlorosphaeropsidone 4k.

cyclohexenone cis-diol metabolites 4g_R–4j_R, 5g_Rand 5i_R

derived from phenol precursors (Scheme 3). The cis,trans meta-

bolite 5k was found to epimerize to cis,cis isomer 4k via the

undetected enol tautomer 10k.⁶The relative conﬁguration of

compound 5k in the solid state was provided by X-ray crystallo-

graphy with (pseudo)equatorial C5–OH and C6–Cl substituents

and a (pseudo)axial C4–OH substituent. Based on NMR coup-

ling constants, this also appeared to be the preferred confor-

mation in CDCl₃solution.^6a

Fig. 1 Cyclohexenone cis-diol relative (4g_R) and absolute (4g_R-cam

and 4i_R) conﬁguration assignments by X-ray crystallography.

,

The preference for cis,cis isomers 4g–4k at equilibrium may

result from reduced repulsive interactions of the 4,6-(pseudo)

diaxial C4–H and C6–H atoms compared with the stronger 4,6-

(pseudo)diaxial C4–OH and C6–H interactions associated with

the preferred conformation of cis,trans isomers 5g–5k.

interactions in crystal growth are likely to dominate. Thus, all

hydroxyl, and some keto, groups are involved in extensive inter-

molecular hydrogen bonds.

Earlier attempts to detect the initial cis-dihydrodiol (enol)

metabolite 10a from TDO-catalysed cis-dihydroxylation of

phenol 1a, either by formation of a diene cycloadduct (using 4-

phenyl-1,2,4-triazoline-3,5-dione as dienophile), or by trapping

as an enol ether using diazomethane as methylating agent, were

unsuccessful.^2aThe preferential formation of cis,cis diastereo-

isomers 4g–4k and epimerization of the corresponding cis,trans

isomers 5g–5k provide strong evidence that the undetected enols

10g–10k were indeed the initial phenol metabolites and were

responsible for cis/trans isomerization.

In all cases the new cyclohexenone cis-diol metabolites 4d–

4g, 4i, 4j, 5g and 5j were found to be enantiopure (>98% ee) by

¹H NMR analysis of the corresponding chiral boronate deriva-

tives which formed readily when mixed with (R)- and (S)-2−(1-

methoxyethyl)benzeneboronic acid. This method has been used

successfully for other cyclohexenone cis-diols 4^2a–cand also for

a wide range of benzene cis-dihydrodiols 6.^7a–c

(ii) Electronic circular dichroism spectroscopy (ECD) and

speciﬁc optical rotation (OR) measurements. The absolute

conﬁgurations of the new metabolites 4a–4g, 4i, 5g and 5i were

independently assigned using ECD spectroscopy. While the

ECD spectra and absolute conﬁgurations of cyclohexenone cis-

diols 4h_Rand 4j_Rhave been reported,^2bfull characterization data

for compound 4j_Rwas not available earlier, and has now been

included in the experimental section. It has been demonstrated

that electronic circular dichroism spectra and optical rotation

values, obtained by comparison of experimental measurements

and by calculations provide a reliable and generally applicable

method for the determination of absolute conﬁgurations of syn-

thetic and natural chiral compounds including cis-dihydro-

diols.^7a–c

Compounds having an enone chromophore occupy a special

position, due to their occurrence in many important polycyclic

molecules, e.g. steroidal hormones. For correlation of the

observed chiroptical phenomena with the structure, two funda-

mental contributions have to be taken into account: ﬁrst – the

intrinsic helicity of the enone chromophore, and second – the

extrachromophoric perturbation (substitution pattern). As both

the helicity of the chromophore and the substituents contribute

to the Cotton effect, it is important to determine the dominant

contribution. During the last decades various chirality rules have

been proposed to correlate the chiroptical properties of α,β-un-

saturated ketones with their stereochemistry.^8a–vHowever, until

now there has been no general empirical rule which unambigu-

ously accounts for all chiroptical phenomena observed for

enones, thus theoretical support seems to be mandatory.

2.2 Absolute conﬁguration determination of cyclohexenone

cis-diols 4a_S–4f_S, 4g_R–4j_R, 5g_Rand 5i_Rby X-ray crystallography

and chiroptical methods

(i) X-ray crystallography. Only the relative conﬁguration of

cyclohexenone cis-diol 4g_Rwas assigned by X-ray crystallogra-

phy (Fig. 1). The absolute conﬁguration was obtained from

monocamphanate derivative 4g_R-camfollowing reaction with

(−)-(S)-camphanic chloride. The cyclohexenone cis-diols 4g_R

and 4i_R, were assigned 4R,5R,6R and 4R,5R,6S conﬁgurations

respectively by X-ray crystallography (Fig. 1). While compounds

4g_Rand 4i_Rwere shown to have opposite absolute conﬁgurations

at both C4 and C5, application of the Sequence Rule resulted in

an R conﬁguration at C5 in each case due to a priority change.

Compounds 4g_R, 4i_Rand 4g_R-camdo not show any intramolecu-

lar hydrogen bonding in the solid state, where intermolecular

We have successfully applied this experimental/theoretical

approach to several alkyl-substituted cyclohexenone-cis-diol

metabolites 4_R(R = Me, Et, i-Pr, t-Bu, R′ = H), 4h_Rand 4j_R.^2b

The latter study focused mostly on the factors that control the

structure and chiroptical properties of substituted 2-

6220 | Org. Biomol. Chem., 2012, 10, 6217–6229

This journal is © The Royal Society of Chemistry 2012

View Article Online

the M and P diastereoisomeric structures. Intramolecular hydro-

gen bond patterns of the hydroxy groups further increase the

number of distinct stereoisomeric structures (Scheme 4). Thus,

either C4–OH or C5–OH can be a hydrogen bond donor and the

orientation of the O–H bond against the vicinal C–H bond can

be either syn or anti (as shown by the corresponding torsion

angles α and β). Up to eight conformers (M1–M4, P1–P4) are

available but this number can still be larger if one includes the

rotamers due to non-spherical substituents in the ring (torsion

angle ϕ), e.g. phenyl (4c) or methoxy (4f). Calculated torsion

angles α and β fall into the range (155°–180°) for anti rotamers

and the range is wider for the syn rotamers (15°–87°). For the

majority of cases the intramolecular hydrogen bond O–H⋯O is

weak (calculated length 2.50 0.15 Å). However, in several con-

formers of enones this bond is substantially shorter

(2.12–2.34 Å). In these conformers the equatorial hydroxy group

is a donor to the axial oxygen atom. Another exception from the

general scheme was found in the cases of 4a, ent-4i, ent-5i and

P3–P4 conformers of 4c, where again the hydrogen bond is

shorter (the shortest value 2.12 Å was calculated for ent-5i) and

thus it is stronger.

Scheme 4 Diastereoisomeric P and M conformers of cis-ketodiols 4a–

4g, ent-4j, 5g and ent-5i and the deﬁnition of torsion angles α, β, ϕ and

ω, τ (see ESI‡ for details).

The distribution of conformers for each ketodiol has been cal-

culated for a polar medium i.e. acetonitrile or methanol, since all

the measurements of chiroptical properties were done in polar

solvents.

cyclohexenones and resulted in redeﬁnition of the role of substi-

tuents and 2-cyclohexenone conformation in electronic circular

dichroism spectra. It has been found that non-planarity of the

CvC double bond and the presence of an equatorial hydroxy

group in the allylic position affected the ECD spectra most sig-

niﬁcantly. Another result of our study was the determination of

the nature of the ﬁrst three bands in the ECD spectra of 2-cyclo-

hexenones. In the case of hydroxy-substituted 2-cyclohexenones,

the long-wavelength electronic transition originates from the

In contrast with the C3-alkyl-substituted 2-cyclohexenone-cis-

diols having 4R,5S conﬁguration shown in Scheme 4 (R′ and

R′′ = H), where calculations indicate a strong preference of ring

P helicity, 3-heteroatom-substituted analogs do not follow any

general trend. It is apparent that the presence of a heteroatom at

the C3 position affects the conformational equilibrium. Confor-

mers of P-type were found as the energetic minima in the case

of 4a, 4c–4g, ent-4i and ent-5i whereas for the other cyclohexe-

none cis-diols, conformers of M-type dominate. The presence of

a polar group next to the allylic hydroxy group suggests that the

interaction between both groups affects the conformation. Attrac-

tive dipole–dipole interactions between the (pseudo)axial

hydroxy group and a polar substituent were found in the cases of

4f(M2), ent-5i(M2) and 4g(P2), whereas in the case of 4b(M2)

the structure of this conformer appears to be stabilized by the

formation of a diad of the hydrogen bonds O_eqH⋯O_axH⋯FCF₂,

as in the case of other arene metabolites.^7aThe formation of

hydrogen bond diads O_axH⋯O_eqH⋯F shifts the conformational

equilibrium to M3 in the case of 5g

ECD spectra of substituted 2-cyclohexenones 4a–4j, 5g and

5i, of assumed 5S (or 5R in the case of 4g and 5g) absolute

conﬁguration, were calculated at the PCM/TDDFT/B2LYP/Aug-

cc-pVTZ level of theory for each conformer of the relative

energy within a 0–2 kcal mol⁻¹window (see ESI‡). After Boltz-

mann averaging and Gaussian curve ﬁtting^9a,bthe calculated

ECD spectra were compared with the experimental ones, as

shown in Fig. 2. The calculated ECD curves were wavelength-

corrected to match the experimental and calculated UV maxima

of the strong π–π* absorption at ca. 220 nm (scaling factors

ranged from 1.11 for 4a to 1.15 for 4c). This procedure still left

several of the calculated ECD curves 10 nm blue-shifted, again

in agreement with the previous ﬁndings. In general, the calcu-

lated ECD spectra of the majority of cyclohexenone cis-diols

were quite similar to the experimental ones, allowing an

n

_CvO–π_CvO* and the second from the π_CvC–π_CvO* electronic

transition whereas the third transition is due to the presence of

the substituent and is of the n_OH–π_CvO* type.^2bIt seemed

reasonable that this approach could also be applied to the assign-

ment of absolute conﬁgurations of the new members of the

cyclohexenone cis-diol family 4a_S–4f_S, 4g_R–4j_R, 5g_Rand 5i_R

with either heteroatom or π-conjugated substituents in C3 or C6

positions.

In order to determine the chiroptical properties of enones 4a–

4g, ent-4i, 5g and ent-5i with a sufﬁcient level of conﬁdence,

their structures and conformational equilibria were calculated at

PCM/B2PLYP/Aug-cc-pVTZ//PCM/B3LYP/6311++G(2d,2p)

level (see ESI,‡ for details). Calculations included both the ring

conformers and the substituent rotamers. As shown in Scheme 4

for the assumed 5S (or 5R in the case of compounds 4g and 5g)

absolute conﬁguration, each ring conformation could be

described as either M (torsion angles C3–C4–C5–C6 negative,

C4–C5–C6–C1 positive, (pseudo)axial C4–OH, (pseudo)equa-

torial C5–OH or P (torsion angle C3–C4–C5–C6 positive, C4–

C5–C6–C1 negative, (pseudo)equatorial C4–OH, (pseudo)axial

C5–OH), according to the previous proposal.^2bCalculated struc-

tures of individual low-energy conformers of these enones (see

ESI‡) are described as sofa S(5) of either M or P helicity

(Scheme 4), with various degrees of distortion from the ideal

form, in the direction of a distorted half-chair (HC) conformer.

As in the case of non-phenolic arene-cis-dihydrodiol metab-

olites 6,^7athe number of available conformers is not limited to

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 6217–6229 | 6221

View Article Online

Fig. 2 ECD spectra of cis-ketodiols 4a–4j, 5g and 5i, experimental (in acetonitrile solutions, solid lines) and ΔE_B2PLYP(D)Boltzmann averaged calcu-

lated at PCM/B2LYP/Aug-cc-pVTZ level (dash-dot-dot lines). Rotatory strengths R were calculated in dipole-velocity representation. All calculated

spectra were wavelength-corrected to match the experimental short-wavelength UV λ_max. Note that ECD spectra for 4h, 4i, 4j and 5i were calculated

for the enantiomers. Experimental and calculated spectra for 4h and 4j were taken from ref. 2b.

unambiguous assignment of the absolute conﬁguration of the

molecules studied. However, neither experimental nor theoretical

ECD spectra for individual keto-cis-diols were similar between

various compounds. This shows the effect of the polar substitu-

ents at C3 on the electronic properties of the enone chromo-

phore. For example, the position of λ_maxfor 3-substituted enones

in relation to hydrogen substituent changes in the sequence: Ph

(+67) > I (+47) > OMe (+31) > Br (+18) ≥ Cl (+18) > Me (+14)

> H (210 nm) > CF₃(−12).

The electron withdrawing properties of the triﬂuoromethyl

group are responsible for the unique 12 nm blue-shift of the UV

maximum of keto cis-diol 4b. The effect of the alkyl substituent

(i.e. the methyl group) on the energies of the π–π* electronic

Scheme 5 Synthesis of cyclohexenone cis-diol metabolites 4c_S–4e_S

transition is comparable to the effect of chlorine substituent. In

the case of cyclohexenone cis-diols 4a, 4d and 4e the substituent

effect on the energy of the π–π* electronic transition correlates

well with the halogen polarizability and is most signiﬁcant for

the iodine substituent. Substitution of the hydrogen atom at C3

in the 2-cyclohexenone skeleton by a methoxy group forms a

classical push–pull system and this is reﬂected in the red-shift of

the position and enhanced magnitude of the π–π* absorption

maximum of ketodiol 4g.

obtained from the corresponding cis-dihydrodiols 6c_S–6e_S.

does not satisfactorily ﬁt the experimental one. The absolute

conﬁguration of compound 4c_Swas however, independently

established by the stereochemical correlation sequence shown in

Scheme 5. As is usually the case, the discrepancy between the

experiment and the computation of chiroptical properties resulted

from either the incorrect computational reproduction of confor-

mer population or from the method of ECD calculation, usually

performed for “static” conformers. In the real molecule the

Cotton effects originating from different rotamers of the phenyl

The conjugative effect of the phenyl substituent is clearly

visible in the electronic spectrum of keto-cis-diol 4c_S. This is

also the most complex case, since the calculated ECD spectrum

6222 | Org. Biomol. Chem., 2012, 10, 6217–6229

This journal is © The Royal Society of Chemistry 2012

View Article Online

Table 1 Speciﬁc optical rotations for cyclohexenone cis-diols 4a–4j, 5g and 5i measured in methanol solution and calculated at the PCM/B3LYP/

Aug-cc-pVTZ level (values in parentheses)

Diol

589 nm

578 nm

546 nm

436 nm

4a

4b

4c

4d

4e

4f

4g

4h

4i

−38^a

−82

(−224)

(−99)

−37

−86

−21

−54

−47

−127

−109

+234

+88

(−238)

(−104)

(−79)

−42

−98

−21

−63

−55

−145

−129

+273

+110

+128

n.a.

(−290)

(−119)

(−93)

−61

(−922)

(−181)

(−203)

(−237)

(−104)

(−264)

(−370)

(−814)^cd

(−331)^c

(−248)^cd

(−81)

−147

−20^b

−52

(−74)

+32

− 109

−96

−219

−286

+540

+219

+259

n.a.

(−110)

(−60)

(−116)

(−62)

(−133)

(−70)

−45

−120

−103

+223

+59

(−129)

(−128)

(−264)^cd

(−105)^c

(−108)^c

(−92)

(−133)

(−136)

(−340)^cd

(−111)^c

(−151)^cd

(−95)

(−154)

(−163)

(−399)^cd

(−135)^c

(−171)^cd

(−102)

(−181)^c

4j

+104^d

−92

+109

n.a.

5g

5i^c

+92

(−151)^c

(−158)^c

n.a.

(−301)^c

^aRef. 2a. ^bRef. 2c. ^cCalculated for the enantiomer. ^dRef. 2b.

ring are mutually cancelled, since the barrier to aromatic ring

rotation around the C3–C_Arbond is low. In the calculated ECD

spectra the effect of the phenyl group is clearly visible because

of the ﬁxed conformation of the aromatic ring.

OMe; R′ = H) conﬁrmed that all are of identical (5S)-absolute

conﬁguration. Conversely, the three cyclohexenone cis-diols (4

and 5) formed from 2,5-disubstituted phenols (1 R = Me, R′ =

Cl; R = Me, R′ = Et; R = R′ = Me) were found to have opposite

cis-diol absolute conﬁgurations at C4 and C5. However, cyclo-

hexenone cis-diols 4g and 5g derived from the 2,5-disubstituted

phenol 1g, had the same conﬁguration as those obtained from 3-

substituted phenols 1. This result could be explained in terms of

similar relatively small size of the C6-F atom in cis-diols 4g and

5g and a C6-H atom in cis-diols 4a–4f.

A further difﬁcult case is represented by keto-cis-diol 4i. The

ﬁrst and the third Cotton effects calculated for ent-4i are of

opposite sign to these measured. The second Cotton effect is

absent in the calculated spectrum due to mutual cancellation of

π–π* electronic transition rotatory strengths of M and P confor-

mers after Boltzmann averaging (see ESI‡). This example

clearly shows that the populations of conformers P1 and P2

were underestimated. It is worth noting that the OR method for

stereochemical assignment worked well in this case where

measured OR values for compound 4i and calculated OR values

for compound ent-4i were found to be of opposite sign

(Table 1).

The effect of conformation of the substituent at C3 is distinc-

tively seen in the calculated optical rotations. An excellent

example is the phenyl-substituted cyclohexenone cis-diol 4c (see

ESI, Table C2‡). Whereas OR values for the majority of confor-

mers of the alkyl-substituted keto-cis-diols are negative (regard-

less P or M conformation),^2bin the case of metabolite 4c the

rotation changes sign depending on the conformation of the

phenyl group and assumes a negative sign for a positive value of

angle ϕ and vice versa.

The results of measurement and calculation of optical rotation

values for cyclohexenone-cis-diols are in agreement with

conﬁguration assignments obtained by ECD. Thus, the calcu-

lated and measured signs and magnitudes of speciﬁc optical

rotations at four different wavelengths for 4a–4i are in agreement

with the assumed 5S conﬁguration (Table 1, and also Table C2

in ESI‡).

This computational study shows that ECD spectra of cyclo-

hexenone cis-diols 4a–4j, 5g, and 5i reﬂect primarily the absol-

ute conﬁguration of these molecules at C4 and C5 and to a much

lesser extent the preferred conformation of the molecules, in

agreement with our previous results. Thus, a comparison of the

results obtained from experimental and calculated ECD spectra

of alkyl substituted cyclohexenone cis-diols 4 derived from the

corresponding 3-substituted phenols (1 R = Me, Et, i-Pr, t-Bu; R′

= H) ^2bwith those of the current study (1 R = Cl, Br, I, CF₃, Ph,

2.3 Chemoenzymatic synthesis of the cyclohexenone cis-diols

4c_S, 4d_Sand 4e_Sfrom cis-dihydrodiols 6

The cyclohexenone cis-diols 4 have generally proved to be more

stable than the corresponding benzene cis-dihydrodiols 6 and

most can be stored at ambient temperatures for extended periods

without signiﬁcant decomposition. Although a range of cyclo-

hexenone cis-diols 4 and 5 is now available from TDO-catalysed

biotransformation of substituted phenols, the isolated yields of

cyclohexenone cis-diols vary due to competing biosynthetic

pathways.^2a–cThus the yield of cis-diol metabolite 4c_S(28%)^2c

was reduced by formation of the alternative cis-dihydrodiol

product 6 (R = 3–HO·C₆H₄, 10%).^2cSimilarly cyclohexenone

cis-diols 4d_Sand 4e_Sfound during the current study, proved to

be minor metabolites (≤5% isolated yield) compared with the

corresponding dominant catechol bioproducts. In order to gener-

ate sufﬁcient quantities of metabolites 4c_S, 4d_Sand 4e_Sfor

stereochemical correlation studies, a chemoenzymatic route,

based on the more readily available substituted benzene cis-dihy-

drodiols 6, was developed (Scheme 5).

The cis-tetrahydrodiols 11c–11e were obtained in good yield

(>85%) by partial hydrogenation from the corresponding cis-

dihydrodiols 6c_S–6e_Susing Pd/C, Rh/Al₂O₃or Rh/graphite cata-

lysts and were available from earlier studies.^4,10a–cWhile prefer-

ential protection of the pseudoaxial C1–OH group was found

using tert-butyldimethylsilyl chloride, silylation of both OH

groups in compounds 11c–11e was achieved in excellent yields

(>90%) by treatment with tert-butyldimethylsilyl triﬂate in the

presence of triethylamine base to yield the diTBDMS derivatives

12c–12e.

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 6217–6229 | 6223

View Article Online

Using the method of Zhao and Yeung,¹¹oxidation at the

allylic (C5) position of the protected cis-tetrahydrodiols 12c–12e

gave the corresponding protected ketones 13c–13e. This allylic

oxidation step was achieved using a combination of diacetoxy-

iodobenzene and tert-butyl hydroperoxide [PhI(OAc)₂,

t-BuO₂H], to generate tert-butylperoxy radical as in situ oxidant

but the oxidation proved difﬁcult to drive to completion.

Although the protected cyclohexenone cis-diols 13c–13e were

only isolated in relatively low yields (37–42%), based on recov-

ered starting material 12c–12e after chromatography, the yields

were estimated to be ca. 60%.

Scheme 6 TDO-catalysed oxidation of meta-phenols (S = H) or 2-

ﬂuoro-5-methyl phenol (S = F) to yield cyclohexenone cis-diols.

The ﬁnal step involved deprotection of silyl ethers 13c–13e to

give cyclohexenone cis-diols 4c_S–4e_Swhich proved to be identi-

cal to the metabolites derived from the corresponding phenols

1c–1e. Although the cyclohexenone cis-diols 4c_S–4e_Swere

much more stable than the corresponding dihydrodiols 6c–6e at

ambient temperature, under the deprotection conditions used for

diTBDMS derivatives (tetrabutylammonium ﬂuoride/THF), they

proved to be less stable and this resulted in only modest isolated

yields (29–52%). To improve the yield in the ﬁnal step alterna-

tive cis-diol protecting groups are currently under investigation.

The stereochemical correlation of cis-dihydrodiols 6c_S–6e_S

with the corresponding cyclohexenone cis-diols 4c_S–4e_S, and

X-ray crystallographic analyses of cyclohexenone cis-diols

4a_S,^2a4h_R,^2a4g_Rand 4i_R, provide unequivocal methods for the

assignment of their stereochemistry. As the same absolute

conﬁgurations were also assigned by the chiroptical methods, the

validity of the ECD and OR approach for the determination of

conﬁguration of these, and other members of the cyclohexenone

cis-diol family of metabolites e.g. 4f_S, has been further conﬁrmed.

When combined with the earlier reports of the dioxygenase-

catalysed formation of cyclohexenone cis-diol bacterial metab-

olites 4 from the corresponding monophenols,^2a–cand a biphenol

(3,3′-biphenol),^12a,baddition of three further family members (R

= H, CN, CO₂Me) obtained by chemoenzymatic synthesis,^2aand

the isolation of the fungal metabolites chlorosphaeropsidone and

epi-chlorosphaeropsidone,⁶more than twenty ﬁve metabolites of

this family have now been identiﬁed. From this study, it appears

that this is a relatively common dioxygenase-catalysed metabolic

pathway for 3-substituted and 2,5-disubstituted phenols.

Although cyclohexenone cis-diol metabolites have also been iso-

lated from the biotransformation of 2-substituted phenols (ref. 2a

and unpublished data), to date none have yet been isolated from

4-substituted phenols.

Scheme 7 TDO-catalysed oxidation of 2,5-disubstituted phenols to

yield cyclohexenone cis-diols.

Simple predictive models have been developed for the TDO-

catalysed cis-dihydroxylation of a large number of mono- and

di-substituted benzenes, polycyclic arenes and azaarenes,^3f,kand

meta-phenols,^2cto yield enantiopure cis-dihydrodiols of identical

absolute conﬁguration. It is, however, more difﬁcult to rational-

ize the enantiomeric preference shown by the TDO enzyme

when producing cyclohexenone cis-diol metabolites of opposite

absolute conﬁgurations (e.g. 4a_S–4g_S, 5g_Rand 4h_R–4j_R, 5i_R).

Our initial attempts to account for the enantiomeric preferences

are shown in Schemes 6 and 7. These models are based on the

assumption that dominant factors include: (i) the differential in

the relative size of spherically symmetrical substituents or con-

formational preference of planar substituents i.e. large (L),

medium (M) and small (S), (ii) ﬂexibility in the relative position

of the phenolic OH group which may interact with the proximate

positively charged imidazole ring of histidine 311 in the active

site of TDO^2c,13a–dand (iii) the approach of the oxidant^13c,14

resulting in cis-dihydroxylation occurring on the lower face of

the benzene ring. In the absence of any unequivocal evidence

for the precise nature of the TDO oxidant, the (hydroxo)oxo

iron (^V) intermediate [HO–Fe^VvO] species appears to be one of

the more favoured structures^13c,14and has been used in Schemes

6 and 7.

The preferred absolute conﬁgurations of the cyclohexenone

cis-diols shown in Scheme 6, are identical to those found for cis-

dihydrodiol metabolites derived from both monocyclic and poly-

cyclic arenes using TDO where a marked difference in large (L)

and small (S) substituent size is also the dominant feature.^3f,k

Similarly, in 2,5-disubstituted phenol 1g, where the smaller sub-

stituent was a ﬂuorine atom, the cyclohexenone cis-diol product

4g had the same absolute conﬁguration as those derived from 3-

substituted phenols. To date three examples of cyclohexenone

cis-diols 4h–4j (Scheme 7), having the opposite absolute

conﬁgurations, have been isolated and in each case the differen-

tial between medium sized substituents e.g. Me, Et and Cl, was

either small (M and M′ in 4i and 4j) or absent (MvM′, 4h). In

2.4 Predictive models for TDO-catalysed cis-dihydroxylation of

phenols

The phenol metabolites 4 and 5 discussed herein can be divided

into two groups each with opposite absolute cis-diol conﬁgur-

ations i.e. 4a_S–4g_S, 5g_Rand 4h_R–4j_R, 5i_R(Scheme 1). In con-

trast

the

cis-dihydrodiol

metabolites

derived

from

monosubstituted (6) and ortho or meta disubstituted benzenes

have consistently been found to possess the same absolute

conﬁguration. They were also found to be enantiopure (>98%

ee) with the only exception being those derived from ﬂuoroben-

zene or diﬂuorobenzene substrates with slightly lower ee

values.^3a–n

6224 | Org. Biomol. Chem., 2012, 10, 6217–6229

This journal is © The Royal Society of Chemistry 2012

View Article Online

(4S,5S)-3-Chloro-4,5-dihydroxycyclohex-2-enone

4d_S. Pale

yellow semi solid (0.038 g, 4%); R_f(0.29, 75% EtOAc in

hexane); [α]_D− 52 (c 1.58, MeOH); HRMS (EI): Found M⁺,

this case other factors including substrate orientation in the TDO

active site relative to histidine 311 and the (hydroxo)oxo iron (^V)

oxidant may also be important.

162.0071 requires C₆H₇O₃³⁵Cl 162.0078; H-NMR (400 MHz,

1

CD₃OD) δ_H2.61 (1 H, dd, J 16.5, 3.9, 6-H), 2.69 (1 H, dd, J

16.5, 7.2 Hz, 6′-H), 4.24 (1 H, ddd, J 7.2, 3.9, 3.6, 5-H), 4.44 (1

H, dd, J 3.6, 0.9, 4-H), 6.23 (1H, d, J 0.9, 2-H); ¹³C-NMR

(100 MHz, CD₃OD) δ_C43.2, 69.6, 72.3, 129.4, 159.8, 197.2;

LRMS (EI): 162 (M⁺, 5%), 144 (18), 131 (53), 120 (32), 118

(100); IR (KBr) ν_max/cm⁻¹3400 (OH), 1676 (CvO).

3. Conclusion

A series of 3-substituted (1d–1f) and 2,5-disubstituted phenols

(1g, 1i and 1j) have been cis-dihydroxylated to yield the corre-

sponding enantiopure cyclohexenone cis-diols i.e. 4d_S–4f_S, 4g_R,

4i_R, 4j_R, 5g_Rand 5i_Rwith TDO as biocatalyst in P. putida UV4.

All cyclohexenone cis-diols were found to be enantiopure with

some being isolated as diastereoisomers e.g. 4 and 5 and some

as enantiomers e.g. cis-diols 4_Sand 4_Rwith opposite absolute

conﬁgurations. The structures and absolute conﬁgurations of

these chiral metabolites were determined using a combination of

NMR and ECD spectroscopy, OR measurements, X-ray crystal-

lography and stereochemical correlation methods. A chemoenzy-

matic synthesis route to cyclohexenone cis-diols 4, from the

more readily available non-hydroxylic monosubstituted benzene

cis-dihydrodiols 6, has been developed. An empirical model has

been proposed, to allow prediction of the absolute conﬁguration

of cyclohexenone cis-diols 4 and 5 expected from the TDO-

catalysed cis-dihydroxylation of the corresponding phenol

substrates 1.

(ii) Biotransformation of 3-bromo-phenol 1e

The major metabolite isolated after PLC separation of the crude

mixture of bioproducts was catechol 2e and cyclohexenone cis-

diol 4e_Sas a minor metabolite.

(4S,5S)-3-Bromo-4,5-dihydroxycyclohex-2-enone 4e_S. White

crystals (0.054 g, 6%); m.p. 82–83 °C (CHCl₃–hexane); R_f0.31

(75% EtOAc in hexane); [α]_D− 45 (c 1.08, MeOH); HRMS

(EI): Found M⁺, 205.9575 requires C₆H₇O₃⁷⁹Br 205.9579;

¹H-NMR (400 MHz, CD₃OD) δ_H2.62 (1 H, dd, J 16.4, 4.1, 6-

H), 2.68 (1 H, dd, J 16.4, 7.1, 6′-H), 4.24 (1 H, ddd, J 7.1, 4.1,

3.5, 5-H), 4.50 (1 H, dd, J 3.5, 1.0, 4-H), 6.48 (1 H, d, J 1.0, 2-

H); ¹³C-NMR (100 MHz, CD₃OD) δ_C43.2, 69.6, 73.5, 133.6,

152.9, 196.7; LRMS (EI): 208 (M⁺, 3%), 206 (3), 190 (15), 188

(16), 164 (97), 162 (100), 136 (62), 134 (65); IR (KBr) ν_max

/

cm⁻¹3399 (OH), 1654 (CvO).

4. 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. Accurate mol-

ecular weights were determined by the peak matching method,

with perﬂuorokerosene as the standard. ECD spectra were

recorded in spectroscopic grade acetonitrile using a JASCO

J-720 instrument. A PerkinElmer 341 polarimeter was used for

optical rotation ([α]_D) measurements (ca. 20 °C). The methods

used and results obtained from the calculations of ECD spectra

and OR values are presented in the ESI.‡

Flash column chromatography and preparative layer chromato-

graphy (PLC) were performed on Merck Kieselgel type 60

(250–400 mesh) and PF_254/366plates respectively. Merck Kiesel-

gel type 60F₂₅₄analytical plates were employed for TLC. All

phenol substrates and catechol bioproducts were available from

commercial sources or from past studies.^4,5The general pro-

cedure reported earlier for the biotransformation and isolation of

metabolites of phenols 1a–1c, 1h and other phenol substrates,

using whole cells of P. putida UV4,^2cwas utilized for small

scale (0.2–1.0 g) biotransformation of phenols 1d–1g, 1i and 1j.

Catechol bioproducts were all known compounds and were

identiﬁed by spectroscopic comparison with authentic samples.

(iii) Biotransformation of 3-methoxyphenol 1f

The major metabolite 4f_Scrystallised out from a mixture of

several uncharacterised minor compounds using ethyl acetate.

(4S,5S)-4,5-Dihydroxy-3-methoxycyclohex-2-enone 4f_S. Col-

ourless crystals (0.7 g, 55%); m.p. 128–129 °C dec. (EtOAc); R_f

0.11 (EtOAc); [α]_D− 120 (c 0.5, MeOH); HRMS (ES): Found

(M + H)⁺, 159.0650 requires C₆H₁₁O₄159.0657; ¹H-NMR

(400 MHz, CD₃OD) δ_H2.50 (1 H, dd, J 16.5, 4.1, 6-H), 2.66 (1

H, dd, J 16.5, 8.6, 6′-H), 3.78 (3 H, s, OMe), 4.12 (1 H, ddd, J

8.6, 4.1, 3.5, 5-H), 4.33 (1 H, d, J 3.5, 4-H), 5.39 (1 H, s, 2-H);

¹³C- NMR (100 MHz, CD₃OD) δ_C42.2, 57.1, 69.0, 70.3, 102.8,

178.5, 200.1; LRMS (ES): 159 ([M + H]⁺, 77%), 181 (45), 317

(18), 339 (100), 497 (15); IR (ﬁlm) ν_max/cm⁻¹3362 (OH), 1605

(CvO).

(iv) Biotransformation of 2-ﬂuoro-5-methylphenol 1g

Biotransformation of phenol 1g gave, after PLC separation, two

major cyclohexenone cis-diol metabolites 4g_Rand 5g_Rin the

ratio 2.2 : 1.

(4R,5R,6R)-6-Fluoro-4,5-dihydroxy-3-methylcyclohex-2-enone

4g_R. Colourless crystalline solid (0.091 g, 14%); m.p.

139–140 °C (CHCl₃–MeOH); R_f(0.16, 40% EtOAc in hexane, 4

elutions); [α]_D− 103.0 (c 0.9, MeOH); HRMS (EI): Found M⁺,

160.0536 requires C₇H₉O₃F 160.0530; ¹H-NMR (400 MHz,

CDCl₃) δ_H2.09 (3 H, dd, J 2.5, 1.2, Me), 2.65 (1 H, d, J 2.7,

OH), 2.83 (1 H, d, J 10.9, OH), 4.46 (1 H, d, J 10.9, 4-H), 4.70

(i) Biotransformation of 3-chlorophenol 1d

The crude mixture of metabolites separated by PLC contained

mainly catechol 2d and cyclohexenone cis-diol 4d_Sas the minor

metabolite.

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 6217–6229 | 6225

View Article Online

(1 H, dddd, J 9.7, 3.0, 3.0, 3.0, 5-H), 4.97 (1 H, dd, J 46.9, 2.6,

6-H), 5.96–5.99 (1 H, m, 2-H); ¹³C-NMR (100 MHz, CDCl₃) δ_C

20.2, 70.1 (d, J 8.6) 72.96 (d, J 17.4), 91.30 (d, J 192.4), 124.4

(d, J 1.4), 160.5 (d, J 1.5), 191.2 (d, J 15.8); ¹⁹F-NMR

(376 MHz, CDCl₃) δ_F202.2 (1 F, dddd, J 46.9, 10.5, 6.1, 1.5);

LRMS (EI): 160 (M⁺, 3%), 142 (45), 125 (15), 98 (100); IR

(KBr) ν_max/cm⁻¹3255 (OH), 1682 (CvO).

< 2θ < 50.0°, measured/independent reﬂections: 2103/1723, R_int

= 0.019, direct methods solution, full-matrix least squares reﬁne-

ment on F_o, anisotropic displacement parameters for non-hydro-

2

gen 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= 0.028 for 1619 data with F_o> 4σ(F_o), 223 par-

ameters, ωR₂= 0.081 (all data), GoF = 1.03, Δρ_min,max= −0.13/

0.17 e Å⁻³. CCDC 852569. The absolute conﬁguration is estab-

lished as (4R,5R,6R) relative to the known absolute conﬁguration

of the (1′S)-camphanate group.

Crystal data for 4g_R. C₇H₉FO₃, M = 160.1, orthorhombic, a

= 4.641(2), b = 10.535(3), c = 15.936(4) Å, U = 779.2(3) Å³, T

= 298(2) K, space group P2₁2₁2₁(no. 19), Mo-Kα radiation, λ =

0.71073Å, Z = 4, F(000) = 336, D_x= 1.365 g cm⁻³, μ =

0.121 mm⁻¹, Bruker SMART CCD diffractometer, ϕ/ω scans,

4.6° < 2θ < 55.9°, measured/independent reﬂections: 3415/1482,

R_int= 0.030, direct methods solution, full-matrix least squares

(4R,5R,6S)-6-Fluoro-4,5-dihydroxy-3-methylcyclohex-2-enone

5g_R. Colourless oil (0.028 g, 4%); R_f(0.21, 40% EtOAc in

hexane, 4 elutions); [α]_D− 92 (c 1.2, MeOH); HRMS (EI):

Found (M − H₂O)⁺, 142.0423 requires C₇H₉O₃F 142.0430;

¹H-NMR (400 MHz, CDCl₃) δ_H2.15 (3 H, d, J 1.4, Me), 3.39

(1 H, br s, OH), 3.51 (1 H, br s, OH), 4.12 (1 H, ddd, J 10.5,

10.3, 4.2, 5-H), 4.41 (1 H, t, J 4.2, 4-H), 5.14 (1 H, dd, J 50.4,

10.3, 6-H), 5.99 (1 H, dq, J 3.8, 1.4, 2-H); ¹³C-NMR (100 MHz,

CDCl₃) δ_C22.4, 70.8 (d, J 8.9), 71.0 (d, J 17.9), 91.4 (d, J

187.3), 126.8, 158.2, 192.3 (d, J 14.59); ¹⁹F-NMR (376 MHz,

CDCl₃) δ_F−214.1 (ddt, J 50.6, 10.4, 4.0); LRMS (EI): 142

([M − H₂O]⁺, 38%), 131 (35), 98 (100), 69 (52); IR (ﬁlm)

ν_max/cm⁻¹3353 (OH), 1687 (CvO).

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= 0.045 for 1205 data with F_o> 4σ(F_o),

103 parameters, ωR₂= 0.115 (all data), GoF = 1.05, Δρ_min,max

=

−0.15/0.15 e Å⁻³. CCDC 852568. The absolute conﬁguration is

not determined in this analysis but is conﬁrmed as (4R,5R,6R)

by correlation with the structure analysis of the camphanate

derivative 4g_R-cam

.

(1S,4R)-1-(1R,2R,6R)-6-Fluoro-2-hydroxy-3-methyl-5-(oxocy-

clohex-3-enyloxymethyl)-4,7,7-trimethyl-2-oxabicyclo[2.2.1]

heptan-3-one 4g_R-cam. A stirred solution of ketodiol 4g_R

(0.040 g, 0.25 mmol), in dry pyridine (0.5 ml), was treated with

(−)-(S)-camphanic chloride (0.070 g, 0.32 mmol) at room temp-

erature. After stirring the reaction mixture for 3 h at room temp-

erature, the pyridine was distilled off under reduced pressure, the

residue extracted with EtOAc (25 ml), the extract successively

washed with 5% aq. NaHCO₃solution (20 ml), water (15 ml)

and then dried (Na₂SO₄). The solvent was removed under

reduced pressure and the residue puriﬁed by PLC (80% EtOAc

in hexane) to yield camphanate 4g_R-camas a colourless crystal-

line solid (0.070 g, 82%); m.p. 125–126 °C (CH₂Cl₂–MeOH);

R_f(0.55, 80% EtOAc in hexane); [α]_D− 46 (c 0.45, MeOH);

HRMS (EI): Found M⁺, 340.1340 requires C₁₇H₂₁O₆F

340.1322; ¹H-NMR (400 MHz, CD₃OD) δ_H0.92 (3 H, s,

CMe₂), 1.00 (3 H, s, CMe₂), 1.06 (3 H, s, MeCCH₂), 1.56–1.64

(1 H, m, MeCCH₂), 1.90–2.03 (2 H, m, MeCCH₂and CH₂CO),

2.06 (3 H, t, J 1.3, CHCMe), 2.29–2.37 (1 H, m, CH₂CO),

4.81–4.84 (1 H, br m, 2-H), 5.38 (1 H, dd, J 45.4, 2.8, 6-H),

5.96–6.00 (1 H, m, 4-H), 6.07 (1 H, dt, J 8.8, 3.1, 1-H);

¹³C-NMR (100 MHz, CD₃OD) δ_C9.8, 16.6, 16.9, 20.1, 30.0,

31.5, 55.6, 56.1, 69.4 (d, J 8.7 Hz), 77.6 (d, J 16.3), 90.4 (d, J

195.2 Hz), 92.9, 124.7 (d, J 1.3), 164.7, 168.1, 180.2, 193.3 (d,

J 15.7 Hz); ¹⁹F-NMR (376 MHz, CD₃OD) δ_F−204.7 (1 F,

dddd, J 45.7, 8.9, 6.0, 2.2); LRMS (EI): 340 (M⁺, 4%), 264

(27), 219 (95), 131 (65), 69 (100); IR (ﬁlm) ν_max/cm⁻¹3275

(OH), 1784 (CvO).

(v) Biotransformation of 5-chloro-2-methylphenol 1i

Phenol substrate 1i gave a mixture of two bioproducts (13 : 1)

which on separation by PLC yielded cyclohexenone cis-diols 4i_R

and 5i_R.

(4R,5R,6S)-3-Chloro-4,5-dihydroxy-6-methylcyclohex-2-enone

4i_R. White crystals (0.058 g, 6%); m.p. 91–92 °C (EtOAc–

hexane); R_f0.17 (25% EtOAc in hexane, 5 elutions); HRMS

(EI): Found M⁺, 176.0225 requires C₇H₉O₃³⁵Cl 176.0234; [α]_D

1

+ 59 (c 0.98, MeOH); H-NMR (500 MHz, CDCl₃) δ_H1.28 (3

H, d, J 6.9, Me), 2.59 (1 H, qd, J 6.9, 2.3, 6-H), 4.34 (1 H, dd, J

3.5, 2.3, 5-H), 4.55 (1 H, dd, J 3.5, 2.0, 4-H), 6.30 (1 H, d, J

2.0, 2-H); ¹³C-NMR (100 MHz, CDCl₃) δ_C11.0, 46.0, 71.2,

73.7, 128.3, 154.5, 195.8; LRMS (EI): 176 (M⁺, 5%), 158 (5),

131 (21), 120 (30), 118 (100), 90 (52); IR (ﬁlm) ν_max/cm⁻¹

3343 (OH), 1677 (CvO).

Crystal data for 4i_R. C₇H₉ClO₃, M = 176.6, monoclinic, a =

12.125(2), b = 4.870(2), c = 14.100(3) Å, β = 92.28(1)°, U =

832.0(3) Å³, T = 293(2) K, space group P2₁(no. 4), Mo-Kα

radiation, λ = 0.71073Å, Z = 4, F(000) = 368, D_x= 1.410 g

cm⁻³, μ = 0.414 mm⁻¹, Bruker P4 diffractometer, ω scans, 4.3°

< 2θ < 50.0°, measured/independent reﬂections: 2129/2024, R_int

= 0.063, direct methods solution, full-matrix least squares reﬁne-

2

ment on F_o, anisotropic displacement parameters for non-hydro-

gen atoms; all hydrogen atoms located in a difference Fourier

synthesis but included at positions calculated from the geometry

of the molecules using the riding model, with isotropic vibration

parameters. R₁= 0.050 for 1400 data with F_o> 4σ(F_o), 205 par-

ameters, ωR₂= 0.127 (all data), GoF = 1.04, Δρ_min,max= −0.20/

0.21 e Å⁻³. CCDC 852570. The absolute conﬁguration is estab-

lished as (4R,5R,6S) from the anomalous scattering arising from

the chlorine atom; Flack parameter x = 0.08(13). The asymmetric

Crystal data for 4g_R-cam. C₁₇H₂₁FO₆, M = 340.3, monoclinic,

a = 8.272(1), b = 10.422(2), c = 10.053(2) Å, β = 106.38(1)°, U

= 831.4(2) Å³, T = 293(2) K, space group P2₁(no. 4), Mo-Kα

radiation, λ = 0.71073Å, Z = 2, F(000) = 360, D_x= 1.359 g

cm⁻³, μ = 0.109 mm⁻¹, Bruker P4 diffractometer, ω scans, 4.2°

6226 | Org. Biomol. Chem., 2012, 10, 6217–6229

View Article Online

unit consists of two molecules, which do not differ signiﬁcantly

in conformation.

(1 H, t, J 3.8, 4-H), 7.21–7.35 (5 H, m, Ar); ¹³C-NMR

(100 MHz, CDCl₃) δ_C−4.9, −4.3, −4.2, −4.1, 18.5, 18.7, 24.3,

26.0 (3C), 26.2, 26.4 (3C), 70.6, 73.5, 126.8 (2C), 126.9, 127.1,

128.3 (2C), 140.3, 141.5; LRMS (EI): 403 ([M − Me]⁺, 2%),

361 (30), 260 (19), 203 (40), 155 (58), 147 (100).

(4R,5R,6R)-3-Chloro-4,5-dihydroxy-6-methylcyclohex-2-enone

5i_R. Colourless oil (0.044 g, 0.5%); R_f0.21 (25% EtOAc in

hexane, 5 elutions); [α]_D+ 92 (c 0.65, MeOH); HRMS (EI):

Found M⁺, 176.0226 requires C₇H₉O₃³⁵Cl 176.0234; H-NMR

(1S,2S-3-Chlorocyclohex-3-ene-1,2-diyl)bis(oxy)bis(tert-butyl-

dimethylsilane) 12d. (1S,2S)-3-Chlorocyclohex-3-ene-1,2-diol

11d (0.250 g, 1.68 mmol) gave di-TBDMS ether 12d as a col-

1

(500 MHz, CDCl₃) δ_H1.24 (3 H, d, J 7.2, Me), 2.67 (1 H, br s,

OH), 2.75 (1 H, dq, J 8.4, 7.2, 6-H), 3.11 (1 H, br s, OH), 3.94

(1 H, dd, J 8.4, 3.8, 5-H), 4.49 (1 H, d, J 3.8, 4-H), 6.28 (1 H, s,

2-H); ¹³C-NMR (100 MHz, CDCl₃) δ_C11.3, 44.8, 70.6, 72.8,

129.0, 154.0, 196.9; LRMS (EI): 176 (M⁺, 3%), 142 (10), 131

(20), 120 (35), 118 (100), 92 (28), 90 (56); IR (ﬁlm) ν_max/cm⁻¹

3318 (OH), 1677 (CvO).

ourless oil (0.580 g, 91%); R_f0.67 (5% Et₂O in hexane); [α]_D

−

84 (c 1.02, CHCl₃); HRMS (ES): Found (M + H)⁺, 377.2115

requires C₁₈H₃₈O₂Si₂³⁵Cl 377.2099; ¹H-NMR (400 MHz,

CDCl₃) δ_H0.07 (3 H, s, SiMe₂), 0.08 (3 H, s, SiMe₂), 0.12 (3 H,

s, SiMe₂), 0.15 (3 H, s, SiMe₂), 0.906 (9 H, s, CMe₃), 0.914 (9

H, s, CMe₃), 1.47–1.53 (1 H, m, 6-H), 1.91–2.12 (2 H, m, 5-H,

6′-H), 2.20 (1 H, dddd, J 18.1, 6.3, 5.0, 1.6, 5′-H), 3.75 (1 H, dt,

J 6.2, 3.2, 1-H), 4.02 (1 H, d, J 3.2, 2-H), 5.79 (H, dd, J 4.8,

2.8, 4-H); ¹³C-NMR (100 MHz, CDCl₃) δ_C−4.5, −4.2, −4.1,

−4.05, 18.6, 18.9, 24.3, 25.3, 26.2 (3C), 26.3 (3C), 72.6, 74.2,

126.7, 132.4; LRMS (EI): 361 ([M − Me]⁺, 2%), 319 (18), 217

(8), 189 (20), 161 (25), 147 (100) 113 (45).

(vi) Biotransformation of 2-ethyl-5-methyl-phenol 1j

PLC separation of the crude mixture of bioproducts gave cyclo-

hexenone cis-diol 4j_Ras the major metabolite and catechol 2j as

the minor metabolite.

(4S,5R,6S)-6-Ethyl-4,5-dihydroxy-3-methylcyclohex-2-enone

4j_R. White crystalline solid (0.035 g, 12%); m.p. 78–79 °C

(EtOAc–hexane); R_f0.3 (75% EtOAc in hexane); [α]_D+ 104 (c

1.1, MeOH); HRMS (LCMS): Found (M + H)⁺, 171.1020

requires C₉H₁₅O₃171.1016; ¹H-NMR (400 MHz, CDCl₃) δ_H

0.98 (3 H, t, J 7.5, CH₂Me), 1.47 (1 H, ddq, J 14.4, 9.1, 7.5,

CH₂Me), 2.02 (3 H, t, J 1.4, CHCMe), 2.04–2.14 (1 H, dqd, J

14.4, 7.5, 5.1, CH₂Me), 2.24 (1 H, ddd, J 9.1, 5.1, 3.2, 6-H),

4.34 (1 H, dd, J 3.5, 2.3, 4-H), 4.39–4.42 (1 H, m, 5-H),

5.88–5.90 (1 H, m, 2-H); ¹³C-NMR (100 MHz, CDCl₃) δ_C11.6,

18.0, 20.1, 53.1, 72.2, 72.3, 126.9, 158.4, 198.8; LRMS

(LCMS): 193 ([M + Na]⁺, 30%), 171 (100), 153 (20); IR (KBr)

ν_max/cm⁻¹3425 (OH), 1656 (CvO).

(1S,2S-3-Bromocyclohex-3-ene-1,2-diyl)bis(oxy)bis(tert-butyl-

dimethylsilane) 12e. (1S,2S)-3-Bromocyclohex-3-ene-1,2-diol

11e (0.5 g, 2.59 mmol) furnished di-TBDMS ether 12e as a col-

ourless oil (0.980 g, 90%); R_f(0.68, 2% Et₂O in hexane); [α]_D−

68 (c 1.0, CHCl₃); HRMS (ES): Found (M + H)⁺, 421.1598

requires C₁₈H₃₈O₂Si₂⁷⁹Br 421.1594; ¹H-NMR (400 MHz,

CDCl₃) δ_H0.08 (3 H, s, SiMe₂), 0.09 (3 H, s, SiMe₂), 0.13 (3 H,

s, SiMe₂), 0.19 (3 H, s, SiMe₂), 0.91 (9 H, s, CMe₃), 0.93 (9 H,

s, CMe₃), 1.49–1.55 (1 H, m, 6-H), 1.93–2.03 (1 H, m, 5-H),

2.04–2.10 (1 H, m, 6′-H), 2.15–2.24 (1 H, m, 5′-H), 3.78 (1 H,

dt, J 11.4, 3.2, 1-H), 4.12 (1 H, d, J 3.2, 2-H), 6.02 (1 H, dd, J

4.8, 2.5, 4-H); ¹³C-NMR (100 MHz, CDCl₃) δ_C−4.5, −4.2,

−4.0, −4.0, 18.6, 18.7, 24.0, 26.3 (3C), 26.35 (3C), 26.9, 72.7,

t

75.8, 122.7, 131.0; LRMS (EI): 365 ([M − Bu]⁺, 8%), 262 (5),

207 (15), 157 (18), 147 (100).

Synthesis of cyclohexenone cis-diols 4c–4e

Di-tert-butyldimethylsilyl ethers 12c–12e. NEt₃(3 equiva-

lents) was added to a solution of each cis-tetrahydrodiol (11c–

11e, 1 equivalent) in dry CH₂Cl₂(∼5 ml), maintained at 0 °C,

followed by the drop-wise addition of tert-butyldimethylsilyl

triﬂate (2.2 equivalents). The reaction mixture was stirred at

room temperature for 2 h, diluted with CH₂Cl₂(15 ml) and then

washed with ice cold water. The organic phase was separated,

dried (Na₂SO₄), concentrated under reduced pressure and the

residue obtained was puriﬁed by column chromatography (2%

→ 10% Et₂O in hexane) to yield di-TBDMS ethers (12c–12e).

Di-tert-butyldimethylsilyl enones 13c–13e. Tert-butylhydroper-

oxide (6 M in decane, 5.5 equivalents) was added drop-wise to a

stirred mixture of each di-TBDMS ether (12c–12e, 1 equivalent),

butyl butyrate (∼2 ml), diacetoxyiodobenzene (3 equivalents)

and potassium carbonate (0.5 equivalents), maintained at 0 °C,

and the solution was stirred over a period of 8 h. The reaction

mixture was diluted with 5% Et₂O in hexane mixture (25 ml),

the insoluble material ﬁltered off and the ﬁltrate concentrated.

The residue left after distilling off the solvent was puriﬁed by

column chromatography (2% → 5% Et₂O in hexane) followed

by PLC to yield di-TBDMS enone (13c–13e).

(1S,2R-3-Phenylcyclohex-3-ene-1,2-diyl)bis(oxy)bis(tert-butyl-

dimethylsilane) 12c. (1S,2R)-3-Phenylcyclohex-3-ene-1,2-diol

11c (0.250 g, 1.31 mmol) yielded di-TBDMS ether 12c as a col-

ourless oil (0.510 g, 93%); R_f0.4 (5% Et₂O in hexane); HRMS

(EI): Found (M − Me)⁺, 403.2499 requires C₂₃H₃₉O₂Si₂

403.2483; [α]_D− 118 (c 1.11, CHCl₃); ¹H-NMR (400 MHz,

CDCl₃) δ_H−0.32 (3 H, s, SiMe₂), −0.04 (3 H, s, SiMe₂), 0.14

(3 H, s, SiMe₂), 0.15 (3 H, s, SiMe₂), 0.71 (9 H, s, CMe₃), 0.97

(9 H, s, CMe₃), 1.53–1.61 (1 H, m, 6-H), 2.05–2.16 (1 H, m, 5-

H), 2.19–2.30 (1 H, m, 6′-H), 2.36 (1 H, ddd, J 18.4, 5.1, 6.4,

5′-H), 3.83–3.88 (1 H, m, 1-H), 4.55–4.57 (1 H, m, 2-H), 5.86

(4R,5S)-4,5-bis(tert-Butyldimethylsilyloxy)-3-phenylcyclohex-

2-enone 13c. Di-TBDMS 12c (0.5 g, 1.19 mmol) gave enone

13c as colourless crystalline solid (0.210 g, 41%); m.

p. 74–75 °C (hexane); R_f0.43 (10% Et₂O in hexane); [α]_D− 86

(c 0.9, CHCl₃); HRMS (ES): Found (M + H)⁺, 433.2578

requires C₂₄H₄₀O₃Si₂433.2594; ¹H-NMR (400 MHz, CDCl₃)

δ_H−0.24 (3 H, s, SiMe₂), 0.08 (3 H, s, SiMe₂), 0.13 (3 H, s,

SiMe₂), 0.15 (3 H, s, SiMe₂), 0.72 (9 H, s CMe₃), 0.94 (9 H, s,

CMe₃), 2.50 (1 H, dd, J 16.9, 4.6, 6-H), 2.99 (1 H, dd, J 16.9,

11.6, 6′-H), 4.18 (1 H, ddd, J 11.6, 4.6, 2.5, 5-H), 4.79 (1 H, d,

Org. Biomol. Chem., 2012, 10, 6217–6229 | 6227

View Article Online

J 2.5, 4-H), 6.23 (1 H, s, 2-H), 7.41–7.51 (5 H, m, Ar);

¹³C-NMR (100 MHz, CDCl₃) δ_C−5.0, −4.4, −4.3, −4.2, 18.4,

18.5, 25.8 (3C), 26.2 (3C), 41.0, 70.7, 70.8, 126.6, 127.1 (2C),

129.0 (2C), 130.2, 137.5, 158.8, 199.5; LRMS (EI): 417 ([M −

Me]⁺, 5%), 375 (51), 274 (20), 243 (19), 147 (100), 141 (30);

IR (KBr) ν_max/cm⁻¹1665 (CvO).

Acknowledgements

We gratefully acknowledge ﬁnancial support from DEL

(PBAM^cI), and thank Sheoliang Guan and Dany Leborgne for

assistance with synthetic aspects of the programme. All calcu-

lations were performed at the Poznan Supercomputing and Net-

working Center.

(4S,5S)-4,5-bis(tert-Butyldimethylsilyloxy)-3-chlorocyclohex-2-

enone 13d. Di-TBDMS derivative 12d (0.540 g, 1.4 mmol)

gave enone 13d as colourless crystalline solid (0.235 g, 42%);

m.p. 59 °C (Et₂O–hexanes); R_f0.49 (5% Et₂O in hexanes); [α]_D

− 33 (c 1.03, CHCl₃); HRMS (EI): Found (M − CH₃)⁺,

375.1579 requires C₁₇H₃₂O₃Si₂³⁵Cl 375.1573; ¹H-NMR

(400 MHz, CDCl₃) δ_H0.08 (3 H, s, SiMe₂), 0.09 (3 H, s,

SiMe₂), 0.15 (3 H, s, SiMe₂), 0.17 (3 H, s, SiMe₂), 0.88 (9 H, s,

CMe₃), 0.90 (9 H, s, CMe₃), 2.43 (1 H, dd, J 16.6, 3.9, 6-H),

2.84 (1 H, dd, J 16.6, 10.2, 6′-H), 4.12 (1 H, dt, J 10.2, 3.2, 5-

H), 4.23–4.32 (1 H, m, 4-H), 6.17 (1 H, s, 2-H); ¹³C-NMR

(100 MHz, CDCl₃) δ_C−4.6, −4.5, −4.3, −4.25, 18.4, 18.5, 26.0

(3C), 26.0 (3C), 41.8, 70.3, 74.5, 128.9, 157.6, 195.7; LRMS

(EI): 375 ([M − CH₃]⁺, 3%), 333 (23), 232 (9), 147 (100); IR

(KBr) ν_max/cm⁻¹1676 (CvO).

Notes and references

1 (a) B. L. Goodwin, Handbook of Biotransformations of Aromatic Com-

pounds, CRC Press LLC, Boca Raton, 2005, pp. 109–124; (b) R.

C. Bayley, S. Dagley and D. T. Gibson, Biochem. J., 1966, 101, 293;

(c) D. T. Gibson, V. Mahadevan and J. F. Davey, J. Bacteriol., 1974, 119,

930; (d) J. A. Buswell, J. Bacteriol., 1975, 124, 1399; (e) J. C. Spain and

D. T. Gibson, Appl. Environ. Microbiol., 1988, 54, 1399; (f) F. K. Higson

and D. D. Focht, Appl. Environ. Microbiol., 1989, 55, 946; (g) J.

C. Spain, G. J. Zylstyra, C. K. Blake and D. T. Gibson, Appl. Environ.

Microbiol., 1989, 55, 2648; (h) C. Hinterregger, R. Leitner, M. Loidl,

A. Ferschl and F. Streichsbier, Appl. Microbiol. Biotechnol., 1992, 37,

252; (i) G. Bestetti, E. Galli, B. Leoni, F. Pelizzoni and G. Sello, Appl.

Microbiol. Biotechnol., 1992, 37, 260; ( j) K. Lee, FEMS Microbiol.

Lett., 2006, 255, 316; (k) D. Kim, J. S. Lee, K. Y. Choi, Y.-S. Kim, J.

N. Choi, S.-K. Kim, J.-C. Chae, G. Zlystra, C. H. Lee and E. Kim,

Enzyme Microb. Technol., 2007, 41, 221.

2 (a) N. D. Sharma, C. C. R. Allen, J. F. Malone and D. R. Boyd, Chem.

Commun., 2009, 3633; (b) M. Kwit, J. Gawronski, D. R. Boyd, N.

D. Sharma and M. Kaik, Org. Biomol. Chem., 2010, 8, 5635; (c) D.

R. Boyd, N. D. Sharma, P. Stevenson, C. McRoberts, J. T. G. Hamilton,

J. M. Argudo, H. Mundi, L. A. Kulakov and C. C. R. Allen, Org.

Biomol. Chem., 2011, 9, 1479.

(4S,5S)-3-Bromo-4,5-bis(tert-butyldimethylsilyloxy)cyclohex-2-

enone 13e. Di-TBDMS derivative 12e (0.750 g, 1.78 mmol)

yielded enone 13e as colourless crystalline solid (0.286 g, 37%);

m.p. 58 °C (Et₂O–hexane); R_f0.41 (7.5% Et₂O in hexane); [α]_D

− 26 (c 0.86, CHCl₃); HRMS (ES): Found (M + H)⁺, 435.1393

requires C₁₈H₃₆O₃Si₂⁷⁹Br 435.1386; ¹H-NMR (400 MHz,

CDCl₃) δ_H0.09 (3 H, s, SiMe₂), 0.10 (3 H, s, SiMe₂), 0.17 (3 H,

s, SiMe₂), 0.21 (3 H, s, SiMe₂), 0.89 (9 H, s, CMe₃), 0.92 (9 H,

s, CMe₃) 2.45 (1 H, dd, J 16.5, 3.8, 6-H), 2.85 (1 H, dd, J 16.5,

10.3, 6′-H), 4.14 (1 H, br d, J 9.2, 5-H), 4.41 (1 H, d, J 1.9, 4-

H), 6.43 (1 H, s, 2-H); ¹³C-NMR (100 MHz, CDCl₃) δ_C−4.5,

−4.3, −4.25, −4.1, 18.4, 18.6, 26.0 (3C), 26.1 (3C), 41.3, 70.4,

76.2, 128.5, 133.3, 195.4; LRMS (EI): 421 ([M − CH₃]⁺, 3%),

379 (18), 278 (10), 213 (8), 147 (100); IR (KBr) ν_max/cm⁻¹

1674 (CbO)).

3 (a) D. A. Widdowson, D. W. Ribbons and S. D. Thomas, Janssen Chim.

Acta, 1990, 8, 3; (b) H. A. J. Carless, Tetrahedron: Asymmetry, 1992, 3,

795; (c) G. N. Sheldrake, in Chirality and Industry, ed. A. N. Collins,

G. N. Sheldrake and J. Crosby, John Wiley Ltd., Chichester, 1992, 127;

(d) S. M. Brown and T. Hudlicky, in Organic Synthesis: Theory and

Applications, ed. T. Hudlicky, JAI Press, Greenwich, 1993, vol. 2, p. 113;

(e) S. M. Resnick, K. Lee and D. T. Gibson, J. Ind. Microbiol., 1996, 17,

438; (f) D. R. Boyd and G. N. Sheldrake, Nat. Prod. Rep., 1998, 15, 309;

(g) T. Hudlicky, D. Gonzalez and D. T. Gibson, Aldrichimica Acta.,

1999, 32, 35; (h) D. T. Gibson and R. E. Parales, Curr. Opin. Biotechnol.,

2000, 11, 236; (i) D. R. Boyd, N. D. Sharma and C. C. R. Allen, Curr.

Opin. Biotechnol., 2001, 12, 564; ( j) R. A. Johnson, Org. React., 2004,

63, 117; (k) D. R. Boyd and T. D. H. Bugg, Org. Biomol. Chem., 2006,

4, 181; (l) K. A. B. Austin, M. Matveenko, T. A. Reekie and M.

G. Banwell, Chem. Aust., 2008, 75, 3; (m) T. Hudlicky and J. W. Reed,

Synlett, 2009, 685; (n) T. Hudlicky and J. W. Reed, Chem. Soc. Rev.,

2009, 38, 3117.

4 V. Berberian, C. C. R. Allen, N. D. Sharma, D. R. Boyd and C. Hardacre,

Adv. Synth. Catal., 2007, 349, 727.

5 D. R. Boyd, J. Blacker, B. Byrne, H. Dalton, M. V. Hand, S. Kelly, R.

A. More O’Ferrall, S. N. Rao, N. D. Sharma, G. N. Sheldrake and

H. Dalton, J. Chem. Soc., Chem. Commun., 1994, 313.

6 (a) A. Evidente, L. Sparapano, A. Andolﬁ, G. Bruno, F. Giordano and

A. Motta, Phytopathol. Mediterr., 2000, 39, 299; (b) A. Evidente,

L. Sparapano, O. Fierro, G. Bruno, F. Giordano and A. Motta, Phyto-

chemistry, 1998, 48, 1139.

7 (a) J. Gawronski, M. Kwit, D. R. Boyd, N. D. Sharma, J. F. Malone and

A. Drake, J. Am. Chem. Soc., 2005, 127, 4308; (b) D. R. Boyd, N.

D. Sharma, G. P. Coen, P. Gray, J. F. Malone and J. Gawronski, Chem.–

Eur. J., 2007, 13, 5804; (c) M. Kwit, N. D. Sharma, D. R. Boyd and

J. Gawronski, Chirality, 2008, 20, 609.

Cyclohexenone diols 4c_S–4e_S. A solution of each enone (13c–

13e, 1 equivalent) in dry THF (∼4 ml) was treated with tetra-

butylammonium ﬂuoride (1 M, THF, 3 equivalents) at 0 °C.

After stirring the reaction mixture at room temperature for 3 h

the solvent was removed under reduced pressure and the residue

puriﬁed by PLC (75% EtOAc in hexanes) to yield cyclohexe-

none diol (4c_S–4e_S). In each case the spectral data and speciﬁc

rotation ([α]_D) value for the synthesised sample was identical to

that of the corresponding metabolite (4c_S–4e_S).^2c

(4R,5S)-4,5-Dihydroxy-3-phenylcyclohex-2-enone 4c_S. Enone

13c (0.2 g, 0.46 mmol) furnished cyclohexenone diol 4c_Sas col-

ourless crystalline solid (0.049 g, 52%).

8 (a) R. D. Burnett and D. N. Kirk, J. Chem. Soc., Perkin Trans. 1, 1981,

1460; (b) J. K. Gawronski, Tetrahedron, 1982, 38, 3; (c) N. Kirk, Tetra-

hedron, 1986, 42, 777; (d) J. Gawronski, Conformations, Chiroptical and

Related Spectral Properties of Enones, in The Chemistry of Enones, ed. S.

Patai and Z. Rappoport, J. Wiley, 1989, ch. 3; (e) D. A. Lightner and J.

E. Gurst, Organic Conformational Analysis and Stereochemistry from

Circular Dichroism Spectroscopy, Wiley-VCH, 2000; (f) G. Snatzke,

Angew. Chem., Int. Ed. Engl., 1979, 18, 363; (g) G. Snatzke, in Funda-

mental Aspects and Recent Developments in Optical Rotatory Dispersion

and Circular Dichroism, ed. F. Ciardelli, P. Salvadori, Heyden, London,

1973, pp. 109–124; (h) G. Snatzke, Tetrahedron, 1965, 21, 413;

(4S,5S)-3-Chloro-4,5-dihydroxycyclohex-2-enone 4d_S. Enone

13d (0.2 g, 0.51 mmol) yielded cyclohexenone diol 4d_Sas col-

ourless crystalline solid (0.024 g, 29%).

(4S,5S)-3-Bromo-4,5-dihydroxycyclohex-2-enone 4e_S. Enone

13e (0.280 g, 0.64 mmol) gave cyclohexenone diol 4e_Sas col-

ourless crystalline solid (0.040 g, 30%).

6228 | Org. Biomol. Chem., 2012, 10, 6217–6229

View Article Online

(d) G. Snatzke, Tetrahedron, 1965, 21, 421; ( j) C. Djerassi, R. Records,

E. Bunnenberg, K. Mislow and A. Moscowitz, J. Am. Chem. Soc., 1962,

84, 870; (k) W. B. Whalley, Chem. Ind., 1962, 1024; (l) W. Hug and

G. Wagniere, Helv. Chim. Acta, 1971, 54, 633; (m) T. Liljefors and N.

L. Allinger, J. Am. Chem. Soc., 1976, 98, 2745; (n) J. K. Gawronski,

T. Liljefors and B. Nordén, J. Am. Chem. Soc., 1979, 101, 5515;

(o) G. Snatzke, Tetrahedron, 1965, 21, 439; (p) G. Snatzke, in Optical

Rotatory Dispersion and Circular Dichroism in Organic Chemistry, ed.

G. Snatzke, Sadtler Research Labs, Inc., Philadelphia, 1967, pp.

208–223; (q) H.-G. Kuball, S. Neubrech and A. Schönhofer, Chem.

Phys., 1992, 163, 115; (r) H.-G. Kuball, B. Schultheis, M. Klasen,

J. Frelek and A. Schönhofer, Tetrahedron: Asymmetry, 1993, 4, 517;

(s) J. Frelek, W. J. Szczepek, H. P. Weiss, G. J. Reiss, W. Frank,

J. Brechtel, B. Schultheis and H.-G. Kuball, J. Am. Chem. Soc., 1998,

120, 7010; (t) J. Frelek, W. J. Szczepek, S. Neubrech, B. Schultheis,

J. Brechtel and H.-G. Kuball, Chem.–Eur. J., 2002, 8, 1899; (u) A.

W. Burgstahler and R. C. Barkhurst, J. Am. Chem. Soc., 1970, 92, 7601;

(v) M. Kwit, P. Skowronek, J. Gawronski, J. Frelek, M. Woznica and

A. Butkiewicz, Some Inherently Chiral Chromophores – Empirical Rules

and Quantum Chemical Calculations in Comprehensive Chiroptical

Spectroscopy, ed. N. Berova, R. Woody, K. Nakanishi and P. Polavarapu,

Wiley, 2012, vol. 2, pp. 39–72.

9 (a) N. Harada and P. Stephens, Chirality, 2010, 22, 229; (b) N.

M. O’Boyle, A. L. Tenderholt and K. M. Langner, J. Comput. Chem.,

2008, 29, 839.

10 (a) D. R. Boyd, N. D. Sharma, G. P. Coen, V. Ljubez, P. K. M. McGeehin

and C. C. R. Allen, Org. Biomol. Chem., 2007, 5, 514; (b) D. R. Boyd,

N. D. Sharma, M. V. Berberian, K. Dunne, C. Hardacre, M. Kaik,

B. Kelly, J. F. Malone, S. T. McGregor and P. J. Stevenson, Adv. Synth.

Catal., 2010, 352, 855; (c) M. Bell, D. R. Boyd, K. S. Dunne, J.

F. Malone, B. Kelly and P. J. Stevenson, Org. Biomol. Chem., 2012, 10,

1388, DOI: 10.1039/c1ob06599h.

11 Y. Zhao and Y.-Y. Yeung, Org. Lett., 2010, 12, 2128.

12 (a) M. Sondossi, B. A. Lloyd, D. Barriault, M. Sylvestre and M. Simard,

Acta Crystallogr., Sect. C: Cryst. Struct. Commun., 1995, 51, 491;

(b) M. Sondossi, D. Barriault and M. Sylvestre, Appl. Environ. Micro-

biol., 2004, 70, 174.

13 (a) A. Karlsson, J. V. Parales, R. E. Parales, D. T. Gibson and

H. Ramaswamy, Science, 2003, 299, 1309; (b) T. D. Bugg, Tetrahedron,

2003, 59, 7075; (c) T. D. Bugg and S. Ramaswamy, Curr. Opin. Chem.

Biol., 2008, 12, 134; (d) R. Friemann, K. Lee, E. N. Brown, D.

T. Gibson, H. Eklund and S. Ramaswamy, Acta Crystallogr., Sect. D:

Biol. Crystallogr., 2009, 65, 24.

14 A. R. McDonald and L. Que, Nat. Chem., 2011, 3, 762.

Org. Biomol. Chem., 2012, 10, 6217–6229 | 6229

Article Doi

Structure, stereochemistry and synthesis of enantiopure cyclohexenone cis-diol bacterial metabolites derived from phenols

DOI: 10.1039/c2ob25079a

Source and publish data:

Authors:

Article abstract of DOI:10.1039/c2ob25079a

Full text of DOI:10.1039/c2ob25079a

Products guided by the article

R&D Labs maybe for 1392229-34-3

Relevant to this article

Hot Product