bis-cis-Dihydrodiols: A New Class of Metabolites Resulting from Biphenyl Dioxygenase-Catalyzed Sequential Asymmetric cis-Dihydroxylation of Polycyclic Arenes and Heteroarenes

Article abstract of DOI:10.1021/jo982517n

The biphenyl dioxygenase-catalyzed asymmetric mono-cis-dihydroxylation of the tetracyclic arenes chrysene 1A, benzo[c]phenanthridine 1B, and benzo[b]naphtho[2,1-d]thiophene 1C, has been observed to occur exclusively at the bay or pseudo-bay region using the bacterium Sphingomonas yanoikuyae B8/36. The mono-cis-dihydrodiol derivatives 2A and 2C, obtained from chrysene 1A by oxidation at the 3,4-bond (2A) and benzo[b]naphtho[2,1-d]thiophene 1C by oxidation at the 1,2-bond (2C), respectively, have been observed to undergo a further dioxygenase-catalyzed asymmetric cis-dihydroxylation at a second bay or pseudo-bay region bond to yield the corresponding bis-cis-dihydrodiols (cis-tetraols) 4A and 4C, the first members of a new class of microbial metabolites in the polycyclic arene series. The enantiopurities and absolute configurations of the new mono-cis-dihydrodiols 2B, 2C, and 3B were determined by ¹H NMR analyses of the corresponding (R)- and (S)-2-(1-methoxyethyl)benzeneboronate (MPBA) ester derivatives. The structure and absolute configurations of the bis-cis-dihydrodiols 4A and 4C were unambiguously determined by spectral analyses, stereochemical correlations, and, for the metabolite 4C, X-ray crystallographic analysis of the bis-acetonide derivative 7C. These results illustrate the marked preference of biphenyl dioxygenase for the cis-di- and tetra-hydroxylations of polycyclic arenes, at the more hindered bay or pseudo-bay regions, by exclusive addition from the same (si:si) face, to yield single enantiomers containing two and four chiral centers.

Full text of DOI:10.1021/jo982517n

J . Org. Chem. 1999, 64, 4005-4011
4005
bis-cis-Dih yd r od iols: A New Cla ss of Meta bolites Resu ltin g fr om
Bip h en yl Dioxygen a se-Ca ta lyzed Sequ en tia l Asym m etr ic
cis-Dih yd r oxyla tion of P olycyclic Ar en es a n d Heter oa r en es
Derek R. Boyd,* Narain D. Sharma, Francis Hempenstall, Martina A. Kennedy,
J ohn. F. Malone, and Christopher C. R. Allen†
School of Chemistry and the QUESTOR Centre, The Queen’s University of Belfast, Belfast BT9 5AG
Sol M. Resnick and David T. Gibson*
Department of Microbiology and the Center for Biocatalysis and Bioprocessing, The University of Iowa,
Iowa City, Iowa 52242
Received December 29, 1998
The biphenyl dioxygenase-catalyzed asymmetric mono-cis-dihydroxylation of the tetracyclic arenes
chrysene 1A, benzo[c]phenanthridine 1B, and benzo[b]naphtho[2,1-d]thiophene 1C, has been
observed to occur exclusively at the bay or pseudo-bay region using the bacterium Sphingomonas
yanoikuyae B8/36. The mono-cis-dihydrodiol derivatives 2A and 2C, obtained from chrysene 1A by
oxidation at the 3,4-bond (2A) and benzo[b]naphtho[2,1-d]thiophene 1C by oxidation at the 1,2-
bond (2C), respectively, have been observed to undergo a further dioxygenase-catalyzed asymmetric
cis-dihydroxylation at a second bay or pseudo-bay region bond to yield the corresponding bis-cis-
dihydrodiols (cis-tetraols) 4A and 4C, the first members of a new class of microbial metabolites in
the polycyclic arene series. The enantiopurities and absolute configurations of the new mono-cis-
1
dihydrodiols 2B, 2C, and 3B were determined by H NMR analyses of the corresponding (R)- and
(S)-2-(1-methoxyethyl)benzeneboronate (MPBA) ester derivatives. The structure and absolute
configurations of the bis-cis-dihydrodiols 4A and 4C were unambiguously determined by spectral
analyses, stereochemical correlations, and, for the metabolite 4C, X-ray crystallographic analysis
of the bis-acetonide derivative 7C. These results illustrate the marked preference of biphenyl
dioxygenase for the cis-di- and tetra-hydroxylations of polycyclic arenes, at the more hindered bay
or pseudo-bay regions, by exclusive addition from the same (si:si) face, to yield single enantiomers
containing two and four chiral centers.
In tr od u ction
occurring within one arene ring followed by a second
epoxidation of a different ring or sequential bis-epoxida-
tion of two different rings4 has been reported as an
example of multiple site oxidation of PAHs during
mammalian metabolism. The monooxygenase-catalyzed
epoxidation in two different arene rings may thus account
for the formation of bis-trans-dihydrodiol,5 arene oxide
trans-dihydrodiol,6 arene oxide phenol,7 and phenol trans-
dihydrodiol metabolites.8 Metabolite instability and/or
insufficient quantities of bioproducts have generally
precluded an unequivocal determination of relative and
absolute stereochemistry of the products formed by
multiple site oxidation of PAHs in mammalian systems.
In procaryotic systems (bacteria), the dioxygenase-
catalyzed dihydroxylation of a PAH to yield a mono-cis-
dihydrodiol is generally the first step in a metabolic
pathway leading to mineralization. Earlier research from
these laboratories, on the dioxygenase-catalyzed cis-
dihydroxylation of PAHs and heterocyclic analogues of
Identification of new metabolites and biodegradation
pathways is of particular importance in the context of
carcinogenic activity of polycyclic aromatic hydrocarbons
(PAHs) and their aza-, thia-, and oxa-analogues, all of
which are found to be prevalent in the environment. The
initial metabolic step for PAHs in eucaryotic systems
(animals, plants, fungi) frequently involves a monooxy-
genase-catalyzed epoxidation followed by isomerization
to yield phenols or by epoxide hydrolase-catalyzed hy-
drolysis to yield trans-dihydrodiols; it is exemplified by
the mammalian metabolism of the PAH chrysene 1A1
and the thiaarene benzo[b]naphtho[2,1-d]thiophene 1C
2
having a similar carcinogenic potency.3 The enzyme-
catalyzed epoxidation steps in arene 1A and thiaarene
1C were found to occur at both non-K (1,2-/7,8- in 1A,
3,4- and 9,10- in 1C), bay (3,4- and 9,10- in 1A), and
pseudo-bay (1,2- and 7,8- in 1C) region bonds. The
processes of epoxidation/aromatization (phenol formation)
or epoxidation/hydrolysis (trans-dihydrodiol formation)
(4) Agarwal, S. K.; Boyd, D. R.; McGuckin, M. R.; J ennings, W. B.;
Howarth, O. W. J . Chem. Soc., Perkin Trans. 1 1990, 3073.
(5) Platt, K. L.; Reischmann, I. Mol. Pharmacol. 1987, 32, 710
(6) Thakker, D. R.; Yagi, H. J .; Sayer, M.; Kapur, U.; Levin, W.;
Chang, R. L.; Wood, A. W.; Conney, A. H.; J erina, D. M. J . Biol. Chem.
1984, 260, 11249.
† QUESTOR Centre.
(1) Nordqvist, M.; Thakker, D. R.; Vyas, K. P.; Yagi, H.; Levin, W.;
Ryan, D. E.; Thomas, P. E.; Conney, A. H.; J erina, D. M. Mol.
Pharmacol. 1981, 19, 168.
(2) Murphy, S. E.; Amin, S.; Coletta, K.; Hoffmann, D. Chem. Res.
Toxicol. 1992, 5, 491.
(3) Wenzel-Hartung, R.; Brune, H.; Grimmer, G.; Germann, P.;
Timm, J .; Wosniok, W. Exp. Pathol. 1990, 40, 221.
(7) Boroujerdi, M.; Kung H. C.; Wilson A. G. E.; Anderson M. W.
Cancer Res. 1981, 41, 951.
(8) Glatt, H.; Seidel, A.; Ribeiro, O.; Kirkby, C.; Hirom P.; Oesch, F.
Carcinogenesis 1987, 8, 1621.
10.1021/jo982517n CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/30/1999

4006 J . Org. Chem., Vol. 64, No. 11, 1999
Boyd et al.
Sch em e 1
PAHs,9-19 showed a strong preference for oxidation at the
bay or pseudo-bay region.
of growing cultures of S. yanoikuyae B8/36 under the
conditions reported,9 again gave the cis-dihydrodiol 2A
(ca. 1% isolated yield, [R]D +112, THF) as the major
metabolite whose structure, enantiopurity (>98%), and
(3S,4R) absolute configuration had earlier been confirmed
by synthesis, stereochemical correlation, and reaction
with (-)-(S)- and (+)-(R)-2-(1-methoxyethyl)phenyl-
boronic acid (MPBA)20 to yield the corresponding MPBA
esters (2AS and 2AR).9 In the earlier study 9 a more polar
minor metabolite was isolated by PLC but could not be
identified due to the lack of sufficient material. Repeated
biotransfomations of chrysene 1A provided a larger
pooled sample from which the more polar metabolite was
isolated by PLC (silica gel, MeOH:CHCl3 1:9), (Rf 0.15;
0.002 g, ca. 0.3% isolated yield). The new metabolite could
be assigned structure 4A, 5A, or 6A (Scheme 1) on the
basis of chromatographic behavior, NMR spectral char-
acteristics (1H and 13C NMR spectroscopy including δ
values, coupling constants, COSY and NOE correlations),
and MS analysis. Measurement of the optical rotation,
[R]D +135, MeOH, and CD spectrum of the minor
metabolite allowed the cis-anti-cis structures 5A and 6A
to be eliminated since both contain a center of symmetry.
The relative (cis-syn-cis) and absolute (3S,4R,9S,10R)
stereochemistry of the bis-cis-diol 4A was confirmed by
addition of the major (+)-(3S,4R)-cis-dihydrodiol metabo-
lite 2A ([R]D +112, THF), as a substrate (0.003 g) to S.
yanoikuyae B8/36, and isolation of the (+)-(3S,4R,9S,-
10R)-bis-cis-dihydrodiol 4A (0.0015 g, [R]D +135, (MeOH).
The bis-cis-dihydrodiol 4A proved to be less stable than
the corresponding mono-cis-dihydrodiol precursor 2A and
readily aromatized during isolation and workup, yielding
a mixture of bis-phenols (by NMR and GC-MS analysis).
In a recent bacterial study using a mutant strain of
Sphingomonas yanoikuyae (B8/36, deficient in cis-dihy-
drodiol dehydrogenase activity), we reported the metabo-
lism of chrysene 1A,9 a tetracyclic PAH. The study has
been extended to include a tetracyclic azaarene, benzo-
[c]phenanthridine 1B, and a thiaarene, benzo[b]naphtho-
[2,1-d]thiophene 1C, both of which also contain two bay
regions or pseudo-bay regions. Since the monooxygenase-
catalyzed multiple site oxidation of larger PAHs in
mammals has been reported, 5-8 the possibility of similar
dioxygenase-catalyzed multiple site oxidation of PAHs
also occurring in bacterial biodegradation is of particular
interest. The results contained herein indicate that a
previously unobserved reaction sequence i.e., bis-cis-
dihydrodiol or cis-tetraol formation, is available to the
cis-dihydrodiols of PAHs and thia-PAHs which contain
bay regions or pseudo-bay regions.
Resu lts
Biotr a n sfor m a tion of Ch r ysen e 1A To Yield (+)-
(3S,4R)-3,4-Dih yd r oxy-3,4-d ih yd r och r ysen e, 2A, a n d
(+)-(3S,4R,9S,10R)-3,4,9,10-Tet r a h yd r oxy-3,4,9,10-
tetr a h yd r och r ysen e, 4A. Biotransformation of the
weakly carcinogenic PAH chrysene 1A, in the presence
(9) Boyd, D. R.; Sharma, N. D.; Agarwal, R.; Resnick, S. M.;
Schocken, M. J .; Gibson, D. T.; Sayer, J . M.; Yagi, H.; J erina, D. M. J .
Chem. Soc., Perkin Trans. 1 1997, 1715.
(10) J effrey, A. M.; Yeh, H. J . C.; J erina, D. M.; Patel, T. R.; Davey,
J . F.; Gibson, D. T. Biochemistry 1975, 14, 575.
(11) Boyd, D. R.; McMordie, R. A. S.; Porter, H. P.; Dalton, H.;
J enkins, R. O.; Howarth, O. J . Chem. Soc., Chem. Commun. 1987,
1722.
Biotr a n sfor m a tion of Ben zo[c]p h en a n th r id in e 1B
To Yield (+)-(3S,4R)-3,4-Dih yd r oxy-3,4-d ih yd r oben -
zo[c]p h en a n th r id in e, 2B, a n d (+)-(9S, 10R)-9,10-
Dih yd r oxy-9,10-d ih yd r oben zo[c]p h en a n th r en e, 3B.
Addition of benzo[c]phenanthridine 1B (0.4 g) to cultures
of S. yanoikuyae B8/36 and biotransformation over an
18 h period using conditions similar to those employed
for chrysene 1A,9 followed by ethyl acetate extraction of
the aqueous bioextracts, gave a mixture of two isomeric
cis-dihydrodiols, 2B and 3B. The two diols were sepa-
rated by PLC on silica gel (CHCl3:MeOH 1:9) and
(12) J erina, D. M.; Selander H.; Yagi, H.; Wells, M. C.; Davey, J .
F.; Mahadevan, V.; Gibson, D. T. J . Am. Chem. Soc. 1976, 98, 5988.
(13) Akhtar, M. N.; Boyd, D. R. Thompson, N. J .; Koreeda, M.;
Gibson, D. T.; Mahadevan, V.; J erina, D. M. J . Chem. Soc., Perkin
Trans. 1 1975, 2506.
(14) Koreeda, M.; Akhtar, M. N.; Boyd, D. R.; Neill, J . D.; Gibson
D. T. J . Org. Chem. 1978, 43, 1023.
(15) Gibson, D. T.; Mahadevan, V.; J erina, D. M.; Yagi, H.; Yeh, H.
J . C. Science 1975, 189, 295.
(16) J erina, D. M.; van Bladeren, P. J .; Yagi, H.; Gibson, D. T.;
Mahadevan, V.; Neese, A. S.; Koreeda, M.; Sharma, N. D.; Boyd D. R.
J . Org. Chem. 1984, 49, 3621.
(17) Gibson, D. T.; Subramanian, V. In Microbial Degradation of
Organic Compounds; Gibson, D. T., Ed.; Marcel Dekker: New York,
1984; p 181.
(18) Resnick, S. M.; Lee, K.; Gibson, D. T. J . Ind. Microbiol. 1996,
17, 438.
(19) Boyd, D. R.; Sheldrake, G. N. Nat. Prod. Rep. 1998, 309.
(20) Resnick, S. M.; Torok, D. S.; Gibson, D. T. J . Org. Chem. 1995,
60, 3546.

bis-cis-Dihydrodiols
J . Org. Chem., Vol. 64, No. 11, 1999 4007
Ta ble 1. Ap p lica tion of 1H NMR Ch em ica l Sh ift Va lu es
for th e MeO Sign a ls of th e Bor on a te Der iva tives 2AR,
2AS, 2BR, 2BS, 3BR, 3BS, 2CR, a n d 2CS in th e
Deter m in a tion of En a n tiom er ic Excess a n d Absolu te
Con figu r a tion
compd
δMeO(RMPBA) δMeO (S-MPBA) ∆δMeO % ee confign
(+)-2A
(+)-2B
(+)-3B
(+)-2C
2.97 (2AR)
3.15 (2BR)
3.08 (3BR)
3.06 (2CR)
3.11 (2AS)
3.26 (2BS)
3.23 (3BS)
3.109 (2CS)
0.14 >98% 3S,4R
0.11 >98% 3S,4R
0.15 >98% 9S,10R
0.49 >98% 1R,2S
identified from spectral (IR, NMR, MS) data as cis-
dihydrodiol 2B (0.036 g, Rf 0.23, [R]D +114, CHCl3; 8%
isolated yield) and the less polar cis-dihydrodiol 3B (0.030
g, Rf 0.36, [R]D +94, CHCl3; 7% isolated yield). Both cis-
dihydrodiols 2B and 3B were found to be enantiopure
1
by H NMR analyses of the corresponding MPBA deriva-
tives (2BR, 2BS, 3BR, and 3BS) using (+)-(R)- and (-)-
(S)-MPBA. Furthermore, on the basis of the general
trend,20 shown to be applicable in the bay region cis-
dihydrodiols of the phenanthrene18 and chrysene9 series,
that MPBA derivatives formed from (+)-(R)-MPBA (2BR
and 3BR) and a bay region cis-dihydrodiol having a
benzylic (R) and an allylic (S) configuration will have a
F igu r e 1. X-ray crystal structure analysis of compound 7C
indicating the (1R,2S,7R,8S) absolute configuration.
guishable. To determine the relative and absolute ster-
eochemistry of the bis-cis-dihydrodiol metabolite, it was
converted to the crystalline bis-acetonide 7C ([R]D +466,
CHCl3). An X-ray crystal structure analysis of compound
7C established the (1R,2S,7R,8S) configuration for the
acetonide 7C and hence for the parent bis-cis-diol 4C
(Figure 1). A minor byproduct, from the synthesis of the
bis-acetonide 7C, was found to be the phenolic acetonide
8C, presumably derived from the corresponding cis-diol
9C formed by selective acid-catalyzed dehydration of the
bis-cis-dihydrodiol 4C.
1
smaller δ value for the methoxyl signal in the H NMR
spectrum (Table 1), the bioproducts were identified as
(+)-(3S,4R)-3,4-dihydroxy-3,4-dihydrobenzo[c]phenan-
thridine 2B and (+)-(9S,10R)-9,10-dihydroxy-9,10-dihy-
drobenzo[c]phenanthridine 3B. It is noteworthy that both
cis-dihydrodiols were formed in similar proportions due
to cis-dihydroxylation in a bay region. When individual
cis-dihydrodiols 2B or 3B were added as substrates to
S. yanoikuyae B8/36, several minor unidentified peaks
were observed by HPLC analysis but no unequivocal
evidence of the bis-cis-dihydrodiol structure 4B could be
obtained.
Discu ssion
Studies of the biodegradation of larger members of the
PAH series by bacterial systems, and identification of
metabolites, have been restricted by their low aqueous
solubility resulting in the low bioavailability of such
substrates and thus the isolation of metabolites in very
low yields. A further limitation has been the inability of
bacterial systems willing to accept larger PAHs as
substrates. The biphenyl dioxygenase (BPDO) present in
S. yanoikuyae B8/36 (previously described as Beijerinckia
strain B8/36) has however proved to be exceptional; it
has the ability to accept tetracyclic PAHs, benz[a]-
anthracene16 and chrysene,9 and pentacyclic PAHs, ben-
zo[a]pyrene,15 as substrates for cis-dihydroxylation. Use
of strain B8/36, a mutant deficient in cis-dihydrodiol
dehydrogenase activity, has allowed the initial cis-
dihydrodiol metabolites to be isolated, albeit in low yield.
Regioselectivity for a bay region bond has emerged as a
common feature of BPDO-catalyzed cis-dihydroxylation
of these larger PAHs. The increased water solubility of
tetracyclic arenes, containing a heteroatom, allied to the
presence of two bay or pseudo-bay regions, prompted the
investigation of the azaarene, benzo[c]phenanthridine
1B, and thiaarene, benzo[b]naphtho[2,1-d]thiophene 1C,
as substrates for BPDO.
Biotr a n sfor m a tion of Ben zo[b]n a p h th o[2,1-d ]-
th iop h en e, 1C, To Yield (+)-(1R,2S)-1,2-Dih yd r oxy-
1,2-d ih yd r oben zo[b]n a p h th o[2,1-d ]th iop h en e, 2C,
a n d (+)-(1R,2S,7R,8S)-1,2,7,8-Tetr a h yd r oxy-1,2,7,8-
t et r a h yd r ob en zo[b]n a p h t h o [2,1-d ]t h iop h en e, 4C.
Metabolism of benzo[b]naphtho[2,1-d]thiophene 1C, us-
ing S. yaniokuyae B8/36 under conditions similar to those
used for substrates 1A and 1B, followed by ethyl acetate
extraction and PLC separation (CHCl3:MeOH 9:1) of the
aqueous bioextracts, yielded a major, less polar (Rf 0.33)
mono-cis-dihydrodiol ([R]D +146, MeOH; 7% isolated
yield) and a minor, more polar (Rf 0.18) metabolite ([R]D
+270, MeOH; 3% isolated yield). On the basis of NMR
and MS data, and comparison with the spectral data
reported for the mammalian metabolite, trans-1,2-dihy-
droxy-1,2-dihydrobenzo[b]naphtho[2,1-d]thiophene,2 the
mono-cis-dihydrodiol metabolite was identified as (+)-
(1R,2S)-1,2-dihydroxy-1,2-dihydrobenzo[b]naphtho[2,1-d]-
thiophene 2C. The enantiopurity of compound 2C (>98%)
and the absolute configuration of (+)-(1R,2S) were de-
termined by formation and 1H NMR analyses of the
corresponding MPBA esters, 2CR and 2CS.
The second more polar metabolite was identified as a
bis-cis-dihydrodiol having structure 4C, 5C, or 6C (Scheme
2). Time course studies of the biotransformation of
thiaarene 1C using S. yanoikuyae showed that cis-
dihydrodiol 2C was the initial product and that the
proportion of this metabolite decreased in concert with
an increasing proportion of the bis-cis-dihydrodiol me-
tabolite. The latter bioproduct, formed from either the
thiaarene 1C or the cis-dihydrodiol 2C, was indistin-
Chrysene 1A is the smallest member of the PAH series
to contain two bay regions. When used as a substrate
for the bacterium S. yanoikuyae (B8/36), enzyme-cata-
lyzed cis-dihydroxylation occurs exclusively at a bond
proximate to a bay region.9 In principle, the 3,4-, 5,6-,
9,10-, and 11,12-bonds are all adjacent to a bay region
and might be expected to form cis-dihydrodiol metabolites

4008 J . Org. Chem., Vol. 64, No. 11, 1999
Boyd et al.
Sch em e 2
preferentially. However, in practice, dioxygenase-cata-
lyzed cis-dihydroxylation of chrysene (and other PAHs
containing a bay region, including phenanthrene and
benz[a]anthracene) occurs almost exclusively at the
positions of lower electron density (3,4- or 9,10-bonds in
1A) rather than the K region bonds (5,6- or 11,12-bonds
in 1A). During the course of the earlier study,9 the cis-
dihydrodiol 2A was found to be the major metabolite,
accompanied by a very minor unidentified metabolite
isolated by PLC in quantities insufficient for character-
ization. In continuation of this investigation, the extracts,
obtained from several identical biotransformation runs,
were combined and the minor bioproduct isolated by PLC
was spectrally identified as 3,4,9,10-tetrahydroxy-3,4,9,-
10-tetrahydrochrysene, 4A. This is the first example of
a novel type of dioxygenase metabolite formed by se-
quential bay region cis-dihydroxylation of PAHs, i.e., the
bis-cis-dihydrodiol series (cis-tetrahydrotetraols). The
symmetrical nature of compound 4A resulted in a rela-
tively simple NMR spectrum in which the number of
signals was halved. The proximity of protons H-6 and
H-7 (H-12 and H-1) in structure 4A was evident from
the NOE signal enhancement. While the NMR data did
not allow a distinction to be made between the cis-syn-
cis (4A) and cis-anti-cis (5A/6A) tetraol isomers (Scheme
1), the presence of optical activity ([R]D +135, MeOH) and
a circular dichroism spectrum can only be consistent with
the cis-syn-cis isomer.
The enantiopurity and absolute configuration of the
mono-cis-diol metabolite 2A9 has been unequivocally
assigned by stereochemical correlation and the formation
of MPBA esters (Table 1). When mono-cis-diol 2A, the
initial metabolite from chrysene 1A, was in turn added
as a substrate to S. yanoikuyae B8/36, the bis-cis-diol 4A
was isolated as the only metabolite. This sample proved
to be spectrally indistinguishable (NMR, CD) from that
originally obtained from chrysene 1A. The formation of
bis-cis-diol 4A as a metabolite of chrysene 1A is, to our
knowledge, a new reaction sequence for mono-cis-dihy-
drodiol derivatives of PAHs (Scheme 1). Enzyme-cata-
lyzed dehydrogenation (cis-diol dehydrogenase) to yield
a catechol in wild type bacterial strains or spontaneous
dehydration to yield a phenol appear to be the only
reported reactions of mono-cis-diols that occur during the
bacterial biodegradation of PAHs.17-19
The bis-cis-dihydrodiol 4A was found to be less stable
than the precursor cis-diol 2A; it readily aromatized to
the corresponding bis-phenols during attempted purifica-
tion. This instability is consistent with the loss of
aromaticity across two benzene rings when chrysene is
tetrahydroxylated and the concomitant gain in resonance
energy upon dehydration. The successful interception of
the first bis-cis-tetrahydrodiol metabolite 4A appears to
have been facilitated by having (i) two bay regions in the
PAH, (ii) a mutant strain deficient in cis-diol dehydro-
genase activity, thus preventing dehydrogenation of the

bis-cis-Dihydrodiols
J . Org. Chem., Vol. 64, No. 11, 1999 4009
cis-dihydrodiol, and (iii) a bay region bis-cis-diol metabo-
lite of sufficient stability.
Benzo[c]phenanthridine 1B, having bay region bonds
in the naphthalene (3,4-bond) and isoquinoline (9,10-)
portions of it, was synthesized by the literature method21
and used as a substrate for the BPDO enzyme present
in S. yanoikuyae B8/36. Earlier studies of the dioxyge-
nase-catalyzed cis-dihydroxylation of naphthalene and
isoquinoline individually have shown that the former is
a much better substrate. Thus, it was anticipated that
cis-dihydroxylation would occur preferentially at the 3,4-
bond to yield mono-cis-dihydrodiol 2B, but surprisingly
the mono-cis-dihydrodiol 3B was also found in almost
equal proportions. The new cis-dihydrodiols 2B and 3B
were identified by spectral comparison with the mono-
cis-diol 2A derived from chrysene 1A. NOE interactions
between protons H-6 T H-7 (2%) in cis-diol 2B and H-6
T H-7 (3%) in cis-diol 3B were consistent with the
assigned structures. Formation of the MPBA derivatives
2BR, 2BS, 3BR, and 3BS from cis-diols 2B and 3B
indicated that both metabolites were enantiopure (>98%
ee). The chemical shift positions of the OMe signals
(Table 1) in each case allowed the absolute configurations
to be assigned as (+)-(3S,4R)-3,4-dihydroxy-3,4-dihy-
drobenzo[c]phenanthridine, 2B, and (+)-(9S,10R)-9,10-
dihydroxy-9,10-dihydrobenzo[c]phenanthrene, 3B. The
formation of single enantiomers (2B and 3B) by oxidation
of bay region bonds (3,4- and 9,10-) from the si:si face of
benzo[c]phenthridine 1B is in accord with similar results
obtained during the cis-dihydroxylation of chrysene 1A.
When small samples of enantiopure cis-diol metabolites
2B and 3B were in turn used as substrates for S.
yanoikuyae B8/36, HPLC analysis showed that each was
being biotransformed and several minor products ap-
peared to be formed. Due to the insufficient quantities
of metabolites, neither the anticipated bis-cis-dihydrodiol
4B nor any of the other metabolites could be identified.
It was assumed that the presence of an isoquinoline ring
in benzo[c]phenthridine 1B would make the putative bis-
cis-dihydrodiol 4B even less stable than the analogous
metabolite 4A obtained from chrysene. Thus, the pos-
sibility that bis-cis-dihydrodiol 4B was indeed formed as
an unstable metabolite, which decomposed to a range of
phenolic products, cannot at present be eliminated.
Benzo[b]naphtho[2,1-d]thiophene 1C is a tetracyclic
thiaarene containing two regions each of which has
geometry similar to a bay region. The term “pseudo-bay
region” has been adopted for benzo[b]naphtho[2,1-d]-
thiophene 1C as the terminology “bay region” has gener-
ally been applied to alternant members of the PAH series.
Earlier studies2 on the metabolism of benzo[b]naphtho-
[2,1-d]thiophene 1C, using rat liver enzymes, have
resulted in the detection of trans-dihydrodiols and phe-
nols due to initial monooxygenase-catalyzed epoxidation
at pseudo-bay region (1,2-, and 7,8-) and non-bay region
(3,4-, and 9,10-) bonds. Using the bacterium S. yanoikuyae
B8/36, the initial metabolic step appears to be cis-
dihydroxylation at the pseudo-bay region (1,2-bond) of
the naphthyl ring of thiaarene 1C rather than the
pseudo-bay region (7,8-bond) of the phenyl ring which
would yield the cis-dihydrodiol 3C (Scheme 1). The
structure of the mono-cis-dihydrodiol 2C was determined
F igu r e 2. X-ray crystal structure analysis of compound 7C
indicating the overall curvature of the molecule.
at the 1,2-bond was inferred from the NOE interactions
between H-4 and H-5 (5%). The enantiopurity was
determined to be >98% by formation of the corresponding
MPBA esters 2CR and 2CS. Applying the empirical
method earlier applied to the mono-cis-diols 2A, 2B, and
3B, based upon the chemical shift values of the OMe
1
signals in the H NMR spectra of MPBA esters 2CR and
2CS (Table 1), the absolute configuration (+)-(1R,2S) was
assigned to cis-diol 2C.
A second more polar metabolite from benzo[b]naphtho-
[2,1-d]thiophene 1C was identified from spectral data as
the bis-cis-diol 4C rather than the isomers 5C or 6C
(Scheme 1). Thus, coupling constants of 5.2 Hz (J 1,2) and
6.0 Hz (J 7,8) are consistent with the presence of cis-diol
groups at the 1,2- and 7,8-bonds. The formation of an
identical sample of the bis-cis-diol 4C when the (+)-
(1R,2S)-mono-cis-diol 2C (>98% ee) was used as sub-
strate for S. yanoikuyae B8/36 allowed the enantiopurity
of compound 4C to be established as >98% and the
absolute configuration at two of the four chiral centers
to be confirmed as (1R,2S). To establish that the relative
and absolute configuration of the bis-cis-diol metabolite
was consistent with structure 4C, and to obtain a more
stable derivative, the bis-acetonide 7C was prepared.
Fortunately the bis-acetonide 7C turned out to be a
crystalline compound, mp 136-137 °C, [RD] +466 (CHCl3).
The crystal structure analysis of compound 7C, employ-
ing anomalous dispersion methods, established the rela-
tive stereochemistry of the hydroxyl groups in metabolite
4C as cis-syn-cis and the absolute configurations at the
four chiral centers as (1R,2S,7R,8S), Figure 1. It also
confirmed that the cis-dihydroxylation steps had occurred
at the pseudo-bay region bonds (1,2- and 7,8-) and on the
same (si:si) face to yield a single enantiomer of tetraol
4C. X-ray crystallography indicated that rings A, B, and
C are essentially planar but ring D is puckered, with the
tetrahedral carbons C1 below (-0.22 Å) and C2 above
(+0.15 Å) the mean plane defined by the atoms of the
diene moiety C3, C4, C4a, C11b. This diene is itself
skewed, with torsion angle -7.6°. There is very little
deviation from planarity in ring A, with the tetrahedral
carbons C7 and C8 only +0.08 and -0.03 Å, respectively,
out of the mean plane defined by the planar diene C6b,
C10a, C10, C9 (Figure 2). At each ring fusion along the
A-D sequence there is a slight fold, 5.8°at junction A/B,
5.4° at B/C, and 2.8° at C/D (where the D plane is defined
by atoms C1, C11b, C4a, C4 only). This produces an
overall curvature, shown in Figure 2. The differences in
the conformations of rings A and D may result from the
differing geometries at the adjacent pseudo-bay regions.
cis-Tetrahydrodiol 4C is thus the second identified
member of this new class of bacterial metabolites from
the corresponding polycyclic arenes.
1
by H NMR analysis. The coupling constant (J 1,2 ) 5.1
Hz) was indicative of a cis-diol moiety, and its location
(21) Hall, G. E.; Walker, J . Chem. Soc. 1968, 2237.

4010 J . Org. Chem., Vol. 64, No. 11, 1999
Boyd et al.
1
analyzed by H NMR spectroscopy (CDCl3, see Table 1). The
diagnostic OMe signals of the boronate esters formed with (-)-
(S)-MPBA, 2AS (δH 3.11), and (+)-(R)-MPBA, 2AR (δH 2.97),
suggested an (R)-configuration for the benzylic C-4 position.
Thus, the cis-dihydrodiol metabolite 2A was predicted to have
the (3S,4R) absolute configuration. Since only one OMe signal
was observed in the 1H NMR spectrum of either the (R)- (2AR)
or the (S)-MPBA ester (2AS), the cis-dihydrodiol metabolite
2A was assumed to be enantiopure.
The isolation of the phenolic acetonide 8C as a byprod-
uct from the reaction mixture of the bis-acetonide 7C
demonstrates the difference in stability between the two
cis-dihydrodiol moieties. The cis-dihydrodiol formed in
the benzothiophene ring appears to be more susceptible
to acid-catalyzed dehydration, thus forming the phenolic
cis-diol 9C and its acetonide derivative 8C. The chemoen-
zymatic formation of the trihydroxylated compound 9C
suggests that similar phenolic cis-dihydrodiol metabolites
could also be isolated directly from the bacterial metabo-
lism of other polycyclic arenes containing two bay or
pseudo-bay regions. This type of metabolite would be
analogous to the reported examples of phenolic trans-
dihydrodiols formed during biotransformation of PAHs
using monooxygenase enzymes.8 The isolation of com-
pounds 4A and 4C provides the first unequivocal evi-
dence that procaryotic (dioxygenase-catalyzed) metabo-
lism of PAHs and HPAHs occurs by multiple site oxidation
of different rings to yield polyoxygenated metabolites if
two bay regions or pseudo-bay regions are present in the
substrate.
The more polar metabolite (Rf 0.15) was isolated as a
semisolid and was characterized as the bis-cis-diol 4A (0.002
g, 0.3% yield): [R]D +135 (c ) 0.4, MeOH); 1H NMR (500 MHz,
(CD3)2CO) δ 3.62 (2H, bs, OH), 4.02 (2H, bs, OH), 4.62 (2H,
ddd, J 1,3 ) J 7,9 ) 2.8, J 2,3 ) J 8,9 ) 1.6, J 3,4 ) J 9,10 ) 5.2, 3-H
and 9-H), 5.24 (2H, d, J 3,4 ) J 9,10 ) 5.2, 4-H and 10-H), 5.97
(2H, dd, J 2,1 ) J 7,8 ) 9.6, J 2,3 ) J 8,9 ) 1.6, 2-H and 8-H), 6.53
(2H, dd, J 1,2 ) J 7,8 ) 9.6, J 1,3 ) J 7,9 ) 2.8, 1-H and 7-H), 7.42
(2H, d, J 6,5 ) J 11,12 ) 8.6, 6-H and 12-H), 8.18 (2H, d, J 5,6
)
J 11,12 ) 8.6, 5-H and 11-H); 13C NMR (125 MHz, CD3COCD3)
δ 65.88, 70.91, 124.69, 126.44, 126.76, 130.04, 131.00, 132.33,
and 133.26; m/z 295(M - H)- (16%); CD 368.1 nm ∆ꢀ +0.416,
348.9 nm ∆ꢀ +0.481, 272.60 nm ∆ꢀ +4.24, 262.9 nm ∆ꢀ +1.91,
243.80 nm ∆ꢀ -0.775, 211.4 ∆ꢀ -3.603.
Bip h en yl Dioxygen a se-Ca ta lyzed cis-Dih yd r oxyla tion
of Ben zo[c]p h en a n th r id in e 1B To Yield (+)-(3S,4R)-3,4-
Dih yd r oxy-3,4-d ih yd r oben zo[c]p h en a n th r id in e, 2B, a n d
(+)-(9S,10R)-9,10-Dih ydr oxy-9,10-dih ydr oben zo[c]ph en an -
th r en e, 3B. A biotransformation of benzo[c]phenanthridine
1B (0.40 g, 1.75 mmol, 18 h) was carried out using S.
yanoikuyae strain B8/36 in the manner described for chrysene
1A.9 The ethyl acetate extract was dried (Na2SO4), and the
residue obtained after removal of solvent was separated by
PLC on silica gel using CHCl3:MeOH (90:10). A small propor-
tion of the substrate 1B (0.10 g) was recovered from the
biomass. The more polar metabolite proved to be (+)-(3S,4R)-
3,4-d ih yd r oxy-3,4-d ih yd r oben zo[c]p h en a n th r id in e, 2B
(0.036 g, 8%; Rf 0.23): mp 63-64 °C (from CHCl3/MeOH); [R]D
+114 (c ) 0.2, CHCl3); 1H NMR (500 MHz, CDCl3) δ 4.49 (1H,
dd, J 3,2 ) 5.4, J 3,4 ) 5.0 3-H), 5.64 (1H, d, J 3,4 ) 5.0, 4-H), 6.37
(1H, dd, J 2,1 ) 9.5, J 2,3 ) 5.4, 2-H), 6.77 (1H, d, J 1,2 ) 9.4,
1-H), 7.50 (1H, d, J 12,11 ) 8.3, 12-H), 7.74 (1H, dd, J 8,7 ) 7.8,
J 8,9 ) 7.0, 8-H), 7.90 (1H, dd, J 9,8 ) 7.0, J 9,10 ) 8.2, 9-H), 8.07
(1H, d, J 7,8 ) 7.9, 7-H), 8.49 (1H, d, J 11,12 ) 8.3, 11-H), 8.59
(1H, d, J 10,9 ) 8.2, 10-H), 9.20 (1H, s, 6-H); 13C NMR (125 MHz,
CD3COCD3) 65.60, 70.54, 121.89, 122.11, 124.10, 125.57,
126.83, 127.85, 128.81, 129.19, 129.89, 130.78, 131.78, 132.59,
132.91, 143.26, 151.53; m/z 263 (35), 245 (40), 256 (100) (found
M+ 263.094901, C10H12O2 requires 263.094621); CD 317.43 nm
∆ꢀ +1.844, 285.60 nm ∆ꢀ -2.21, 264.60 nm ∆ꢀ +1.965, 234.80
nm ∆ꢀ -0.681, 226.20 nm ∆ꢀ +4.027, 207.60 nm ∆ꢀ -3.619.
Con clu sion
Evidence is presented for the formation of a new class
of metabolites (bis-cis-dihydrodiols) during the bacterial
biodegradation of carcinogenic PAHs. mono-cis-Dihy-
drodiols (from chrysene, benzo[c]phenanthridine, and
benzo[b]naphtho[2,1-d]thiophene), and bis-cis-dihydrodi-
ols from chrysene and benzo[b]naphtho[2,1-d]thiophene),
have been isolated and stereochemically assigned. The
formation of bis-cis-dihydrodiols as procaryotic metabo-
lites from the exclusively cis-tetrahydroxylation of chry-
sene and benzo[b]naphtho[2,1-d]thiophene) is comple-
mentary to earlier reports of bis-trans-dihydrodiol for-
mation as a result of multiple site oxidation of PAHs by
eucaryotic systems. It is probable that a much larger
number of bis-cis-dihydrodiol metabolites have previously
gone unreported during bacterial metabolism of PAHs
due to increased instability and water solubility. Recent
investigations from these laboratories have already
identified two further members of this new family of bis-
cis-dihydrodiol metabolites but from the tricyclic azaare-
ne series (acridine and phenazine). The structure and
reactivity of these bis-cis-dihydrodiol metabolites of acri-
dine and phenazine will be reported elsewhere.
The less polar metabolite (+)-(9S,10R)-9,10-d ih yd r oxy-
9,10-d ih yd r oben zo[c]p h en a n th r id in e, 3B (0.030 g, 7%; Rf
Exp er im en ta l Section
0.36): mp 83-85 °C (from CHCl3/MeOH); [R]D +94 (c ) 0.2,
1
CHCl3); H NMR (500 MHz, CDCl3) δ 4.77 (1H, ddd, J 9,10
)
Bip h en yl Dioxygen a se-Ca ta lyzed cis-Dih yd r oxyla tion
of Ch r ysen e 1A To Yield (+)-(3S,4R)-3,4-Dih yd r oxy-3,4-
d ih yd r och r ysen e 2A a n d (+)-(3S,4R,9S,10R)-3,4,9,10-Tet-
r a h yd r oxy-3,4,9,10-tetr a h yd r och r ysen e 4A. A biotrans-
formation of chrysene 1A (0.5 g) was carried out using S.
yanoikuyae strain B8/36 in the manner described previously.9
The reaction mixture was incubated in the dark at 30 °C with
shaking (220 rpm) for 18 h. The cells were subsequently
removed by centrifugation, and the supernatant was extracted
with ethyl acetate after saturation with sodium chloride. The
dried ethyl acetate extract was concentrated and the residue
subjected to PLC on silica gel (CHCl3:MeOH 9:1), to yield two
compounds. The less polar (Rf 0.6) compound was identified
as cis-dihydrodiol metabolite 2A (0.005 g, ca. 1% yield): mp
241-243 °C (dec) as colorless needles (CHCl3-MeOH), [R]D
+112. (c ) 0.5, THF; lit.9 [R]D +112). The enantiopurity of the
dihydrodiol metabolite 2A (0.0005 g) was established by
treatment with (-)-(S)- and (+)-(R)-2-(1-methoxyethyl)phenyl
boronic acid (0.00034 g) separately in chloroform solution. The
boronates 2AS and 2AR were dried (Na2SO4), concentrated, and
5.2, J 9,8 ) 1.5, J 9,7 ) 2.6, 9-H), 5.38 (1H, d, J 10,9 ) 5.2, 10-H),
6.12 (1H, dd, J 8,7 ) 9.6, J 8,9 ) 1.5, 8-H), 6.68 (1H, dd, J 7,8
)
9.7, J 7,9 ) 2.6, 7-H), 7.72 (2H, m, 2-H and 3-H), 7.87 (1H, d,
J 12,11 ) 9.2, 12-H), 7.89 (1H, d, J 1,2 ) 9.1, 1-H), 8.03 (1H, d,
J 11,12 ) 9.2, 11-H), 8.77 (1H, s, 6-H), 9.22 (1H, d, J 4,3 ) 7.1,
4-H); 13C NMR (125 MHz, CD3COCD3) δ 64.90, 70.18, 120.14,
124.16, 124.19, 124.65, 125.03, 127.42, 127.81, 128.45, 129.07,
131.69, 132.75, 133.19, 137.24, 146.59, 147.34; m/z 263 (40),
245 (100) (found M+ 263.094743, C10H12O2 requires 263.094621);
CD 369.80 nm ∆ꢀ +0.657, 324.80 nm ∆ꢀ -1.410, 281.00 nm
∆ꢀ +8.593, 232.80 nm ∆ꢀ -5.197, 213.80 nm ∆ꢀ -9.347.
Bip h en yl Dioxygen a se-Ca ta lyzed cis-Dih yd r oxyl-
a tion of Ben zo[b]n a p h th o[2,1-d ]th iop h en e 1C To Yield
(+)-(1R,2S)-1,2-Dih yd r oxy-1,2-d ih yd r oben zo[b]n a p h th o-
[2,1-d ]th iop h en e 2C a n d (+)-(1R,2S,7R,8S)-1,2,7,8-Tetr a -
h yd r oxy-1,2,7,8-tetr a h yd r oben zo[b]n a p h th o[2,1-d ]th io-
p h en e 4C.
A biotransformation of benzo[b]naphtho[2,1-
d]thiophene 1C (0.20 g, 0.85 mmol, 18 h) was carried out using
S. yanoikuyae strain B8/36 in the manner described for

bis-cis-Dihydrodiols
J . Org. Chem., Vol. 64, No. 11, 1999 4011
chrysene 1A.9 The ethyl acetate extract was dried, and the
residue obtained after removal of solvent was purified by PLC
on silica gel using CHCl3:MeOH (90:10) to yield the less polar
metabolite (+)-(1R,2S)-1,2-d ih yd r oxy-1,2-d ih yd r oben zo[b]-
n a p h th o[2,1-d ]th iop h en e 2C (0.016 g, 7%; Rf 0.32): mp
103-105 °C (dec, from CHCl3) [R]D +146 (c ) 0.7, MeOH); 1H
NMR (500 MHz, (CD3)2CO) δ 4.18 (1H, d, J 2,OH ) 5.6, OH),
Da ta Collection . Diffraction data were collected on a
Siemens P3 four-circle diffractometer using graphite-mono-
chromated Cu KR radiation (λ ) 1.54184 Å). Unit cell
parameters were determined by least-squares refinement on
the setting angles for 10 automatically centered reflections in
the range 8 < θ < 20°. Data were collected using the ω-scan
mode, ω scan range 2°, for a unique monoclinic set (+h, +k,
(l) and for their Friedel opposites, for 3 < θ < 55°. The
intensity of one control reflection, measured after every 100
reflections, showed a deterioration to 57% of its original value,
and the measured intensities were rescaled accordingly. A total
of 2844 reflections were measured, of which 2426 were
independent. Friedel pairs were not averaged. Lorentz and
polarization factors were applied.
An a lysis a n d Refin em en t. The structure was determined
by direct methods and refined by full-matrix least-squares on
F2. Except for some methyl hydrogens, all hydrogen atoms
were located in a difference Fourier synthesis, but in the final
refinement all hydrogens were included at positions calculated
from the geometry of the molecule and riding on the carbon
atoms to which they were attached, with isotropic displace-
ment parameters kept at 120% of the equivalent isotropic
displacement parameters of the parent carbons for tertiary and
aromatic C-H and at 150% for methyl C-H atoms. Methyl
hydrogen atoms were placed using a rotating group refinement
for the methyl group, and it was confirmed that the resulting
positions agreed with those originally located in the difference
Fourier. Final R1 ) 0.056 for 1953 reflections with I > 2σ(I),
wR2 (all data) ) 0.134. Goodness of fit ) 1.04. Maximum/
minimum residual electron density was +0.17/-0.16 e Å-3. The
Flack absolute structure parameter x ) 0.02(4) established the
configuration of compound 7C as (1R,2S,7R,8S). [Refinement
of the inverse structure gave x ) 0.97(4).] (See Supporting
Information.) All crystallographic computations were carried
out using the SHELX-97 suite of programs.22
4.33 (1H, d, J 1,OH ) 6.8, OH), 4.46 (1H, dd, J 2,3 ) 3.8, J 1,2
)
5.1, 2-H), 4.93 (1H,d, J 1,2 ) 5.1, 1-H), 6.09 (1H, dd, J 3,4 ) 9.7,
J 3,2 ) 3.8, 3-H), 6.64 (1H, dd, J 4,3 ) 9.7, J 4,2 ) 1.3, 4-H), 7.31
(1H, d, J 5,6 ) 7.9, 5-H), 7.47 (2H, m, 8-H and 9-H), 7.94 (1H,
m, 7-H), 8.19 (1H, d, J 6,5 ) 7.9, 6-H), 8.26 (1H, m, 10-H); 13C
NMR (125 MHz, (CD3)2CO) 67.79, 70.46, 121.41, 121.87,
122.95, 124.49 126.30, 127.25, 128.16, 130.46, 130.98, 131.49,
135.55, 136.27, 139.28, 140.89; m/z 268 (57), 250 (100), 222
(82), 196 (20), 149 (25), 111 (34) (found M+ 268.056348, C16H12
-
SO2 requires 268.055802); CD 308.8 nm ∆ꢀ +2.506, 285.1 nm
∆ꢀ +2.429, 234.1 nm ∆ꢀ +1.022, 216.9 nm ∆ꢀ -2.514, 200.9
nm ∆ꢀ +4.334.
The more polar bioproduct was identified as (+)-
(1R,2S,7R,8S)-1,2,7,8-tetr ah ydr oxy-1,2,7,8-tetr ah ydr oben zo
[b]n a p h th o[2,1-d ]th iop h en e 4C. A solid (6 mg, 3%), Rf 0.18
(10% MeOH/CHCl3): mp 65-66 °C (dec, from CHCl3-MeOH);
[R]D +270 (c ) 0.3 in MeOH); νmax/cm-1 3432.9 (4 × OH); δH
(500 MHz, (CD3)2CO) 4.26 (1H, ddd, J 2,1 ) 5.2, J 2,3 ) 4.3, J 2,4
) 1.1, 2-H), 4.45 (1H, ddd, J 8,7 ) 5.8, J 8,9 ) 3.4, J 8,10 ) 2.7,
8-H), 4.75 (1H, d, J 1,2 ) 5.2, 1-H), 4.78 (1H, dd, J 7,8 ) 6.0, J 7,9
) 0.9, 7-H), 5.87 (1H, dd J 2,3 ) 4.1, J 3,4 ) 9.7, 3-H), 5.93 (1H,
ddd, J 9,10 ) 9.8, J 9,8 ) 3.4, J 9,7 ) 0.9, 9-H), 6.41 (1H, dd, J 10,9
) 9.8, J 10,8 ) 2.7, 10- H), 6.47 (1H, dd, J 4,3 ) 9.7, J 4,2 ) 1.0,
4-H), 7.07 (1H, d, J 5,6 ) 8.1, 5-H), 7.65 (1H, d, J 6,5 ) 8.0, 6-H);
13C NMR (125 MHz, CD3OCD3) δ 69.92, 67.25, 70.42, 70.52,
119.88, 120.89, 124.40, 1218.33, 128.74, 129.04, 130.09, 130.77,
133.67, 136.98, 138.78, 139.41; m/z ) 266 (100), 237 (27), 208
(12), 183 (22), 138 (54); CD 276.9 nm ∆ꢀ 1.114, 260.5 nm ∆ꢀ
-0.7359, 232.5 nm ∆ꢀ 0.1888, 210.9 ∆ꢀ -0.7390.
Ack n ow led gm en t. We thank the BBSRC (N.D.S.),
the Queen’s University of Belfast (M.K., F.H.), and the
QUESTOR Centre (C.C.R.A.) for funding the program
at the Queen’s University of Belfast, U.K. We also wish
to thank the U.S. Public Health Service (Grant GM29909
to D.T.G. and a predoctoral fellowship Training Grant
T32GM8365 from the National Institute of General
Medical Sciences to S.M.R.) for financial support which
was used at the Iowa center for Biocatalysis and
Bioprocessing (University of Iowa).
3,4:9,10-bis-Aceton id e of (1R,2S,7R,8S)-1,2,7,8-Tetr a h y-
dr oxy-1,2,7,8-tetr ah ydr oben zo [b]n aph th o[2,1-d]th ioph en e
7C. A catalytic quantity of p-toluenesulfonic acid was added
to a suspension of the bis-cis-diol 4C (0.008 g) in 2,2-
dimethoxypropane (0.4 mL), and the reaction mixture was
stirred for 2 h at room temperature. After removal of the
solvent from the reaction mixture, two compounds were
isolated by PLC on silica gel (diethyl ether:hexane 1:1). The
less polar (Rf 0.57) compound was identified as the bis-
acetonide 7C (0.005 g, 50%) as colorless crystals: mp 136-
137 °C (methanol); [R]D +466 (c ) 0.36, CHCl3) (found M+
382.123881, C22H22SO4 requires 382.123540).
The more polar compound (Rf 0.24) was found to be the
monoacetonide derivative of 1,2,8-trihydroxy-1,2-dihydrobenzo-
[b]naphtho[2,1-d]thiophene 8C (0.003 g, 35%): mp 168-70 °C
(dec, from CH2Cl2-hexane); [R]D +184 (c ) 0.25, CHCl3) (found
M+ 324.082016, C19H16SO3 requires 324.080887).
Su p p or tin g In for m a tion Ava ila ble: Additional experi-
1
mental details, H and 13C NMR spectral data for compounds
2A, 7C, and 8C, and crystallographic details for 7C (coordi-
nates, anisotropic displacement parameters, bond lengths
angles, and torsion angles). This material is available free of
charge via the Internet at http://pubs.acs.org.
X-r a y Cr ysta l Str u ctu r e An a lysis of th e (+)-bis-Ac-
eton id e 7C. Cr ysta l Da ta : C22H22O4S; M ) 382.46, mono-
clinic, a ) 9.524(7), b ) 7.379(5), and c ) 14.253(14) Å, â )
104.73(7)o, V ) 968.7(14) Å3, space group P21 (No. 4), Z ) 2,
Dx ) 1.311 Mg/m3, µ(Cu KR) ) 1.69 mm-1, F(000) ) 404.
J O982517N
(22) Sheldrick, G. M. SHELX-97, University of Go¨ttingen, Germany,
1997.