Toluene dioxygenase-mediated oxidation of aromatic substrates with remote chiral centers

Article abstract of DOI:10.1039/b002143l

Several aromatic substrates containing remote chiral centers were subjected to toluene and naphthalene dioxygenase expressed in blocked mutants and recombinant organisms yielding cis-diol metabolites with little or no kinetic resolution. Substrates were t

Full text of DOI:10.1039/b002143l

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Toluene dioxygenase-mediated oxidation of aromatic substrates
with remote chiral centers
Vu Bui,a Trond Vidar Hansen,a† Yngve Stenstrøm,a† Douglas W. Ribbonsb and
Tomas Hudlickya*
1
a University of Florida, Department of Chemistry, Gainesville, FL 32611-7200, USA.
E-mail: hudlicky@chem.ufl.edu
b SFB Biocatalysis at the Institute for Organic Chemistry, Technical University Graz,
Stremayrgasse 16, A-8010 Graz, Austria. E-mail: sekretariat@orgc.tu-graz.ac.at
Received (in Cambridge, UK) 16th March 2000, Accepted 17th April, 2000
Published on the Web 9th May 2000
Several aromatic substrates containing remote chiral
centers were subjected to toluene and naphthalene
dioxygenase expressed in blocked mutants and
recombinant organisms yielding cis-diol metabolites with
little or no kinetic resolution.
means of parallel experiments with both blocked mutants and
recombinant organisms.7 The results of these experiments are
shown in Table 1.
Our interest in these issues is related to ongoing activities
in the synthesis of morphine alkaloid.8 One such approach
already published9 employed a dienediol generated from
biphenyl. We reasoned that a suitably functionalized phenyl-
cyclohexanone or phenylcyclohexanol of type 18 or 19 would,
if successfully oxidized, furnish a “resolved” synthon in which
the biochemically set chirality would later be destroyed to pro-
vide the dibenzofuran core of the alkaloid (Scheme 2). Clearly,
the best models for such an undertaking are compounds 10
and 12 (Table 1). To provide a background against which any
potential kinetic resolution could be measured we repeated
the published experiments with 1-phenylethan-1-ol and also
compared several organisms used in the biooxidations.
The utility of cis-dihydroxydihydrobenzene metabolites of
aromatic hydrocarbons in asymmetric synthesis is firmly
established. Several recent reviews highlight the growing activ-
ity in this field1 while investigations continue to explore new
compounds as potential substrates for the enzyme(s).2 To date,
over three hundred homochiral diols have been identified from
the oxidations of simple, fused, and biphenyl-type aromatics.1
Reports of metabolites containing additional chiral centers
and arising from kinetic resolution of substrates of type 1
(Scheme 1) are rare. To our knowledge only five instances of
Repetition of Ribbons’ and Ahmed’s experiments with phen-
ethyl alcohol gave confirmation of their earlier observation
except in the case of fermentation with Escherichia coli JM109
(pDTG601A). While Pseudomonas putida F39/D organism
recognizes the R-enantiomer of the alcohol to a lesser extent,
the recombinant clone produced the diol metabolite without
any kinetic resolution. Although surprising, since the same
dioxygenase enzyme is expressed in both organisms, this result
may be rationalized by slight differences in the enzyme
topology resulting from post-translational folding. All of the
fermentations were carried out with freshly grown cells10 of
either organism. For all runs with P. putida F39/D the enzyme
expression was induced with toluene prior to substrate add-
ition, while in the fermentations with E. coli such expression
was induced with IPTG. Substrates were added as neat com-
pounds at a concentration of 0.05–0.1%. The disruption of the
aromatic ring was monitored by an increasing absorption
between 260 and 270 nm. In general, work-up was performed
by centrifuging the cells followed by extraction of the super-
natant with ethyl acetate, drying and evaporation. Products
were isolated by recrystallisation and/or flash chromatography
and were fully characterized by spectral and physical methods.
With the E. coli JM109 (pDTG601) both enantiomers of
racemic 1-phenylethan-1-ol (6a,b) were fully converted to
two compounds 4a and 4b in a 1:1 ratio assumed to be the
Scheme 1 Biooxidation of substituents containing benzylic chiral
centers.
biooxidation of such compounds have been reported. Gibson
et al. reported the production of diol 4 in conjunction with their
studies of di- vs. monohydroxylation.3 This has been further
elaborated by Cripps and co-workers.4 Several studies were
recently conducted by Boyd et al. on 1-phenylethan-1-ol and
other substrates.5 Ribbons and Ahmed6 studied the biooxid-
ation of phenyl ethanols (R-, S-, and racemate) with blocked
mutants and reported a slight preference for the S-enantiomer.
Racemic sec-butylbenzene (5) and racemic 1-phenylpropan-1-ol
were also oxidized but the stereochemical outcome was not
reported. With these limited examples we nevertheless investi-
igated several substrates with one or more remote centers by
1
diastereomeric mixture as revealed by both H and 13C NMR
spectroscopy. Analyses of aliquots from fermentations for the
non-converted 1-phenylethan-1-ol on a chiral support GLC did
not reveal any difference in the rate of conversion of the two
enantiomers. Moreover, in separate runs both the R- and the
S-enantiomers were oxidized at the same rate (UV-monitoring
at λ = 266 nm). In contrast, when P. putida F39/D was used the
S-enantiomer was oxidized at a faster rate than the R-antipode
according to UV-monitoring. When the racemic mixture was
used as substrate a 60:40 ratio of diastereomers resulted with
a preference for the dihydroxylation of the S-enantiomer as
evident by 1H NMR spectroscopy as well as analysis of the non
converted compound.
† On leave from Agricultural University of Norway, Department
of Chemistry and Biotechnology, N-1432 Ås, Norway. E-mail:
yngve.stenstrom@ikb.nlh.no
DOI: 10.1039/b002143l
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This journal is © The Royal Society of Chemistry 2000
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Table 1 Comparison of biooxidation of substrates with different organismsa
Entry
JM109 (pDTG601) TDO
JM109 (pDTG141) NDO
Pp F39/D
—
—
—
No conversion
No conversion
—
No conversion
No conversion
No conversion
No conversion
—
—
—
No conversion
—
No conversion
—
—
—
—
a A dash implies that the experiment has not been performed. Numbers in parentheses are ratios, numbers in brackets are yields (g LϪ1) and are not
optimised. For mixtures the numbers refer to the yield for both isomers. b [α]D28 = ϩ47.5 (c 0.05, MeOH). c [α]D30 = Ϫ73.8 (c 1.0, CH2Cl2).
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Scheme 2 Chemoenzymatic approach to morphine synthesis—introduction of catechol to ring A.
Scheme 3 Correlation of absolute stereochemistry of metabolites. Reagents and conditions: a) E. coli JM109 (pDTG602); b) PAD, MeOH, HOAc,
0 ЊC; c) DMP, TsOH, rt; d) Ph3P, CBr4, CH2Cl2, 0–5 ЊC; e) Bu3SnH, AlBN, THF, ∆; f) Mg (act.), ether; g) Li2CuCl4, Ϫ78 ЊC; h) sec-BuLi, THF,
Ϫ78 ЊC; then Me3SnCl, rt; i) (CH3CN)2PdCl2, DMF, rt; j) PCC, CH2Cl2, 0–5 ЊC; k) NaBH4, EtOH, rt.
To explore the potential of the kinetic resolution we tested
other substrates with an oxygen substituent in the benzylic
position. Because of the known overlap in recognition of
certain aromatics by both toluene dioxygenase (TDO) and
naphthalene dioxygenase (NDO), we performed the ferment-
ations of 1,2,3,4-tetrahydro-1-naphthol (7), benzhydryl alcohol
(8), and 1,4-dihydro-1,4-epoxynaphthalene (9) with both
enzymes. However, neither 7, 8 or 9 produced detectable
amounts of dihydroxylated metabolites with TDO or NDO (the
latter expressed by E. coli JM109 (pDTG141)). It is known that
indane, indan-1-ol, indan-2-ol, indan-1-one, and indan-2-one
are not dihydroxylated,11 but undergo benzylic hydroxylation
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instead. It may well be that this is a general trend for the ortho-
fused ring systems, at least with the use of organisms described
in this manuscript, and this may explain why there is no dihy-
droxylation for 1,2,3,4-tetrahydro-1-naphthol and 1,4-dihydro-
1,4-epoxynaphthalene.12 It is however known that P. putida
UV4 oxidizes a number of ortho-fused ring systems.13
Acknowledgements
Financial support from the National Science Foundation
(NSF) (CHE-9615112 and CHE-9910412), the U.S. Environ-
mental Protection Agency (EPA) (R826113) and TDC
Research, Inc., as well as from the Agricultural University of
Norway (T. V. H. and Y. S.) and The Norwegian Research
Council (T. V. H.) is gratefully appreciated.
The substrate of most interest to us is trans-2-phenylcyclo-
hexanol (10) which has two neighboring stereogenic centers and
which most closely resembles the intended morphine models 18
or 19. However, racemic 10 was converted to the corresponding
dienediol as a 1:1 mixture of diastereomers indicating no
preference of the enzyme for either enantiomer. Three other
substrates, 1-phenylcyclohexanone (12), 1-phenylcyclohexene
(14) and phenylcyclohexane (16) were converted to the corre-
sponding dienediols for the purpose of correlation of absolute
stereochemistry in 11a/11b. Even though the kinetic resolution
in 10 did not occur, the utility of this type of substrate in
approaches to morphine is still viable and compounds of this
structural type with additional chiral centers, such as 18 and 19,
will be investigated, if for no other reason, because of the facile
introduction of the catechol unit to the morphine nucleus.
The absolute stereochemistry of the vic-diols in 11a, 11b, and
13a, 13b, 15 and 17 were proven according to the reactions in
Scheme 3. The mixture of triols 11a and 11b was reduced to the
alkenes 23a,b with potassium azodicarboxylate (PAD). Protec-
tion of the vic-diol as the ketals, bromination with CBr4–Ph3P
and reduction of the bromides with Bu3SnH yielded one com-
pound which was identical to 24 as prepared from 17. Since 17
was obtained from the biooxidation of phenylcyclohexane
followed by PAD reduction and protection with 2,2-dimethoxy-
propane (DMP), the absolute stereochemistry of the diol in
both 17 and 11 is identical. It is also of the same absolute
configuration as the diol derived from bromobenzene. Coupling
of bromide 25 of known14 stereochemistry with cyclohexyl
triflate 26 was achieved via the transformation of the bromide
to the Grignard compound in the presence of Li2CuCl4.15 The
product was identical by all means, including optical rotation,
with the material prepared from 11a and 11b, the products of
the biotransformation of 10. The absolute stereochemistry of
15 was proven by a similar sequence using the bromide 25, but
coupling with cyclohexenyl triflate 28 according to the work of
Stille et al.16 giving diene 27, identical in all respects to the one
derived from 15, the biotransformation product of 14. Finally,
the absolute stereochemistry of 13 was proven by conversion
to 24.
References
1 (a) T. Hudlicky, D. Gonzalez and D. T. Gibson, Aldrichimica Acta,
1999, 31, 35; (b) D. R. Boyd and G. N. Sheldrake, Nat. Prod. Rep.,
1998, 15, 309; (c) T. Hudlicky and J. W. Reed, in Advances in
Asymmetric Synthesis, ed. A. Hassner, JAI Press, Greenwich, CT,
1995, vol. 1, p. 271.
2 (a) For listing of the metabolites see refs. 1a and 1b; for recent
reports of new metabolites see (b) T. Hudlicky and B. Novak,
Tetrahedron: Asymmetry, 1999, 10, 2067.
3 D. T. Gibson, B. Gschwendt, W. K. Yeh and V. M. Kobal,
Biochemistry, 1973, 8, 1520.
4 R. E. Cripps, P. W. Trudgill and G. Whateley, Eur. J. Biochem., 1978,
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5 D. R. Boyd, N. D. Sharma, N. I. Bowers, J. Duffy, J. S. Harrison and
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6 S. Ahmed, PhD Thesis, Imperial College of Science, Technology
and Medicine, London, 1991 (D. W. Ribbons, thesis supervisor).
7 The organisms in this study were supplied by David Gibson:
Pseudomonas putida F39/D see: (a) D. T. Gibson, J. R. Koch and
R. E. Kallio, Biochemistry, 1968, 7, 2653; (b) E. coli JM109 (pDTG
601) see G. J. Zylstra and D. T. Gibson, J. Biol. Chem., 1989, 264,
14940; (c) E. coli JM109 (pDTG 141) see M. J. Simon, T. D.
Osslund, R. Saunders, B. D. Ensley, S. Suggs, A. Harcourt, W. C.
Suen, D. L. Cruden and D. T. Gibson, Gene, 1993, 127, 31.
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Environ. Microbiol., 1992, 58, 3407; (b) S. M. Resnick, D. S. Torok,
K. Lee, J. M. Brand and D. T. Gibson, Appl. Environ. Microbiol.,
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It appears that the substrates chosen for this study were not
processed by the enzymes as separate enantiomeric entities.
While no significant kinetic resolution was observed the investi-
gation of compounds of type 18 and 19 remains of interest in
the production of synthons containing the ring A of morphine.
Because compounds of type 19 will be enantiomerically pure
it appears that the merit of their oxidation will be in the bio-
catalytic conversion of the phenyl ring directly to catechol when
the clone containing catechol diol dehydrogenase is used. We
will report on these studies as well as further details connected
to the work presented here in the near future.
12 A good model to test this assumption would be o-methylphenyl-
ethan-1-ol.
13 (a) D. R. Boyd, N. D. Sharma, T. A. Evans, M. Groocock, J. F.
Malone, P. J. Stevenson and H. Dalton, J. Chem. Soc., Perkin Trans.
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Goodrich, M. R. Groocock, A. J. Blacker, P. Goode and H. Dalton,
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14 D. R. Boyd, M. R. Hand, N. D. Sharma, J. Chima, H. Dalton and
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15 M. Tamura and J. Kochi, Synthesis, 1971, 303.
16 W. J. Scott, G. T. Crisp and J. K. Stille, Org. Synth., 1990, 68,
116.
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