Regio- and stereoselective oxidation of unactivated C-H bonds with Rhodococcus rhodochrous

Article abstract of DOI:10.3762/bjoc.8.56

The ability of Rhodococcus rhodochrous (NCIMB 9703) to catalyse the regio- and stereoselective hydroxylation of a range of benzyloxy-substituted heterocycles has been investigated. Incubation of 2-benzyloxytetrahydropyrans with resting cell suspensions of the organism yielded predominantly a mixture of 5-hydroxylated isomers in combined yields of up to 40%. Exposure of the corresponding 2-benzyloxytetrahydrofuran derivatives to the cell suspensions gave predominantly the 4-hydroxylated isomers in yields of up to 26%. Most interestingly, 2-(4-nitrobenzyloxy)tetrahydrofuran and 2-(4-nitrobenzyloxy) tetrahydropyran were transformed in high yields to the 4-hydroxylated and 5-hydroxylated products, respectively.

Full text of DOI:10.3762/bjoc.8.56

Regio- and stereoselective oxidation of unactivated
C–H bonds with Rhodococcus rhodochrous
Elaine O’Reilly1, Suzanne J. Aitken1, Gideon Grogan2, Paul P. Kelly1,
Nicholas J. Turner1 and Sabine L. Flitsch*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 496–500.
1School of Chemistry, Manchester Interdisciplinary Biocentre, The
University of Manchester, 131 Princess Street, Manchester, M1 7ND,
UK, Fax: +44 (0)161 2751311, Tel: +44 (0)161 3065172 and
2Department of Chemistry, University of York, Heslington, York, YO10
5DD, UK, Tel: +44 (0)1904 328256
doi:10.3762/bjoc.8.56
Received: 06 October 2011
Accepted: 22 March 2012
Published: 03 April 2012
Email:
Associate Editor: D. Trauner
Sabine L. Flitsch* - sabine.flitsch@manchester.ac.uk
© 2012 O’Reilly et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
biocatalysis; cytochrome P450; hydroxylation; Rhodococcus
rhodochrous; selective C–H activation
Abstract
The ability of Rhodococcus rhodochrous (NCIMB 9703) to catalyse the regio- and stereoselective hydroxylation of a range of
benzyloxy-substituted heterocycles has been investigated. Incubation of 2-benzyloxytetrahydropyrans with resting cell suspensions
of the organism yielded predominantly a mixture of 5-hydroxylated isomers in combined yields of up to 40%. Exposure of the
corresponding 2-benzyloxytetrahydrofuran derivatives to the cell suspensions gave predominantly the 4-hydroxylated isomers in
yields of up to 26%. Most interestingly, 2-(4-nitrobenzyloxy)tetrahydrofuran and 2-(4-nitrobenzyloxy)tetrahydropyran were trans-
formed in high yields to the 4-hydroxylated and 5-hydroxylated products, respectively.
Introduction
Selective C–H activation remains a challenge for synthetic Many hydroxylating enzymes have been identified within
chemists, who often rely on differences in the steric and elec- bacteria belonging to the genus Rhodococcus. These organisms
tronic properties of bonds to achieve regioselectivity [1]. The are ubiquitous in the environment and their ability to activate
preparative-scale generation of hydroxylated intermediates can recalcitrant compounds and xenobiotics with an array of mono-
often provide synthetically useful derivatives as well as pharma- and dioxygenases has made them the focus of research into
ceutically important drug metabolites and lead compounds bioremediation and biocatalyst development [12]. The soil
[2-6]. Difficulties associated with chemical routes for the bacterium Rhodococcus rhodochrous (also referred to as
stereo- and regioselective preparation of such compounds has Corynebacterium sp. 7E1C, Rhodococcus rhodochrous ATCC
led to the exploitation of natural and recombinant whole-cell 19067 and Gordonia rubripertinctus 7E1C) is known to oxidise
biocatalysts as tools for their preparation [7-11].
a wide variety of aliphatic hydrocarbons, including n-alkanes
496

Beilstein J. Org. Chem. 2012, 8, 496–500.
and isoalkanes, and has been reported to catalyse the terminal panel of compounds and to investigate whether these hydroxy-
hydroxylation of chloroalkanes and long-chain alkenes [13]. It lations proceeded with regio-, diastereo- and enantioselectivity.
has been suggested that this strain contains an alkane-inducible We were also interested in establishing whether the oxidising
NADH-dependent hydroxylase that catalyses the oxidation of capabilities of this organism were the result of cytochrome
n-octane to 1-octanol [13,14]. In light of this, we have explored P450 activity.
the ability of this strain to perform regio- and stereoselective
Results and Discussion
oxidations of selected protected heterocyclic substrates.
Protected substrates are of particular interest, as subtle changes Transformations of THP ether derivatives
in the protecting group have been shown to affect the regiose- The results of the biohydroxylations of the p-substituted
lectivity of hydroxylation [10].
2-benzyloxypyrans 3a–d are summarised in Scheme 2.
An initial substrate screen with the organism revealed that The regiochemistry and relative stereochemistry of the biohy-
carbobenzyloxy-protected (Cbz) piperidine 1, which is known droxylated products were determined by analysis of 1H NMR
to be hydroxylated by various microorganisms [15], was coupling constants and by COSY and NOE/NOESY experi-
oxidised selectively in the 4-position, as indicated in Scheme 1 ments, which identified the 5-axial and 5-equatorial hydroxy-
[10].
lated 2-benzopyran isomers 4 and 5 as the major products.
We initially examined the regioselectivity of the biohydroxyla-
tion of tetrahydropyran (THP) derivative 3a and found that an
inseparable mixture of hydroxylated products was obtained.
Studies have shown that minor alterations in substrate structure
can strongly affect the selectivity of Beauveria bassiana ATCC
7159 [16,17]. Interestingly, the introduction of substituents on
the aryl ring of some substrates can assist in directing hydroxy-
lation away from the substituted position and can improve
regioselectivity. For example, varying the aryl ring substituents
on N-arylpiperidines can direct hydroxylation away from the
substituted position and improve the product yield [18].
Scheme 1: Regioselective hydroxylation of Cbz-piperidine by
Rhodococcus rhodochrous resting cells.
This encouraging result prompted us to challenge this organism Although we did not observe any aromatic hydroxylation of 3a
with a variety of novel heterocyclic substrates. Substrates based during our studies, we observed a significant improvement in
on oxygen heterocycles were thought to be particularly interest- regioselectivity with the THPs 3b–d, in which a substituent was
ing since, upon deprotection, they would yield polyfunction- present on the phenyl ring.
alised molecules that represent valuable chiral intermediates. A
number of p-substituted benzyloxytetrahydropyrans and furans Analysis of the products arising from the biotransformation
were synthesised and incubated with resting cell suspensions of with p-methyl 3b and the p-chloro 3c derivatives revealed that
R. rhodochrous. Our aim in this study was to establish whether the bacterium was capable of catalysing hydroxylation selec-
the organism is capable of catalysing the hydroxylation of this tively in the C5-position, providing the C5-axial isomers 4b and
Scheme 2: Results from the incubation of THP ether derivatives with Rhodococcus rhodochrous showing the isolated yields; *an inseparable mixture
of products was obtained.
497

Beilstein J. Org. Chem. 2012, 8, 496–500.
4c and C5-equitorial hydroxylated isomers 5b and 5c in signifi- dence confirmed that the ether in 3a–d sits in the axial position
cant yields. The p-nitro substituted THP 3d was also regioselec- due to the anomeric effect. In the case of substrate 6, the ether
tively hydroxylated at C5 and this oxidation was considerably sits in the sterically favoured equatorial position. In order to
quicker than that of 3b and 3c, resulting in an appreciably investigate this effect, substrate 8 (Scheme 3), which combines
higher yield of the C5-isomers 4d and 5d (Scheme 2). The the axial positioning with the longer ether chain, was synthe-
transformations were found to be highly reproducible with the sised and subjected to the resting cell suspensions. A complex
exception of the p-chloro substituted benzyloxypyran 3c, in mixture of inseparable products was obtained. This result
which both hydroxylated products were not consistently suggested that the length of the linker was not the most impor-
isolated. In each case, no starting material was recovered, which tant factor influencing the regioselectivity.
suggests further breakdown of the THPs by the organism,
although this was not investigated further.
Transformation of THF ether derivatives
The ability of the organism to catalyse the selective hydroxyla-
As reported previously with other hydroxylating systems, it is tion of the THP series 3b–d, prompted us to investigate the
often possible to modify the selectivity of microbial hydroxyla- biotransformations of the corresponding racemic tetrahydro-
tion by minor alterations of the substrate structure [16,17]. furan (THF) compounds 9a–d. The results, shown in Scheme 4,
Although exposure of 3a to the resting cell suspension resulted were largely analogous to those from the THP series. In the case
in a complex mixture of hydroxylation products, compound 6 of the THF ring systems, the site of hydroxylation was again
demonstrates that insertion of one methylene unit was suffi- determined by NMR, although the relative stereochemistry for
cient to improve the regioselectivity dramatically. THP 7 was all of the derivatives in this series has not yet been determined.
isolated in excellent yield (35%) and as the sole regioisomer In the cases of the substrates 9a and 9b, in which the aryl
(Scheme 3).
substituents are hydrogen and methyl, respectively, single prod-
ucts were isolated and identified as the C4 hydroxylated prod-
At this stage it remained unclear as to whether the gain in ucts 10a and 10b. In the case of the p-chloro substituted
regioselectivity observed with 6 was due to the increased length 2-benzyloxytetrahydrofuran 9c, 14% of mixed hydroxylated
of the ether linkage or due to its spatial orientation. NMR evi- furan derivatives were isolated along with 29% of p-chloroben-
Scheme 3: Influence of substrate structure and linker length on the regioselectivity of hydroxylation.
Scheme 4: Regioselective hydroxylation following incubation of THF ether derivatives with Rhodococcus rhodochrous; *see discussion.
498

Beilstein J. Org. Chem. 2012, 8, 496–500.
zoic acid. The isolation of the benzoic acid derivative suggests Nature of the hydroxylating enzyme
that benzylic hydroxylation takes place, leading to cleavage of One of the aims of this study was to investigate the nature of the
the unstable acetal [19,20]; however, this was not investigated hydroxylating enzyme in an attempt to establish whether the
further and the oxidation products were not characterised. The catalytic protein was a cytochrome P450. Inhibition studies with
most interesting transformation from this series was that of 1-aminobenzotriazole and metyrapone showed that hydroxyla-
2-(p-nitrobenzyloxy)tetrahydrofuran (9d), which was selec- tion was inhibited by both of these compounds. These
tively oxidised to provide product 10d in substantially higher cytochrome P450 inhibitors are known to block the activity of
yield (26%) than the other derivatives in the series.
the enzyme by different mechanisms [10,12] and are taken to be
evidence that a cytochrome P450 system operates in these trans-
Following the apparent increase in regioselectivity induced by formations.
the insertion of a methylene unit into THP 6, the organism was
challenged with the corresponding THF derivative. In this case, Conclusion
however, the transformation produced only a small amount of a Rhodococcus rhodochrous has been shown to mediate the
complex mixture of hydroxylated products, which was not puri- regio-, diastereo- and enantioselective hydroxylation of unacti-
fied.
vated C–H bonds on selected THF and THP derivatives. We
have demonstrated that the introduction of substituents onto the
These transformations demonstrate a definite bias towards C5 aryl ring of the THP ethers can dramatically affect the regiose-
hydroxylation on the THP series and a preference for C4 lectivity of hydroxylation. Most notably, exposure of the p-nitro
hydroxylation on the THF series.
substituted THP ether 3d to resting cells provided the
C5-equatorial and C5-axial regioisomers in 18% and 21%,
respectively. Similarly, the organism catalysed the regioselec-
Enantioselectivity of hydroxylation
The product resulting from the transformation of the p-nitro tive hydroxylation of p-nitro substituted THF ether
substituted THF derivative 9d was chosen for some initial 10d providing the C4-hydroxylated product as the sole regio-
screens with the aim of investigating the enantioselectivity of isomer.
hydroxylation. The MTPA (Mosher) ester 12d was prepared on
reaction of alcohol 10d with (R)-methoxytrifluorophenylacetic Our investigations into the enantioselectivity of hydroxylation
acid in the presence of DCC and pyridine. Only one diastereo- revealed that the p-nitro THF derivative was hydroxylated
mer was clear in the 1H NMR, but examination of the 19F NMR selectively, providing the 2R,4R derivative exclusively. The
clearly showed two fluorine resonances from which an enan- organism catalysed the enantioselective hydroxylation of
tiomeric excess of 93% was calculated. The S-camphanic ester p-nitro THP 3d, providing cis-hydroxylated 5d as the sole dia-
11d was prepared in a 76% yield resulting from the reaction of stereomer. In contrast, our results indicate that the organism
the alcohol 10d with S-camphanic acid chloride in the presence catalyses the axial hydroxylation of both enantiomers of 3d,
of pyridine. 1H NMR analysis revealed that the resulting ester providing 4d as a mixture of enantiomers. As alcohol 4d
was a single diastereomer, and the absolute stereochemistry of showed a strong optical rotation value, this result requires
the THF portion was confirmed by X-ray crystallography and further investigation.
found to be 2R,4R.
Future efforts will focus on the identification, cloning and
The two products arising from the hydroxylation of THP heterologous expression of cytochrome P450 genes from
derivative 3d showed high optical rotation values, and their this organism to allow for more detailed investigations. It is
enantioselectivity was investigated further. Mosher’s esters 13d envisaged that these transformations could be used to
and 14d of THP alcohols 4d and 5d, respectively, were produce important hydroxylated, polyfunctionalised chiral inter-
prepared in good yield under the same conditions as those mediates.
employed for alcohol 10d. The 19F NMR of Mosher’s ester 14d
showed a single peak suggesting an enantiomeric excess of
Supporting Information
greater than 95%. It should be noted, however, that authentic
samples of the enantiomers are not available and it is possible
Supporting Information File 1
that the resonances may overlap. Examination of the Mosher’s
ester 13d revealed two resonances in the 19F NMR with approx-
imately equal peak area. These preliminary results suggest that
the organism catalyses the axial hydroxylation of both enan-
tiomers of 3d, providing 4d as a mixture of enantiomers.
Experimental procedures and characterisation data of
synthesised, previously unknown compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-56-S1.pdf]
499

Beilstein J. Org. Chem. 2012, 8, 496–500.
Acknowledgements
We are grateful to the BBSRC and FP7 for funding. SLF is the
License and Terms
recipient of a Wolfson Merit Award of the Royal Society.
This is an Open Access article under the terms of the
Creative Commons Attribution License
References
1. Chen, M. S.; White, M. C. Science 2007, 318, 783–787.
(http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
doi:10.1126/science.1148597
2. Weis, R.; Winkler, M.; Schittmayer, M.; Kambourakis, S.; Vink, M.;
Rozzell, J. D.; Glieder, A. Adv. Synth. Catal. 2009, 351, 2140–2146.
doi:10.1002/adsc.200900190
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
3. Schroer, K.; Kittelmann, M.; Lütz, S. Biotechnol. Bioeng. 2010, 106,
699–706. doi:10.1002/bit.22775
(http://www.beilstein-journals.org/bjoc)
4. O'Reilly, E.; Köhler, V.; Flitsch, S. L.; Turner, N. J. Chem. Commun.
2011, 47, 2490–2501. doi:10.1039/C0CC03165H
The definitive version of this article is the electronic one
which can be found at:
5. Robin, A.; Köhler, V.; Jones, A.; Ali, A.; Kelly, P. P.; O’Reilly, E.;
Turner, N. J.; Flitsch, S. L. Beilstein J. Org. Chem. 2011, 7,
1494–1498. doi:10.3762/bjoc.7.173
doi:10.3762/bjoc.8.56
6. Robin, A.; Roberts, G. A.; Kisch, J.; Sabbadin, F.; Grogan, G.;
Bruce, N.; Turner, N. J.; Flitsch, S. L. Chem. Commun. 2009,
2478–2480. doi:10.1039/b901716j
7. Peters, M. W.; Meinhold, P.; Glieder, A.; Arnold, F. H.
J. Am. Chem. Soc. 2003, 125, 13442–13450. doi:10.1021/ja0303790
8. Vail, R. B.; Homann, M. J.; Hanna, I.; Zaks, A.
J. Ind. Microbiol. Biotechnol. 2005, 32, 67–74.
doi:10.1007/s10295-004-0202-1
9. Çelik, A.; Flitsch, S. L.; Turner, N. J. Org. Biomol. Chem. 2005, 3,
2930–2934. doi:10.1039/b506159h
10.Flitsch, S. L.; Aitken, S. J.; Chow, C. S.-Y.; Grogan, G.; Staines, A.
Bioorg. Chem. 1999, 27, 81–90. doi:10.1006/bioo.1999.1135
11.Grogan, G.; Roberts, G. A.; Parsons, S.; Turner, N. J.; Flitsch, S. L.
Appl. Microbiol. Biotechnol. 2002, 59, 449–454.
doi:10.1007/s00253-002-1054-0
12.Larkin, M. J.; Kulakov, L. A.; Allen, C. C. R. Curr. Opin. Biotechnol.
2005, 16, 282–290. doi:10.1016/j.copbio.2005.04.007
13.Jurtshuk, P.; Cardini, G. E.; Gibson, D. T. Crit. Rev. Microbiol. 1971, 1,
239–289. doi:10.3109/10408417109104483
14.Cardini, G.; Jurtshuk, P. J. Biol. Chem. 1968, 243, 6070–6072.
15.Chang, D.; Feiten, H.-J.; Engesser, K.-H.; van Beilen, J. B.; Witholt, B.;
Li, Z. Org. Lett. 2002, 4, 1859–1862. doi:10.1021/ol025829s
16.Aitken, S. J.; Grogan, G.; Chow, C. S.-Y.; Turner, N. J.; Flitsch, S. L.
J. Chem. Soc., Perkin Trans. 1 1998, 3365–3370.
doi:10.1039/A805800H
17.Holland, H. L.; Morris, T. A.; Nava, P. J.; Zabic, M. Tetrahedron 1999,
55, 7441–7460. doi:10.1016/S0040-4020(99)00393-2
18.Floyd, N.; Munyeman, F.; Roberts, S. M.; Willetts, A. J.
J. Chem. Soc., Perkin Trans. 1 1993, 881–882.
doi:10.1039/P19930000881
19.Schwaneberg, U.; Schmidt-Dannert, C.; Schmitt, J.; Schmid, R. D.
Anal. Biochem. 1999, 269, 359–366. doi:10.1006/abio.1999.4047
20.Cirino, P. C.; Arnold, F. H. Angew. Chem., Int. Ed. 2003, 42,
3299–3301. doi:10.1002/anie.200351434
500