P450-catalyzed regio- and stereoselective oxidative hydroxylation of disubstituted cyclohexanes: Creation of three centers of chirality in a single CH-activation event This paper is dedicated to the memory of Harry H. Wasserman

Article abstract of DOI:10.1016/j.tet.2014.11.067

Wild-type P450-BM3 is able to catalyze in a highly regio- and diastereoselective manner the oxidative hydroxylation of non-activated disubstituted cyclohexane derivatives lacking any functional groups, including cis- and trans-1,2-dimethylcyclohexane, cis- and trans-1,4-dimethylcyclohexane, and trans-1,4-methylisopropylcyclohexane. In all cases except chiral trans-1,2-dimethylcyclohexane as substrate, the single hydroxylation event at a methylene group induces desymmetrization with simultaneous creation of three centers of chirality. Certain mutants increase selectivity, setting the stage for future directed evolution work.

Full text of DOI:10.1016/j.tet.2014.11.067

Tetrahedron 71 (2015) 470e475
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
P450-catalyzed regio- and stereoselective oxidative hydroxylation
of disubstituted cyclohexanes: creation of three centers of chirality
in a single CH-activation event
,
b
a
,
,
,b,
Adriana Ilie a , Ruben Agudo b, Gheorghe-Doru Roiban a b, Manfred T. Reetz a
*
ꢀ
a
€
Department of Chemistry, Philipps-Universitat Marburg, Hans-Meerwein Str., 35032 Marburg, Germany
Max-Planck-Institut fur Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mulheim an der Ruhr, Germany
b
€
€
a r t i c l e i n f o
a b s t r a c t
Article history:
Wild-type P450-BM3 is able to catalyze in a highly regio- and diastereoselective manner the oxidative
hydroxylation of non-activated disubstituted cyclohexane derivatives lacking any functional groups,
including cis- and trans-1,2-dimethylcyclohexane, cis- and trans-1,4-dimethylcyclohexane, and trans-1,4-
methylisopropylcyclohexane. In all cases except chiral trans-1,2-dimethylcyclohexane as substrate, the
single hydroxylation event at a methylene group induces desymmetrization with simultaneous creation
of three centers of chirality. Certain mutants increase selectivity, setting the stage for future directed
evolution work.
Received 7 September 2014
Received in revised form 25 November 2014
Accepted 26 November 2014
Available online 2 December 2014
This paper is dedicated to the memory of
Harry H. Wasserman
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Regioselectivity
CH-activation
P450
Monooxygenase
Diastereoselectivity
1. Introduction
cytochrome P450 monooxygenases,6 and indeed a number of in-
dustrial processes utilizing natural forms (wild-type, WT) are
Enzymes are being increasingly used as catalysts in the pro-
duction of fine chemicals, stereoselectivity often being the focus of
interest.1 Of particular importance are those transformations, which
are difficult or impossible using state of the art synthetic transition
metal catalysts ororganocatalysts. The toolboxof organic chemists is
greatly enriched by the development of complementary methods.1,2
Until recently the traditional limitations of enzymes as catalysts in
synthetic organic chemistry and biotechnology have prevented
broad application, but fortunately the advent of directed evolution3
as a viable protein engineering technique has changed the situation
so that even retrosynthetic approaches based on enzymes are now
emerging.4 One of the current challenges in synthetic organic
methodology development is regio- and stereoselective CH-
activating oxidative hydroxylation of simple and complex organic
compoundsRH/ROH. Significantprogress usingsyntheticcatalysts
or reagents has been achieved in this exciting area,5 but a number of
problems remain unsolved. A complementary approach is to use
known.7
Whenever WT P450 monooxygenases fail to be regio- and ster-
eoselective in a transformation of synthetic interest, which is re-
quired for applications, two strategies are possible to solve this
problem: (1) Test mutants generated previously for other substrates
and hope that a good catalyst will be found.8 (2) Apply directed
evolution,3 which has a higher probability of success.9 Directed
evolution has been applied to P450 enzymes for more than decade,
but the control of both regio- and stereoselectivity remained elusive for
a long time. In early work, Arnold and co-workers reported the ap-
plication of directed evolution to the P450-catalyzed oxidative hy-
droxylation of linear alkanes such as octane or nonane, which
resulted in some improvement of regioselectivity, but poor to
moderate enantioselectivity.9c Later Zhao et al. utilized saturation
mutagenesis in order to improve and invert the enantioselectivity of
P450(pyr) as a catalyst in the hydroxylation of N-benzylpyrrolidine
at the 2-position, wild-type (WT) being slightly (S)-selective (43%
ee) and the best mutants leading to 65% ee (S) and 83% ee (R).9d Later
Li et al. performed directed evolution in order to boost (S)-selectivity
to 98% ee, but enhanced (R)-selectivity was not reported.9f Recently
we reported laboratory evolution of a P450 monooxygenase
* Corresponding author. Tel.: þ49 (0)6421 28 25500; fax: þ49 (0)6421 28 25620;
e-mail address: reetz@mpi-muelheim.mpg.de (M.T. Reetz).
http://dx.doi.org/10.1016/j.tet.2014.11.067
0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.

A. Ilie et al. / Tetrahedron 71 (2015) 470e475
471
enabling both pronounced regio- and enantioselectivity in oxidative
hydroxylation, both (R)- and (S)-selective mutants being evolved.10
Using cyclohexene carboxylic acid methyl ester as substrate, highly
(R)- and (S)- selective (>95% ee) mutants of P450-BM311 were
evolved using iterative saturation mutagenesis (ISM),2,12 essentially
complete regioselectivity in favor of the 3-hydroxy products being
observed.10 In another recent study, >95% regio- and >95% dia-
stereoselectivity in the oxidative hydroxylation of steroids was en-
such as menthol or stereoisomers thereof. The present study con-
stitutes an exploratory investigation of testing P450-BM3 as a cat-
alyst in the oxidation of substrates 3e5.
sured by 2b- and 15b-selective mutants generated by directed
evolution on the basis of ISM.13 A number of other recent studies
likewise addressed the challenge of regio- and stereoselectivity,
rational design or directed evolution being used to generate ap-
propriate mutants.9 In many cases only one of the two possible
enantiomers was accessed by protein engineering. Complete control
of regio- and stereoselectivity as well as access to both stereoiso-
mers on an optional basis remains a challenge for many types of
substrates, as in the case of such simple molecules as 1-
methylcyclohexene.9l
P450 monoxygenases are Fe-heme dependent enzymes, the
catalytically active high-spin intermediate [heme-Fe]O] (Com-
pound I) inducing H-atom abstraction of the substrate RH with
formation of the radical Rꢀ followed by rapid CeO bond formation
and release of the product ROH.6,14 This means that high regio- and
stereoselectivity requires the compound to be held in a single
specific pose with the respective CeH moiety pointing to the cat-
alytically active species at an optimal angle of 130ꢁ.14b,c Substrates
having functional groups may undergo H-bonding as one of the
determining factors in positioning the compound in the large
binding pockets of CYPs, which means that selective hydroxylation
of substrates lacking such anchor points can be expected to be
difficult, 1-methylcyclohexene being an example.9l
2. Results and discussion
In initial experiments, WT P450-BM3 was used as the biocatalyst
in the oxidation of substrates 3 and 4, which can lead to different
regio-, distereo-, and enantiomers as indicated in Scheme 1.
Hydroxylation at the methylene groups gives rise to molecules,
which have three chiral centers. This ensures greater complexity
and therefore added value to the products, provided some degree of
stereoselectivity is achieved. This would be a highly desirable fea-
ture, but it also makes unambiguous stereochemical assignments
more difficult.
In order to address these challenges, directed evolution has been
applied to small molecules, which are devoid of any functional
groups (not even olefinic bonds), the hydroxylation of achiral
methylcyclohexane 1 being a rare successful example.15 Two dif-
ferent P450-BM3 mutants were evolved utilizing ISM, which led to
regioselective hydroxylation with favored formation of cis-(1S,2R)-
2a (71% regioselectivity; 94% diastereoselectivity; 92% enantiose-
lectivity), and 2b (71% regioselectivity), respectively.15 In the case of
desymmetrization 1/cis-(1S,2R)-2a, two chiral centers are created
in a single activation step, a phenomenon that is of considerable
synthetic value.
Scheme 1. Possible oxidation products by WT P450-BM3 catalyzed oxidation of
substrates 3 and 4.
By GCeMS analyses, we have identified all products arising from
the P450-BM3 catalyzed oxidation of 1,2-dimethylcyclohexane (3)
and 1,4-dimethylcyclohexane (4) (Schemes 2 and 3) Subsequently,
the products were fully characterized by comparison with au-
thentic commercially available or separately prepared samples.
Initially, we used mixtures of cis- and trans-3, but the GC chro-
matograms of the crude product mixtures proved to be too complex
We were interested in the question whether a single oxidative
hydroxylation can lead to the creation of three new centers of
chirality and therefore considered disubstituted cyclohexanes as
model compounds in this challenging endeavor. The selective oxi-
dative functionalization of 1,2-dimethylcyclohexane (3) and 1,4-
dimethylcyclohexane (4) using P450-BM3 as catalyst has not
been attempted thus far. We also surmised that the regioselective
P450-mediated oxidation of 1,4-methylisopropylcyclohexane (5)
may constitute a novel access to biologically active compounds
Scheme 2. Oxidation products obtained by WT P450-BM3 catalyzed oxidation of
substrates cis-3 and rac-trans-3.

472
A. Ilie et al. / Tetrahedron 71 (2015) 470e475
additionally led to small amounts of dihydroxylated products as in
the case of 1-methylcyclohexene.9l
It was also of interest to test compound 5. The use of menthane
as substrate for P450-BM3 catalyzed hydroxylation has been
mentioned in the literature, but no details about the products.16 In
our study, the reaction products were established by performing
experiments using WT P450-BM3 as catalyst (TOF 2.9; TTN 500)
and by comparison with commercial or separately synthesized
chemical controls. To clearly assign the reaction products, and to
easily assign the relative configuration, we used only trans-1,4-
menthane (5) as the achiral starting material. The results of this
analytical work are summarized in Fig. 1, which features the GC
chromatogram of the crude product mixture.
The major product proved to be cis-2-isopropyl-5-
methylcyclohexan-1-ol (8c) (40%), known as isomenthol, the
crude reaction product containing significant amounts of tertiary
alcohols (8ad3% and cis-8bd27%) (Fig. 1, top and Scheme 4). It is
interesting to note that the relative configuration of the product 8c
is cis in accord with hydroxylation of substrates 3 and 4 and with
oxidation of substrate 1. However, the product proved to be race-
mic. Several mutants previously evolved for the regio- and ster-
eoselective hydroxylation of compound 115 were tested in the
oxidation of substrate 5, but only a moderate increase in selectivity
for neomenthol 8c was observed.
Scheme 3. Oxidation products obtained by WT P450-BM3 catalyzed oxidation of
achiral substrates cis- and trans-4.
due to several overlapping peaks. Therefore, cis- and trans-3 were
reacted separately. When achiral cis-3 was used as starting mate-
rial, WT P450-BM3 proved to be cis-selective with respect to the
adjacent methyl group, in accord with the previously observed
reaction pattern of methylcyclohexane15 [Scheme 2; turnover fre-
quency (TOF) 2.6; total turnover number (TTN) 400]. Upon sub-
jecting racemic trans-3 to WT P450-BM3 catalyzed oxidation, high
diastereoselective preference for the formation of 1,2-cis-2,3-trans
6a resulted (Table 1; TOF 3.2; TTN 530). The fact that the product is
not quite racemic (5% ee) may be due to incomplete conversion and
some degree of kinetic resolution. The oxidations of achiral cis- and
trans-4 also proceeded regio- and diastereoselectively, although
with poor enantioselectivity (Table 1; TOF 6.1; TTN 970 and TOF 9.4;
TTN 1900 using as starting materials cis- and trans-4, respectively).
Of the four possible hydroxylation products, only compounds 6a,b
and 7a,b were detected. In this sense P450-BM3 is much more
diastereo- and regioselective than other catalysts.5e Similar results
were observed when subjecting cis- and trans-4 to P450-BM3 cat-
alyzed oxidative hydroxylation (Table 1).
From the mutants screened, only A328F, a well-known variant,9
proved to be considerably more selective than WT P450-BM3, the
GC chromatogram of the crude product mixture indicating only
the presence of only one compound, namely 2-(4-
methylcyclohexyl)propan-2-ol 8e (Fig. 1, bottom). In order to ob-
tain enough products for structural analysis, reaction 5/8 was
performed using 180 mg of substrate employing mutant A328F
(TOF 1.7; TTN 150), pure products being successfully isolated fol-
lowing column chromatography (5e10% yield). The procedure was
not optimized for preparative purposes. It should be noted that
excessively longer reaction times when using WT or mutants al-
low the formation of the dihydroxylated species as observed by
GCeMS analysis.
Table 1
Oxidative hydroxylation of 1,2- and 1,4-dimethylcyclohexanes (3 and 4) catalyzed by WT P450-BM3a
Entry
P450 Variant
Substrate
1
2
3
4
WT
WT
WT
WT
cis-3
trans-3
cis-4
6a, 23%, dr 83:17 (1,2-trans/1,2-cis)b
6b, 77%, trans (er¼56:42)
6b, 11%, cis (er¼53:47)
7b, 70%, trans
6a, 89%, dr 98:2 (1,2-cis/1,2-trans), (er¼55: 45)
7a, 30%, dr 80:20 (1,2-cis/2,5-cis)b
trans-4
7a, 81%, dr >99 (1,2-cis/2,5-trans), (er¼64: 36)
7b, 19%, cis
a
Values obtained from average of at least three independent experiments performed with resting cells using NADPþ 50
m
M. %-values refer to regioselectivity of
hydroxylation. Owing to the tendency of 3 and 4 to evaporate under the reaction conditions, it is difficult to measure the exact %-conversion, which may vary when the
reaction is performed in plastic or glass plates.
b
Enantiomeric ratio (er) not measured.
In an attempt to increase regio- and enantioselectivity, some of
our libraries of P450-BM3 mutants evolved for methylcyclohex-
ane15 were tested for substrates 3 and 4 without performing ad-
3. Conclusions and perspectives
It is well-known that achieving high regio- and stereoselectivity
in P450-catalyzed oxidative hydroxylation of ‘small’ molecules
lacking any functional groups is a daunting task,9l,15 as it is when
attempting to apply synthetic catalysts.5 In the present study we
have identified all products arising from the P450-BM3 catalyzed
oxidation of cyclic disubstituted alkanes 3e5, including the assign-
ment of relative stereochemistry. Several of them contain three new
centers of chirality. Whereas bioprocess optimization was not per-
formed for increasing isolated yields, the findings set the stage for
future directed evolution work in the quest to control this type of
ditional
mutagenesis
experiments.
Approximately
300
transformants were screened, but better catalyst in terms of en-
hanced regio- and enantioselectivities were not discovered, al-
though several mutants showed similar selectivities. Owing to the
tendency of 3 and 4 to evaporate under the reaction conditions, as
in the case of methylcyclohexane,15 it is difficult to measure the
exact %-conversion, which may vary considerably when the re-
action is performed in plastic or glass plates. Reactions were
allowed to run for 3e5 h, while longer reaction times (18 h)

A. Ilie et al. / Tetrahedron 71 (2015) 470e475
473
Fig. 1. GC chromatograms of the trans-5 oxidation catalyzed by WT P450-BM3 (top) and mutant A328F (bottom) using resting cells (3 h of reaction time). GC conditions: 30 m
DBWaxetr, inner diameter 0.25 mm; pressure: 0.8 bar H2; injector: 230 ꢁC; oven: temperature gradient: from 60 to 180 ꢁC with 6 ꢁC/min, then from 180 ꢁC to 250 ꢁC with 18 ꢁC/min
FID detector: 350 ꢁC.
frequency; TOF (calculated as mol of product/nmol of protein/
min during the first hour of reaction); and total turnover number;
TTN (calculated as mol of product/nmol of after 1200 min of re-
action) have been calculated as previously reported15 in reactions
containing 1.5 nmol of P450-BM3 and different amounts of starting
materials 3, 4 or 5 (20, 20 or 10 mmol, respectively).
Scheme 4. Oxidation products formed by WT P450-BM3 catalyzed oxidation of sub-
strate trans-5.
4.2. Chemistry
4.2.1. General
dimethylcyclohexane,
dimethylcyclohexane,
remarks. cis-,
trans-,
trans-,
and
and
cisþtrans-1,2-
cisþtrans-1,4-
desymmetrization more precisely. The creation of two, three or
more centers of chirality by a single oxidative hydroxylation of
functionalized or non-functionalized substrates in desymmetriza-
tion events with formation of value-added products opens a new
door in asymmetric catalysis. It will also be interesting to see if
synthetic catalysts can be designed that make such transformations
possible.
cis-,
trans-1-isopropyl-4-methylcyclohexane,
(ꢂ)-terpinen-4-ol,
a
-terpineol, (þ)-neomenthol, (þ)-menthol,
(ꢃ)-menthol, (ꢂ)-menthone, and 3,4-dimethylcyclohexan-1-ol
were purchased from SigmaeAldrich, Alfa Aesar, ABCR or TCI and
used without further purification. NMR spectra were recorded on
a Bruker Avance 300 (1H: 300 MHz, 13C: 75 MHz) spectrometer
using TMS as internal standard (d¼0). Analytical thin layer chro-
matography was performed on Merck silica gel 60 F254 while
for column chromatography Merck silica gel 60 was used. Conver-
sion and enantiomeric excess were determined by achiral and
chiral gas chromatography. Determination of the relative configu-
ration was performed after comparison with commercial or
separately prepared racemic samples. Diastereoisomeric mixtures
of cisþtrans-1-isopropyl-4-methylcyclohexan-1-ol (8a),18 cisþ-
trans-4-isopropyl-1-methylcyclohexan-1-ol (8b),19 cis-(1S,2S,5R)þ
4. Experimental section
4.1. Molecular biology
4.1.1. Reagents. E. coli BOU730 cells used are described elsewhere.9l
Electro-competent cells were prepared in-house according to
standard protocols.17 Scaled up biohydroxylation using P450 mu-
tants was performed as described previously.15 Turnover

474
A. Ilie et al. / Tetrahedron 71 (2015) 470e475
trans-(1R,2S,5R)-2-isopropyl-5-methylcyclohexan-1-ol (8c),20 cis-
(1R,2R,5S)-2-isopropyl-5-methylcyclohexan-1-ol, and trans-
References and notes
(1R,2S,5R)-2-isopropyl-5-methylcyclohexan-1-ol (8c)20 were pre-
pared according to described literature protocols. (1R,2S,5S)-5-Iso-
propyl-2-methylcyclohexan-1-ol (8d) and (1S,2S,5S)-5-isopropyl-2-
methylcyclohexan-1-ol (8d) were prepared according to a protocol
described by Negoro et al.21 and Pavia et al.22 starting from (S)(þ)-
carvone and (R)(þ)-carvone. Their spectroscopic data are identical
to the published ones. Authentic samples of known products were
obtained as follows:
€
1. (a) Enzyme Catalysis in Organic Synthesis, 3rd ed.; Drauz, K., Groger, H., May, O.,
Eds.; Wiley-VCH: Weinheim, Germany, 2012; (b) Faber, K. Biotransformations in
Organic Chemistry, 6th ed.; Springer: Heidelberg, Germany, 2011; (c) Wells, A.;
Meyer, H.-P. ChemCatChem 2014, 6, 918.
2. Reetz, M. T. J. Am. Chem. Soc. 2013, 135, 12480.
3. Reviews of directed evolution: (a) Directed Evolution Library Creation In
Methods in Molecular Biology, 2nd ed.; Gillam, E. M. J., Copp, J. N., Ackerley, D. F.,
Eds.; Humana: Totowa, 2014; (b) Widersten, M. Curr. Opin. Chem. Biol. 2014, 21,
42; (c) Siloto, R. M. P.; Weselake, R. J. Biocatal. Agric. Biotechnol. 2012, 1, 181; (d)
Goldsmith, M.; Tawfik, D. S. Curr. Opin. Struct. Biol. 2012, 22, 406; (e) Cobb, R. E.;
Chao, R.; Zhao, H. AIChE J. 2013, 59, 1432; (f) Reetz, M. T. Directed Evolution of
€
Enzymes InDrauz, K., Droge, H., May, O., Eds.. Enzyme Catalysis in Organic Syn-
thesis; Wiley-VCH: Weinheim, Germany, 2012; Vol. 1, pp 119e190; (g)
Bommarius, A. S.; Blum, J. K.; Abrahamson, M. J. Curr. Opin. Chem. Biol. 2011, 15,
194; (h) Quin, M. B.; Schmidt-Dannert, C. ACS Catal. 2011, 1, 1017; (i) Brustad, E.
4.2.2. 1,2-cis-2,3-trans-2,3-Dimethylcyclohexan-1-ol (6a). A com-
mercial available mixture of four diastereoisomers (TCI) was sepa-
rated on column chromatography to afford 1,2-cis-2,3-trans-6a as
€
M.; Arnold, F. H. Curr. Opin. Chem. Biol. 2011, 15, 201; (j) Jackel, C.; Hilvert, D.
crude oil. 1H NMR (300 MHz, CDCl3)
d 3.80e3.77 (m, 1H), 1.89e1.74
Curr. Opin. Biotechnol. 2010, 21, 753; (k) Turner, N. J. Nat. Chem. Biol. 2009, 5,
567; (l) Lutz, S.; Bornscheuer, U. T. Protein Engineering Handbook; Wiley-VCH:
Weinheim, Germany, 2009, Vols. 1 and 2.
(m, 1H), 1.68e0.76 (10H). 13C NMR (75 MHz, CDCl3)
d 71.9, 42.9, 35.1,
33.6, 32.2, 20.3, 20.7, 16.4.
4. Turner, N. J.; O’Reilly, E. Nat. Chem. Biol. 2013, 9, 285.
5. Recent representative studies of oxidative hydroxylation utilizing synthetic
reagents or catalysts for the preparation of simple and complex organic com-
pounds: (a) Newhouse, T.; Baran, P. S. Angew. Chem., Int. Ed. 2011, 50, 3362; (b)
White, M. C. Science 2012, 335, 807; (c) Neufeldt, S. R.; Sanford, M. S. Acc. Chem.
4.2.3. cisþtrans-1,2-Dimethylcyclohexan-1-ol (6b). A mixture of
cisþtrans-1,2-dimethylcyclohexan-1-ol was prepared starting from
2-methylcyclohexan-1-one and CH3MgCl in dry THF according to a
published protocol used for the synthesis of 1-isopropylcyclohexan-
1-ol.23 The crude reaction product was loaded on a column chro-
matography and trans-1,2-dimethylcyclohexan-1-ol separated.
Spectroscopic data are in agreement with published data.24 cisþtrans
€
Res. 2012, 45, 936; (d) Adam, W.; Zhao, C.-G.; Saha-Moller, R. R.; Jakka, K. Ox-
idation of Organic Compounds with Dioxiranes; Wiley-VCH: New York, NY, 2009;
(e) Kamata, K.; Yonehara, K.; Nakagawa, Y.; Uehara, K.; Mizuno, N. Nat. Chem.
2010, 2, 478; (f) Periana, R. A. Acc. Chem. Res. 2012, 45, 885; (g) Sun, M.; Zhang,
J.; Putaj, P.; Caps, V.; Lefebvre, F.; Pelletier, J.; Basset, J. M. Chem. Rev. 2014, 114,
€
981; (h) Roduner, E.; Kaim, W.; Sarkar, B.; Urlacher, V. B.; Pleiss, J.; Glaser, R.;
Einicke, W.-D.; Sprenger, G. A.; Beifuß, U.; Klemm, E.; Liebner, C.; Hieronymus,
H.; Hsu, S.-F.; Plietker, B.; Laschat, S. ChemCatChem 2013, 5, 82.
6b: 1H NMR (300 MHz, CDCl3)
d
1.77e1.52 (m, 4H), 1.44 (m, 4H), 1.22
(m, 3H), 0.90 (m, 3H). cis 6b: 13C NMR (75 MHz, CDCl3)
73.1, 42.4,
41.5, 32.2, 25.4, 24.3, 21.0, 15.4. trans 6b: 13C NMR (75 MHz, CDCl3)
71.1, 40.4, 40.1, 30.7, 28.7, 26.0, 22.2, 15.3.
d
6. Reviews of structure, mechanism and applications of P450 enzymes: (a) Ortiz
de Montellano, P. R. Cytochrome P450: Structure, Mechanism, and Biochemistry,
3rd ed.; Springer: Berlin, Germany, 2005; (b) Isin, E. M.; Guengerich, F. P. Bio-
chim. Biophys. Acta Gen. Subj. 2007, 1770, 314; (c) Munro, A. W.; Girvan, H. M.;
McLean, K. J. Nat. Prod. Rep. 2007, 24, 585; (d) Ortiz de Montellano, P. R. Chem.
Rev. 2010, 110, 932; (e) Shaik, S.; Cohen, S.; Wang, Y.; Chen, H.; Kumar, D.; Thiel,
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2006, 24, 324; (g) Kadish, K. M.; Smith, K. M.; Guilard, R. Handbook of Porphyrin
Science; World Scientific Publishing, 2010, Vol. 5; (h) Meunier, B.; de Visser, S.
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L.-L. Chem. Soc. Rev. 2012, 41, 1218; (j) Lewis, J. C.; Coelho, P. S.; Arnold, F. H.
d
4.2.4. 1,2-cis-2,5-trans-2,5-Dimethylcyclohexan-1-ol (7a). Compound
1,2-cis-2,5-trans-1,4-dimethylcyclohexan-1-ol 7a was separated from
a commercial diastereoisomeric mixture and characterized by NMR.
1H NMR (300 MHz, CDCl3)
d
3.80 (s, 1H), 1.83 (d, 3J¼13.6 Hz, 1H),
1.73e164 (s, 2H), 1.55e1.04 (m, 6H), 0.91 (m, 6H). 13C NMR (75 MHz,
€
Chem. Soc. Rev. 2011, 40, 2003; (k) O’Reilly, E.; Kohler, V.; Flitsch, S. L.; Turner, N.
CDCl3)
d 71.3, 42.3, 36.1, 34.9, 28.2, 25.7, 22.4, 18.3.
J. Chem. Commun. 2011, 2490; (l) Fasan, R. ACS Catal. 2012, 2, 647; (m) Khatri, Y.;
Hannemann, F.; Girhard, M.; Kappl, R.; Meme, A.; Ringle, M.; Janocha, S.; Leize-
Wagner, E.; Urlacher, V. B.; Bernhardt, R. Biotechnol. Appl. Biochem. 2013, 60, 18;
(n) Fifty Years of Cytochrome P450 Research; Yamazaki, H., Ed.; Springer: Berlin,
Germany, 2014.
4.2.5. trans-1,4-Dimethylcyclohexan-1-ol(7b). A mixture of cisþtrans-
1,4-dimethylcyclohexan-1-ol was prepared starting from 4-
methylcyclohexan-1-one and CH3MgCl in dry THF according to
a published protocol used for the synthesis of 1-isopropylcyclohexan-
1-ol.23 The crude reaction product was loaded on a column chro-
matography and trans-1,4-dimethylcyclohexan-1-ol separated.
Spectroscopic data are in agreement with published data.25 1H NMR
7. Review of early steroid oxidations practiced by: (a) Upjohn Company/USA:
Hogg, J. A. Steroids 1992, 57, 593; (b) Schering Corporation: Herzog, H.; Oliveto,
P. Steroids 1992, 57, 617.
€
8. See for example: Landwehr, M.; Hochrein, L.; Otey, C. R.; Kasrayan, A.; Backvall,
J.-E.; Arnold, F. H. J. Am. Chem. Soc. 2006, 128, 6058.
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Dennig, A.; Marienhagen, J.; Ruff, A. J.; Guddat, L.; Schwaneberg, U. Chem-
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(300 MHz, CDCl3)
d 1.77e1.52 (m, 4H), 1.44 (m, 4H), 1.22 (m, 3H),
0.90 (m, 3H). 13C NMR (75 MHz, CDCl3)
21.6.
d 70.9, 39.9, 32.4, 31.9, 26.1,
4.2.6. cisþtrans-2-(4-Methylcyclohexyl)propan-2-ol (8e).
a-Terpineol
(300 mg, 1.94 mmol) was reduced using the same protocol as above,
to afford a mixture of cisþtrans 2-(4-methylcyclohexyl)propan-2-ol. A
GC sample of the crude reaction product was compared with products
resulting from hydroxylation reaction of trans-1-isopropyl-4-
methylcyclohexane catalyzed by WT P450. trans-8e was obtained
after hydroxylation reaction of trans-5, catalyzed by mutant P450-
A328F, and was scaled-up according to a general procedure de-
scribed elsewhere.5e After extraction and concentration using a rota-
vap, the crude reaction product was purified by column
chromatography (EA/PE¼1:4, Rf¼0.55) to afford compound trans-8e
€
14310; (n) Bogazkaya, A. M.; von Buhler, C. J.; Kriening, S.; Busch, A.; Seifert, A.;
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J.; Hollmann, F. Chem. Commun. 2014, 50, 13180; (q) Neufeld, K.; Henßen, B.;
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as a colorless oil, yield <10%. 13C NMR (75 MHz, CDCl3)
d 71.8, 49.0,
35.5 (2C), 27.5 (2C), 27.1 (2C), 22.7, 14.3.
Acknowledgements
Financial support by the Max-Planck-Society and the Arthur C.
Cope Foundation is gratefully acknowledged.
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