Cytochrome P450 catalyzed oxidative hydroxylation of achiral organic compounds with simultaneous creation of two chirality centers in a single C-H activation step

Article abstract of DOI:10.1002/anie.201310892

Regio- and stereoselective oxidative hydroxylation of achiral or chiral organic compounds mediated by synthetic reagents, catalysts, or enzymes generally leads to the formation of one new chiral center that appears in the respective enantiomeric or diastereomeric alcohols. By contrast, when subjecting appropriate achiral compounds to this type of C-H activation, the simultaneous creation of two chiral centers with a defined relative and absolute configuration may result, provided that control of the regio-, diastereo-, and enantioselectivity is ensured. The present study demonstrates that such control is possible by using wild type or mutant forms of the monooxygenase cytochrome P450 BM3 as catalysts in the oxidative hydroxylation of methylcyclohexane and seven other monosubstituted cyclohexane derivatives.

Full text of DOI:10.1002/anie.201310892

Angewandte
Chemie
DOI: 10.1002/anie.201310892
Biocatalysis
Cytochrome P450 Catalyzed Oxidative Hydroxylation of Achiral
Organic Compounds with Simultaneous Creation of Two Chirality
ꢀ
Centers in a Single C H Activation Step**
Gheorghe-Doru Roiban, Rubꢀn Agudo, and Manfred T. Reetz*
Abstract: Regio- and stereoselective oxidative hydroxylation
of achiral or chiral organic compounds mediated by synthetic
reagents, catalysts, or enzymes generally leads to the formation
of one new chiral center that appears in the respective
enantiomeric or diastereomeric alcohols. By contrast, when
R1CH2R2!R1CH(OH)R2, whereas chiral compounds pro-
vide a pair of diastereomers in the otherwise similar process
R*CH2R!R*CH(OH)R. The first examples of the use of
directed evolution to produce cytochrome P450 variants that
induce high regioselectivity while also controlling stereose-
lectivity were recently reported with functionalized substrates
such as steroids,[6,7] 1-cyclohexene carboxylic acid ester,[8] or
N-benzyl pyrrolidine,[5c,9] compounds that may undergo bind-
ing interactions at the respective hetero-atoms. Such regio-
and enantioselectivity has not been achieved in reactions of
alkanes that lack functional groups.
ꢀ
subjecting appropriate achiral compounds to this type of C H
activation, the simultaneous creation of two chiral centers with
a defined relative and absolute configuration may result,
provided that control of the regio-, diastereo-, and enantiose-
lectivity is ensured. The present study demonstrates that such
control is possible by using wild type or mutant forms of the
monooxygenase cytochrome P450 BM3 as catalysts in the
oxidative hydroxylation of methylcyclohexane and seven other
monosubstituted cyclohexane derivatives.
Oxidative hydroxylation produces a different stereochem-
ical outcome when appropriate achiral substrates are used, in
ꢀ
which case a single C H-activating process induces the
concomitant creation of more than one center of chirality.
Consider, for example, the reaction of achiral compounds of
the type I, in which oxidation at the four stereotopic H atoms
of the two methylene units flanking the X-bearing C atom
leads to four different stereoisomers: II, III, IV, and V, each of
which has two new chirality centers (Scheme 1). Depending
upon the nature of the R groups, other regioisomeric alcohols
can also be formed, a fact that contributes to the overall
challenge.
ꢀ
O
xidative C H activation of structurally simple and com-
plex organic compounds that leads to the regio- and
stereoselective introduction of hydroxy groups at predeter-
mined non-activated sites constitutes a difficult yet rewarding
goal in synthetic organic chemistry.[1] Numerous synthetic
reagents and catalysts have been developed for achieving this
kind of selective functionalization but there are still problems
regarding control of regio-, diastereo- and enantioselectiv-
ity.[1] The use of cytochrome P450 enzymes constitutes an
alternative approach.[2] The mechanism of these heme
dependent monooxygenases involves radical abstraction at
ꢀ
a C H site with formation of the respective alkyl radical
ꢀ
followed by rapid C O bond formation. When selectivity is
poor, protein engineering based on directed evolution[3,4]
provides a means to generate improved cytochrome P450
mutants.[5] In the case of most achiral substrates, oxidative
hydroxylation leads to enantiomers according to the process
[*] Dr. G.-D. Roiban,[+] Dr. R. Agudo,[+] Prof. Dr. M. T. Reetz
Max-Planck-Institut fꢀr Kohlenforschung
Kaiser-Wilhelm-Platz 1, 45470 Mꢀlheim an der Ruhr (Germany)
and
Department of Chemistry, Philipps-Universitꢁt Marburg
Hans-Meerwein-Strasse, 35032 Marburg (Germany)
E-mail: reetz@mpi-muelheim.mpg.de
Scheme 1. The stereochemical consequences of the oxidative hydroxyl-
ation of prochiral compounds of type I.
[+] These authors contributed equally to this work.
[**] Financial support by the Max-Planck-Society and the Arthur C. Cope
Foundation is gratefully acknowledged. We thank Dr. Pamela Torres-
Salas for assistance in TON and TTN measurements, Stephanie
Dehn and Corinna Heidgen for numerous GC analyses, Julian
Kuttner (group of Prof. G. Hilt) for providing several starting
cyclohexane derivatives, and Jonas Schwaben (group of Prof. U.
Koert) for support with optical rotation measurements.
We chose methylcyclohexane (1a), which is devoid of any
functional groups, as the model substrate and cytochrome
P450 BM3 (CYP102A1) from Bacillus megaterium as the
enzyme.[2,10,11] P450 BM3 is a self-sufficient fusion protein
composed of a cytochrome P450 monooxygenase and an
NADPH diflavin reductase for cofactor regeneration. Hy-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201310892.
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
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droxylation at the two methylene units flanking the CH3-
bearing C atom provides four possible products, cis-(1S,2R)-
2a, cis-(1R,2S)-2a, trans-(1R,2R)-2a and trans-(1S,2S)-2a,
with regioisomers 2b–e resulting from oxidative attack at
other C atoms also being possible (Scheme 2).
Scheme 2. Possible products of the P450 BM3 catalyzed oxidative
hydroxylation of methylcyclohexane (1a).
Figure 1. Selected CAST sites A (V78/T88; dark blue/green), B (V78/
L181; dark blue/yellow), C (T268/A328; brown/red), and D (F87; light
blue) in P450 BM3, chosen with the help of the crystal structure (PDB:
1JPZ).[11]
Wild type (wt) P450 BM3 was found to be notably regio-
(78%), diastereo- (ꢁ 97%), and enantioselective (e.r. = 91:9)
in favor of cis-(1S,2R)-2a (Table 1, entry 1). This is a remark-
able result not possible when utilizing synthetic catalysts or
oxidants.[12] In the hope of boosting enantioselectivity in the
regioselective formation of cis-(1S,2R)-2a, directed evolution
by using the combinatorial active-site saturation test (CAST)
was applied.[4] Accordingly, four CAST sites were chosen for
saturation mutagenesis (Figure 1): A (V78/T88), B (V78/
L181) and C (T268/A328), which were randomized by using
a highly reduced amino acid alphabet composed of six
building blocks (Phe, Tyr, Trp, Lys, Arg, His) and the
corresponding wt amino acids, and site D (F87) for which
NNK codon degeneracy was chosen to enode all 20 canonical
amino acids. Upon screening a total of 760 transformants,
variant F87A (identified from library D), which is the
“standard” mutant generated in previous protein engineering
studies involving other substrates,[2,10] led to an undesired shift
in regioselectivity (71%) in favor of the tertiary alcohol 2b
with enhanced enantioselectivity (e.r. = 96:4) at the expense
of a slight decrease in regioselectivity (71%, Table 1, entry 3).
We then subjected substrates 1b–h to oxidative hydrox-
ylation using wt P450 BM3 and some of the mutants
previously screened for 1a (Table 2). The results point to
remarkable effects, the most prominent of which is consis-
tently high diastereoselectivity in favor of the cis configu-
ration. This diastereoselectivity is often accompanied by high
enantioselectivity, although at the expense of regioselectivity,
as in the case of cis-2-halocycloalkanols (1S,2R)-3a and
(1S,2R)-4a. Synthetic strategies that lead to the enantiose-
lective production of cis halo alcohols in a single step from
halocycloalkanes have not been considered to date; such
compounds are generally synthesized by the reduction of a-
halo ketones.[13] When comparing the stereochemical results
with the absolute configuration of compound 2a, it can be
seen that the sense of the enantioselectivity does not change.
Mutants F87A and A328F do not result in appreciable
improvements in regio- or enantioselectivity. Cyanocyclohex-
ane (1d) is also hydroxylated with cis diastereoselectivity at
the same enantiotopic H atom with preferential formation of
(1S,2S)-5a, with the stereochemical nomenclature changing in
ꢀ
(Table 1, entry 2). This reaction involves the weakest C H
bond in the molecule and is more easily achieved by synthetic
reagents such as dioxiranes.[1a,c] Several improved variants
were detected (see the Supporting Information), the best of
which was A328F[2,10] from library C. It affords cis-(1S,2R)-2a
Table 1: Oxidative hydroxylation of methylcyclohexane (1a) catalyzed by wt P450 BM3 or mutants thereof.[a]
Entry
P450 Variant
1
2
3
wt
F87A
A328F
2a/78, d.r. =97:3 (cis), (1S,2R), (e.r.=91:9)
2a/13, d.r. =97:3 (cis), (1S,2R), (e.r.=68:32)
2a/71, d.r. =97:3 (cis), (1S,2R), (e.r.=96:4)
2b/17
2b/71
2b/20
2c/4, d.r.=95:5 (trans)
2c/8, d.r.>99:1 (trans)
2c/9, d.r.=56:44 (cis)
2d/1, d.r.=69:31 (cis)
2d/8, d.r.=68:32 (cis)
–
–
–
–
[a] Values were obtained by averaging at least three independent experiments performed as described in the Supporting Information. The numbers
following the slash after the product designation represent % regioselectivity. Owing to the tendency of methylcyclohexane to evaporate under the
reaction conditions, it is difficult to measure the exact % conversion, which may vary considerably when the reaction is performed in plastic or glass
plates (see the Supporting Information).
2
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Table 2: Oxidative hydroxylation of compounds 1b–h by using wt P450 BM3 and mutants evolved for the reaction of 1a.[a]
P450
variant
Other oxid.
prod. [%]
Entry
1
R
Conv. [%]
1
2
3
1b Cl
1c Br
1d CN
wt
wt
wt
3a/49, d.r.=96:4 (cis),
(1S,2R), (e.r.=96:4)
4a/67, d.r.=94 :6 (cis),
(1S,2R), (e.r.=98:2)
5a/47, d.r.=95:5 (cis),
(1S,2S), (e.r.=96:4)
5a/4
6a/77, d.r.=97:3 (cis),
(1S,2S), (e.r.=63:37)
6a/48, d.r.=98:2 (cis),
(1S,2S), (e.r.=96:4)
7a/59, d.r.=98:2 (cis),
(1R,2R), (e.r.=95:5)
8a/58, d.r.=95:5 (cis),
(1R,2S), (e.r.=77:23)
8a/78, d.r.=98:2 (cis),
(1R,2S), (e.r.=98:2)
8a/2
–(7)[b]
–(13)[c]
–(23)[d]
3c/21, d.r.=
99:1 (trans)
4c/11, d.r.=
99:1 (trans)
5c/16
3d/18, d.r.=
99:1(cis)
4d/7, d.r.=
99:1 (cis)
5d/10
–
–
–
5
2
4
58
86
62
4
5
1d CN
1e iPr
F87A
wt
–(86)[d]
6b/8
5c/3
6c/7
5d/2
6d/2
–
5
3
10
12
6e/3[f]
6
7
8
9
1e iPr
A328F
wt
6b/3
7b/8
8b/34
8b/7
6c/18
7c/25
n.a.[e]
n.a.[e]
6d/9
6e/18[f]
4
1
30
19
1 f tBu
7d/7, d.r.=
54:46 (cis)
n.a.[e]
–
–
–
1g COCH3
1g COCH3
1g COCH3
wt
8
88
A328F
n.a.[e]
15[g]
85 (97)[h]
10
11
F87A
8b/84
9b/14
n.a.[e]
n.a.[e]
n.a.[e]
9d/13, d.r.=
62:38 (cis)
–
–
14[g]
6
14
1h CH2COCH3 wt
9a/67, d.r.=97:3 (cis),
(1R,2R), (e.r.=78:22)
86 (98)[h]
[a] Values were obtained by averaging at least three independent experiments performed with resting cells as described in the Supporting Information.
The numbers following the slash after the product designation represent % regioselectivity. [b] The crude reaction products contain 7%
cyclohexanone, which is formed by dehydrohalogenation of cis-3a and/or 3b. [c] The crude reaction products contain 13% cyclohexanone, which is
formed by dehydrohalogenation of cis-4a and/or 4b. [d] The crude reaction products contain 23% (entry 3)/86% (entry 4) cyclohexanone, which is
derived from the tertiary cyanohydrin. [e] n.a.=not assigned (concentration too low/control not available). [f] 6e=2-cyclohexyl-2-propanol. [g] The
other oxidation products consist mainly of cis/trans 3- and 4-hydroxy substituted methylcyclohexyl ketone but the exact structure was not identified.
[h] Conversion obtained in a scaled up experiment, thus showing that the screening process leads to lower conversion values.
this case according to the CIP convention. It is also interesting
to note that 86% regioselectivity in favor of hydroxylation at
the cyano-bearing C atom can be achieved by using mutant
F87A, in which case the intermediate cyanohydrin liberates
cyclohexanone (Table 2, entry 4). This is a convenient bio-
catalytic method for transforming an alkylnitrile into the
respective ketone. Reasonable activity was typically
observed, as in the case of substrate 1c, which gave a turnover
frequency (TOF) of 8.5 minꢀ1 and a total turnover number
(TTN) of 1616 (see the Supporting Information).
In the case of isopropylcyclohexane (1e), wt P450 BM3 is
77% regioselective and 97% diastereoselective favoring cis-
(6a) but the enantioselectivity is poor (e.r. = 63:37). When
using mutant A328F, a pronounced increase in enantioselec-
tivity resulted (e.r. = 96:4), albeit at the expense of some
regioselectivity (Table 2, entries 5 and 6). tert-Butylcyclohex-
ane (1 f) is also hydroxylated with cis selectivity, but in this
case the other stereotopic H atom is hydroxylated with
formation of the opposite absolute configuration (1R,2R,
e.r. = 95:5; Table 2, entry 7). Upon changing from wt
P450 BM3 to mutant A328F in the reaction of cyclohexyl
methyl ketone (1g), regioselectivity in favor of alcohol 8a is
boosted from 58% to 78%, with diastereoselectivity in favor
of the cis isomer amounting to 98% with high enantioselec-
tivity (e.r. = 98:2) in favor of (1R,2S)-8a. Catalytic activity is
acceptable (TOF = 11.9 and 12.1 minꢀ1 for wt and mutant,
respectively; TTN = 1535 and 1707 for wt and mutant,
respectively). This result means that the sense of the
enantioselectivity is the same as in the reaction of 2a. The
reaction was scaled up to 1.85 mmol of substrate 1g (97%
conversion; 65% yield of isolated 8a following chromatog-
raphy). By contrast, mutant F87A delivers primarily the
tertiary alcohol 8b in a fairly slow reaction. Wt P450 BM3
catalyzes the hydroxylation of ketone 1h with 67% regiose-
lectivity, 97% cis diastereoselectivity, and reversed enantio-
selectivity in favor of (1R,2R)-9a (e.r. = 78:22; Table 2,
entry 11) with good activity (TOF = 27 minꢀ1; TTN = 1616).
Similarly, a scaled up reaction of 1h (1.85 mmol) led to 98%
conversion and provided 9a with a yield of 60% after column
chromatography. On scaling up the reactions of substrates
1a–e, the conversion values generally proved to be higher
than those observed under screening conditions. However,
this does not apply to compounds 1a, 1e, and 1 f, in which
cases such characteristics as volatility and solubility influence
the yields of the hydroxylated products following column
chromatography. Nevertheless, sufficient amounts of the
respective cis alcohols could be prepared, thus confirming,
even in these cases, the synthetic utility of the catalytic
system. Further directed evolution and appropriate process
engineering constitute ways to optimize the reaction of each
substrate.
In summary, we have shown that the desymmetrization of
prochiral monosubstituted cyclohexane derivatives by means
of oxidative hydroxylation catalyzed by wt P450 BM3 or
mutants thereof can be highly regio-, diastereo- and enantio-
selective, thus leading to the creation of two centers of
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ꢀ
house, S. G. Bell, L.-L. Wong, Chem. Soc. Rev. 2012, 41, 1218 –
1260; j) J. C. Lewis, P. S. Coelho, F. H. Arnold, Chem. Soc. Rev.
2011, 40, 2003 – 2021; k) E. OꢁReilly, V. Kçhler, S. L. Flitsch, N. J.
Turner, Chem. Commun. 2011, 47, 2490 – 2501; l) R. Fasan, ACS
Catal. 2012, 2, 647 – 666; m) Y. Khatri, F. Hannemann, M.
Girhard, R. Kappl, A. Meme, M. Ringle, S. Janocha, E. Leize-
Wagner, V. B. Urlacher, R. Bernhardt, Biotechnol. Appl. Bio-
chem. 2013, 60, 18 – 29.
chirality in a single C H-activation event. The regioselective
formation of the vicinal cis diastereomers appears to be the
rule, with enantioselectivity also being high in many cases.
This constitutes the first strategy for accessing highly enantio-
enriched vicinal disubstituted cis-cyclohexanols in one step,
a method that fulfills the requirements of a “green” process.[14]
Some of the products arising from the hydroxylation of
substrates 1a–h, which have previously been prepared by
multistep routes, are of potential interest as building blocks in
the synthesis of certain biologically active compounds as
[3] Recent reviews of directed evolution:[4] a) S. Lutz, U. T. Born-
scheuer, Protein Engineering Handbook, Vol. 1 – 2, Wiley-VCH,
Weinheim, 2009; b) N. J. Turner, Nat. Chem. Biol. 2009, 5, 567 –
573; c) C. Jꢀckel, P. Kast, D. Hilvert, Annu. Rev. Biophys.
Biomol. Struct. 2008, 37, 153 – 173; d) S. Bershtein, D. S. Tawfik,
Curr. Opin. Chem. Biol. 2008, 12, 151 – 158; e) P. A. Romero,
F. H. Arnold, Nat. Rev. Mol. Cell Biol. 2009, 10, 866 – 876;
f) L. G. Otten, F. Hollmann, I. W. C. E. Arends, Trends Biotech-
nol. 2010, 28, 46 – 54; g) A. V. Shivange, J. Marienhagen, H.
Mundhada, A. Schenk, U. Schwaneberg, Curr. Opin. Chem. Biol.
2009, 13, 19 – 25; h) G. A. Strohmeier, H. Pichler, O. May, M.
Gruber-Khadjawi, Chem. Rev. 2011, 111, 4141 – 4164; i) U. T.
Bornscheuer, G. Huisman, R. Kazlauskas, S. Lutz, J. C. Moore,
K. Robins, Nature 2012, 485, 185 – 194; j) M. T. Reetz, J. Am.
Chem. Soc. 2013, 135, 12480 – 12496.
therapeutic drugs.[15] Although the C H bond strength in all
ꢀ
of the methylene units can be expected to be similar, those
flanking the R-bearing C atom seem to be the preferred sites
for oxidative hydroxylation despite the fact that the reaction
might be expected to be disfavored at these sites owing to
steric effects.
The present work also suggests that in the quest to
transform simple achiral compounds into value-added chiral
products of higher structural complexity, it may be useful to
screen further P450 BM3 mutant libraries as well as other
cytochrome P450 enzymes. By using appropriate substrates, it
should also be possible to use the same type of cytochrome
P450 catalyzed oxidative hydroxylation to bring about the
simultaneous creation of three or more new chirality centers
[4] Review of the directed evolution of stereoselective enzymes as
catalysts in synthetic organic chemistry: M. T. Reetz, Angew.
Chem. 2011, 123, 144 – 182; Angew. Chem. Int. Ed. 2011, 50, 138 –
174.
[5] Recent accounts of protein engineering of P450 enzymes by
using traditional site-specific mutagenesis or directed evolu-
tion:[7] a) B. M. A. van Vugt-Lussenburg, M. C. Damsten, D. M.
Maasdijk, N. P. E. Vermeulen, J. N. M. Commandeur, Biochem.
Biophys. Res. Commun. 2006, 346, 810 – 818; b) S. T. Jung, R.
Lauchli, F. H. Arnold, Curr. Opin. Biotechnol. 2011, 22, 809 –
817; c) W. L. Tang, Z. Li, H. Zhao, Chem. Commun. 2010, 46,
5461 – 5463; d) P. R. Ortiz de Montellano, Chem. Rev. 2010, 110,
932 – 948; e) V. B. Urlacher, M. Girhard, Trends Biotechnol.
2012, 30, 26 – 36; f) A. Dennig, J. Marienhagen, A. J. Ruff, L.
Guddat, U. Schwaneberg, ChemCatChem 2012, 4, 771 – 773;
g) E. Weber, A. Seifert, M. Antonovici, C. Geinitz, J. Pleiss, V. B.
Urlacher, Chem. Commun. 2011, 47, 944 – 946; h) H. Venkatara-
man, S. B. A. de Beer, L. A. H. van Bergen, N. van Essen, D. P.
Geerke, N. P. E. Vermeulen, J. N. M. Commandeur, ChemBio-
Chem 2012, 13, 520 – 523; i) J. C. Lewis, S. M. Mantovani, Y. Fu,
C. D. Snow, R. S. Komor, C. H. Wong, F. H. Arnold, ChemBio-
Chem 2010, 11, 2502 – 2505; j) K. Zhang, S. E. Damaty, R. Fasan,
J. Am. Chem. Soc. 2011, 133, 3242 – 3245; k) G.-D. Roiban, R.
Agudo, M. T. Reetz, Tetrahedron 2013, 69, 5306 – 5311.
[6] S. Kille, F. E. Zilly, J. P. Acevedo, M. T. Reetz, Nat. Chem. 2011,
3, 738 – 743.
[7] a) S. H. Mao, X. J. Hu, B. Y. Hua, N. Wang, X. G. Liu, F. P. Lu,
Biotechnol. Lett. 2012, 34, 2113 – 2117; b) K. T. Nguyen, C. Virus,
N. Gunnewich, F. Hannemann, R. Bernhardt, ChemBioChem
2012, 13, 1161 – 1166; c) A. Braun, M. Geier, B. Buhler, A.
Schmid, S. Mauersberger, A. Glieder, Microb. Cell Fact. 2012, 11,
106; d) M. Geier, A. Braun, P. Fladischer, P. Stepniak, F.
Rudroff, C. Hametner, M. D. Mihovilovic, A. Glieder, FEBS J.
2013, 280, 3094 – 3108; e) P. Bracco, D. B. Janssen, A. Schallmey,
Microb. Cell Fact. 2013, 12, 95.
ꢀ
in a single C H-activation step.
Received: December 16, 2013
Revised: January 22, 2014
Published online: && &&, &&&&
ꢀ
Keywords: biocatalysis · C H activation · cytochrome P450 ·
hydroxylation · stereoselectivity
.
[1] Recent representative studies of oxidative hydroxylation utiliz-
ing synthetic reagents or catalysts for the preparation of simple
and complex organic compounds: a) T. Newhouse, P. S. Baran,
Angew. Chem. 2011, 123, 3422 – 3435; Angew. Chem. Int. Ed.
2011, 50, 3362 – 3374; b) M. C. White, Science 2012, 335, 807 –
809; c) S. R. Neufeldt, M. S. Sanford, Acc. Chem. Res. 2012, 45,
936 – 946; d) W. Adam, C.-G. Zhao, R. R. Saha-Mçller, K. Jakka,
Oxidation of Organic compounds with Dioxiranes, Wiley-VCH,
New York, 2009; e) K. Kamata, K. Yonehara, Y. Nakagawa, K.
Uehara, N. Mizuno, Nat. Chem. 2010, 2, 478 – 483; f) R. A.
Periana, Acc. Chem. Res. 2012, 45, 885 – 898; g) M. Sun, J. Zhang,
P. Putaj, V. Caps, F. Lefebvre, J. Pelletier, J. M. Basset, Chem.
Rev. 2014, 114, 981 – 1019; h) E. Roduner, W. Kaim, B. Sarkar,
V. B. Urlacher, J. Pleiss, R. Glꢀser, W.-D. Einicke, G. A.
Sprenger, U. Beifuß, E. Klemm, C. Liebner, H. Hieronymus,
S.-F. Hsu, B. Plietker, S. Laschat, ChemCatChem 2013, 5, 82 –
112.
[2] Reviews of P450 enzymes: a) P. R. Ortiz de Montellano, Cyto-
chrome P450: Structure, Mechanism, and Biochemistry, 3rd ed.,
Springer, Berlin, 2005; b) E. M. Isin, F. P. Guengerich, Biochim.
Biophys. Acta Gen. Subj. 2007, 1770, 314 – 329; c) A. W. Munro,
H. M. Girvan, K. J. McLean, Nat. Prod. Rep. 2007, 24, 585 – 609;
d) P. R. Ortiz de Montellano, Chem. Rev. 2010, 110, 932 – 948;
e) S. Shaik, S. Cohen, Y. Wang, H. Chen, D. Kumar, W. Thiel,
Chem. Rev. 2010, 110, 949 – 1017; f) V. B. Urlacher, S. Eiben,
Trends Biotechnol. 2006, 24, 324 – 330; g) Handbook of Porphy-
rin Science (Eds.: K. M. Kadish, K. M. Smith, R. Guilard), Vol. 5,
World Scientific Publishing, 2010; h) B. Meunier, S. P. de Visser,
S. Shaik, Chem. Rev. 2004, 104, 3947 – 3980; i) C. J. C. White-
[8] R. Agudo, G.-D. Roiban, M. T. Reetz, ChemBioChem 2012, 13,
1465 – 1473.
[9] S. Q. Pham, G. Pompidor, J. Liu, X. D. Li, Z. Li, Chem. Commun.
2012, 48, 4618 – 4620.
[10] a) L. O. Narhi, A. J. Fulco, J. Biol. Chem. 1986, 261, 7160 – 7169;
b) A. W. Munro, D. J. Leys, K. J. McLean, K. R. Marshall,
T. W. B. Ost, S. Daff, C. S. Miles, S. K. Chapman, D. A. Lysek,
C. C. Moser, C. C. Page, P. L. Dutton, Trends BioChem. Sci.
2002, 27, 250 – 257; c) T. Jovanovic, R. Farid, R. A. Friesner,
4
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
These are not the final page numbers!

Angewandte
Chemie
A. E. McDermott, J. Am. Chem. Soc. 2005, 127, 13548 – 13552;
d) K. H. Clodfelter, D. J. Waxman, S. Vajda, Biochemistry 2006,
45, 9393 – 9407; e) U. Schwaneberg, A. Sprauer, C. Schmidt-
Dannert, R. D. Schmid, J. Chromatogr. A 1999, 848, 149 – 159.
[11] a) D. C. Haines, D. R. Tomchick, M. Machius, J. A. Peterson,
Biochemistry 2001, 40, 13456 – 13465; b) D. C. Haines, B. Chen,
D. R. Tomchick, M. Bondlela, A. Hegde, M. Machius, J. A.
Peterson, Biochemistry 2008, 47, 3662 – 3670; see also c) H. Li,
T. L. Poulos, Nat. Struct. Biol. 1997, 4, 140 – 146.
[12] a) See Ref. [1e]; b) S. I. Murahashi, N. Komiya, Y. Oda, T.
Kuwabara, T. Naota, J. Org. Chem. 2000, 65, 9186 – 9193; c) H.
Fuwa, T. Kobayashi, T. Tokitoh, Y. Torii, H. Natsugari,
Tetrahedron 1984, 40, 4297 – 4312; d) J. R. Lindsay Smith, G. B.
Shulꢁpin, Tetrahedron Lett. 1998, 39, 4909 – 4912; e) M. V.
Kirillova, A. M. Kirillov, D. Mandelli, W. A. Carvalho, A. J. L.
Pombeiro, G. B. Shulꢁpin, J. Catal. 2010, 272, 9 – 17; f) G. B.
Shulꢁpin, C. C. Golfeto, G. Sꢂss-Fink, L. S. Shulꢁpin, D. Mandelli,
Tetrahedron Lett. 2005, 46, 4563 – 4567.
[14] a) B. M. Trost, Angew. Chem. 1995, 107, 285 – 307; Angew. Chem.
Int. Ed. Engl. 1995, 34, 259 – 281; b) R. A. Sheldon, Chem. Soc.
Rev. 2012, 41, 1437 – 1451; c) P. A. Wender, V. A. Verma, T. J.
Paxton, T. H. Pillow, Acc. Chem. Res. 2008, 41, 40 – 49; d) T.
Newhouse, P. S. Baran, R. W. Hoffmann, Chem. Soc. Rev. 2009,
38, 3010 – 3021.
[15] For the use of compound 2a in the synthesis of analgesics: a) Y.
Tsuzuki, T. Morie, T. Nakamura, I. Shimizu, M. Miyauchi,
US2012/28937A1, 2012; b) Dainippon Sumitomo Pharma Co.,
Ltd. EP2277861A1, 2011; synthesis of androgen receptors and
binding affinity substrates: c) Warner-Lambert Company LLC
US2006/9427A1, 2006; compounds 3a and 4a are key inter-
mediates for the synthesis of many bioactive compounds: d) N.
De Kimpe, R. Verhe, The Chemistry of a-Haloketones, a-
Haloaldehydes, and a-Haloimines, Wiley, New York, 1990;
e) “Halohydrins and derivatives”: Fiesersꢁ Reagents for Organic
Synthesis (Eds.: J. G. Smith, M. Fieser), Wiley, New York, 1990;
compound 5a can be used in the synthesis of inhibitors for the
viruses hepatitis B and C, and HIV-1: f) Institute of Medicinal
Biotechnology, EP2253621A1, 2010; g) P. Beaulieu, P. Bonneau,
R. Coulombe, P. Forgione, J. Gillard, A. Jakalian, J. Rancourt,
WO2010/37210A1, 2010; kinase inhibitors: h) D. Kay, R. Kneg-
tel, J.-D. Charrier, H. Twin, US2008/167289A1, 2008; i) Amgen
Inc., WO2009/11871A2, 2009.
[13] a) M. Marigo, S. Bachmann, N. Halland, A. Braunton, K. A.
Jørgensen, Angew. Chem. 2004, 116, 5623 – 5626; Angew. Chem.
Int. Ed. 2004, 43, 5507 – 5510; b) A. Ros, A. Magriz, H. Dietrich,
R. Fernandez, A. Alvarez, J. M. Lassaletta, Org. Lett. 2006, 8,
127 – 130.
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.
Angewandte
Communications
Communications
Biocatalysis
G.-D. Roiban, R. Agudo,
M. T. Reetz*
&&&& — &&&&
Cytochrome P450 Catalyzed Oxidative
Hydroxylation of Achiral Organic
Compounds with Simultaneous Creation
Two birds with one stone: Two new
centers of chirality are created by the
cytochrome P450 catalyzed regio-, dia-
stereo-, and enantioselective oxidative
hydroxylation of appropriate achiral
compounds. When using either wild type
cytochrome P450 BM3 or mutants gen-
erated by directed evolution, a high pref-
erence for one of the four stereotopic H
atoms is observed depending on the
nature of the X moiety in the substrate.
ꢀ
of Two Chirality Centers in a Single C H
Activation Step
6
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These are not the final page numbers!