CH-activating oxidative hydroxylation of 1-tetralones and related compounds with high regio- and stereoselectivity

Article abstract of DOI:10.1039/c4cc04925j

Mutants of P450-BM3 evolved by directed evolution are excellent catalysts in the CH-activating oxidative hydroxylation of 1-tetralone derivatives and of indanone, with unusually high regio- and enantioselectivity being observed. Similar results were achieved in the oxidative hydroxylation of tetralin and indane. The products are useful building blocks in the synthesis of a number of biologically active compounds.

Full text of DOI:10.1039/c4cc04925j

Volume 50 Number 92 28 November 2014 Pages 14275–14464
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Manfred T. Reetz et al.
CH-activating oxidative hydroxylation of 1-tetralones and related
compounds with high regio- and stereoselectivity

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CH-activating oxidative hydroxylation
of 1-tetralones and related compounds
with high regio- and stereoselectivity†
Cite this: Chem. Commun., 2014,
50, 14310
Received 27th June 2014,
Accepted 21st August 2014
Gheorghe-Doru Roiban,‡ab Ruben Agudo,‡ab Adriana Ilie,‡ab Richard Lonsdale‡ab
´
and Manfred T. Reetz*ab
DOI: 10.1039/c4cc04925j
www.rsc.org/chemcomm
Mutants of P450-BM3 evolved by directed evolution are excellent
catalysts in the CH-activating oxidative hydroxylation of 1-tetralone
derivatives and of indanone, with unusually high regio- and enantio-
selectivity being observed. Similar results were achieved in the oxidative
hydroxylation of tetralin and indane. The products are useful building
blocks in the synthesis of a number of biologically active compounds.
4-Hydroxy-1-tetralones of the type 2a–c are valuable constituents
and/or building blocks of a number of biologically active natural
products and pharmaceuticals. Examples include glucosides
from Juglans mandshurica1 containing (S)-2a, which have been
used in Chinese folk medicine to treat cancer, dermatosis and
pain, as well as the fresh pericarps of Juglans sigillata2 also
employed in folk medicine in Asia and Europe.3 More recent
examples are 8MAPK inhibitors as anti-inflammatory agents in
the treatment of respiratory diseases.4 Few catalytic methods for
the asymmetric synthesis of this class of compounds have been
developed.5 We envisioned a one-step access by P450-catalysed
Scheme 1 P450-catalysed oxidative hydroxylation of 1-tetralones (1a–c)
and 1-indanone (3).
CH-activating oxidative hydroxylation6 of readily available 1-tetra- study we first tested WT P450-BM3 and 25 previously evolved
lones 1a–c (Scheme 1), the challenge being the control of regio- and mutants10c in the hydroxylation of the model compound 1a.
enantioselectivity.7 The present study also includes 1-indanone (3) Whereas WT led to essentially complete regioselectivity in favor of
and the saturated analogs tetralin and indane as substrates. The the desired (S)-4-hydroxy-1-tetralone (2a), enantioselectivity proved to
possible use of chiral synthetic catalysts for this type of selective be poor (33% ee). In contrast, several mutants showed excellent
transformation has not been reported to date.8
regio- and enantioselectivity (Table 1, entries 2–5). All of them are
In earlier studies we utilized P450-BM3 (CYP102A1) from characterized by point mutations at residue A328, which shows that
Bacillus megaterium6,9 as the catalyst in the oxidative hydroxylation of this position is a ‘‘hot spot’’ as noted in other studies.6 Indeed, in
steroids10a and of small molecules such as cyclohexene-1-carboxylic the case of the other two substrates 1b–c, the best mutants likewise
acid ester10b and methylcyclohexane,10c regio- and stereoselectivity show amino acid substitutions at position 328. Surprisingly, in the
being controlled by directed evolution11 based on saturation reaction of 6-methoxy-1-tetralone (1b) reversal of enantioselectivity in
mutagenesis at sites aligning the binding pocket.12 In the present favour of (R)-2b was observed (95% ee), while regioselectivity reaches
only 48% (Table 1, entry 7). Therefore, saturation mutagenesis was
a
performed at residue A328 (using NNK degeneration), which
resulted in the identification of a notably improved variant A328P
showing enhanced regioselectivity in favor of the 4-position while
maintaining high enantioselectivity (94% ee) (Table 1, entry 8). This
library was then screened in an attempt to identify a catalyst for the
hydroxylation of substrate 1c that is superior to WT (essentially
racemic 2c; Table 1, entry 9). The best variant proved to be A328I,
¨
Max-Planck-Institut fur Kohlenforschung, Kaiser-Wilhelm-Platz 1,
¨
45470 Mulheim/Ruhr, Germany. E-mail: reetz@mpi-muelheim.mpg.de
b
¨
Fachbereich Chemie, Philipps-Universitat, Hans-Meerwein-Strasse,
35032 Marburg, Germany
† Electronic supplementary information (ESI) available: Compounds character-
ization, copies of NMR, GC spectra and details of docking/MD simulations. See
DOI: 10.1039/c4cc04925j
‡ These authors contributed equally.
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Table 1 P450-BM3 catalysed oxidative hydroxylation of ketones 1a–c and 3 with formation of 2a–c and 4a
Entry
Substrate
P450-BM3
Product
%-Regio.
%-Enantio.
TOFb [minÀ1
]
%-Conv.b,c
1
2
3
4
5
6
7
8
1a
1a
1a
1a
1a
1b
1b
1b
1c
1c
1c
3
WT
2a
2a
2a
2a
2a
2b
2b
2b
2c
2c
2c
4
99
98
99
99
99
97
48
85
91
50
84
98
98
95
98
33, (S)
99, (S)
96, (S)
88, (S)
97, (S)
82, (R)e
95, (R)
94, (R)
1, (S)e
99, (S)
86, (S)
76, (S)
89, (S)
93, (S)
96, (S)
1.9
3.8
—
—
—
2.2
1.3
—
6.2
3.0
—
0.9
1.9
—
86
499
56
59
39
86
64
71
88
75
92
47
96
37
45
A328F
A328K
A328R
A328Y
WT
A328F
A328P
WT
A328F
A328I
WT
A328F
A328K
A328R
d
c
10
11
12
13
14
15
3
3
3
4
4
4
0.2
a
b
Values were obtained by averaging at least three independent experiments performed with resting cells at 5 mM. TOF and conversions were
c
d
e
calculated for WT and the best mutants. Conversion calculated after 20 h. Not determined. Absolute configuration assigned after NMR analysis
of derivatized alcohols 2b–c with Mosher chloride and also comparison of the optical rotation signs of 2b–c with the optical rotation sign of 2a.
which shows enhanced regioselectivity compared with A328F, but at hydroxylation involves H-atom abstraction by the catalytically active
slight expense of enantioselectivity (Table 1, entries 10 and 11). In heme-FeVQO species (Compound I), with intermediate formation
the case of 1-indanone (3), WT P450-BM3 resulted in 98% regio- of an alkyl radical, followed by rapid C–O bond formation.5,8 Using
selectivity but moderate enantioselectivity (76% ee in favor of (S)-4), 1-tetralone (1a) as the model substrate, a docking calculation was
while variant A328R constitutes a nearly perfect catalyst in terms of first performed on the WT enzyme (details in ESI†). Previous
overall selectivity. It is interesting to note that the Pseudomonas sp. computational studies of P450-catalysed hydroxylation of several
strain 9816/11 expressing naphthalene dioxygenase (NDO) leads to different substrates have revealed that the ideal angle of approach
the enantiomeric product (R)-4.13 Thus, in this particular case NDO of the hydrogen atom undergoing abstraction should be at approxi-
and P450-BM3 variant A328R are complementary biocatalysts. The mately 1301 to the FeVQO fragment.16 Several docking poses of
performance of NDO in the oxidation of 1-tetralone derivatives has 1-tetralone (1a) were observed, but only a single pose satisfied this
not been reported to date.
criterion. In that pose, the pro-(S) hydrogen at C4 favors abstraction,
Finally we tested some of the best mutants as catalysts in the consistent with our experimental findings. In this pose, the substrate
oxidative hydroxylation of indane (5a) and tetralin (5b). In the former is positioned within a hydrophobic pocket above the heme, and in
case excellent regio- and enantioselectivity is possible using variants contact with F87. In order to investigate the conformational
A328K or A328Y (Table 2, entries 8 and 10). High regioselectivity was dynamics of 1-tetralone in the active site of the WT enzyme, two
also achieved in the C–H activating hydroxylation of indane, but independent unrestrained MD simulations were performed on this
maximum enantioselectivity did not exceed 83% ee. Noyori-type docked structure (details in ESI†). The simulations reveal significant
Ru-catalyzed reduction of indanone (3) constitutes the superior tumbling of the substrate around the hydrophobic pocket and no
strategy in this case.14 Hydroxylated products 6a–b and their hydrogen bonds are observed between the substrate carbonyl oxygen
derivatives are of great biological importance (Scheme 2).15
and the active site residues. The latter finding is consistent with the
In an attempt to characterize and understand the origin of similar selectivity patterns observed for substrates 1a and 5b, as well
selectivity of some of the variants, kinetic studies and docking/ as 3 and 5a. Substrate 1a rarely gets close enough to the FeVQO
molecular dynamics (MD) experiments were performed (see moiety for reaction to occur, however such events do take place at
ESI†). The accepted mechanism of P450-catalysed oxidative multiple instances during the timescale of the simulations (48 ns)
Table 2 P450-BM3 catalysed oxidative hydroxylation of tetralin (5a) and indane (5b)a
Entry
Substrate
P450-BM3
Product
%-Regio.
%-Enantio.
TOFb [minÀ1
]
%-Conv.b,c
1
2
3
4
5
6
7
8
9
5a
5a
5a
5a
5a
5b
5b
5b
5b
5b
WT
6a
6a
6a
6a
6a
6b
6b
6b
6b
6b
495
495
495
495
90
92
90
97
98
97
o1, (S)
83, (S)
68, (S)
78, (S)
61, (S)
56, (S)
99, (S)
98, (S)
98, (S)
97, (S)
14.9
6.1
—
—
—
4.9
13.9
—
—
—
499
499d
83
59
67
498
499d
499
499
499
A328F
A328K
A328R
A328Y
WT
A328F
A328K
A328R
A328Y
10
a
b
Values were obtained by averaging at least three independent experiments performed with resting cells at 5 mM. TOF and conversions
c
d
were calculated for WT and the best mutants. Conversion calculated after 20 h. Reactions reached total conversion after 1 h.
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P450-BM3 which ensure high degrees of regio- and stereoselectivity.
This strategy is also successful in the oxidative hydroxylation of
indane and tetralin, an approach which is currently not possible
using chiral synthetic CH-activating transition metal catalysts.8
Financial support by the Max-Planck-Society and the Arthur
C. Cope foundation is gratefully acknowledged.
Scheme 2 CH-activating oxidative hydroxylation of indane (5a) and
tetralin (5b).
Notes and references
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